WO2017156594A1 - Use of laminins as biomarkers for cancer diagnosis and prognosis - Google Patents

Use of laminins as biomarkers for cancer diagnosis and prognosis Download PDF

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WO2017156594A1
WO2017156594A1 PCT/AU2017/050245 AU2017050245W WO2017156594A1 WO 2017156594 A1 WO2017156594 A1 WO 2017156594A1 AU 2017050245 W AU2017050245 W AU 2017050245W WO 2017156594 A1 WO2017156594 A1 WO 2017156594A1
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cancer
laminin chain
laminin
expression
chain subunits
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PCT/AU2017/050245
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French (fr)
Inventor
Frederic Philippe Dominique Ghislain HOLLANDE
Sergey Alekseevich RODIN
Oscar Erik SIMONSON
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University Of Melbourne
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Priority claimed from AU2016901024A external-priority patent/AU2016901024A0/en
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Publication of WO2017156594A1 publication Critical patent/WO2017156594A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Abstract

The present disclosure relates generally to methods of measuring laminin subunits as biomarkers for cancer and cancer progression, including methods and kits for diagnosis, prognosis and treatment of cancer progression and response to treatment.

Description

USE OF LAMININS AS BIOMARKERS FOR CANCER DIAGNOSIS AND
PROGNOSIS
Field of the Disclosure
[0001] The present disclosure relates generally to methods for the diagnosis, prognosis and treatment of cancer.
Background of the Disclosure
[0002] According to Cancer Council Australia, an estimated 130,470 new cases of cancer will be diagnosed in Australia this year, with that number set to rise to 150,000 by 2020. Furthermore, 1 in 2 Australian men and 1 in 3 Australian women will be diagnosed with cancer by the age of 85. Around 19,000 more people die each year from cancer than 30 years ago, attributed primarily to population growth and aging.
[0003] The most common cancers in Australia (excluding non-melanoma skin cancer) are prostate, breast, colorectal, melanoma and lung cancer, which account for over 60% of all cancers diagnosed in Australia. From an economic perspective, cancer costs more than $4.5 billion in direct health system costs (6.9% of GDP). Moreover, around $378 million was spent on cancer research in 2000 and 2001, which accounted for 22% of all health research expenditure in Australia in that period.
[0004] Whilst the rate of cancer-related death (number of deaths per 100,000 people) has fallen by more than 16% since 1980, it remains the leading cause of death in Australia, accounting for 3 in every 10 deaths. Many of those deaths are attributed to late diagnosis, noting that prognosis typically worsens with increased cancer progression and seventy. Therefore, there remains an urgent need to improve the diagnosis, prognosis and treatment of cancer.
Summary of the Disclosure
[0005] In one aspect of the present disclosure, there is provided a method of determining whether a subject is at risk of cancer progression, the method comprising:
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and (b) comparing the level of expression of the one or more lamimn chain subunits in the biological sample to a reference value;
wherein said comparison provides an indication as to whether or not the subject is at risk of cancer progression, in an embodiment, the one or more lamimn chain subunits are selected from the group consisting of lamimn chain subunit oc3, laminin chain subunit β3, laminin chain subunit βΐ, laminin chain subunit jl and lamimn chain subunit γ2,
[0006] The inventors' findings enable a therapeutic regimen, which can be adopted or prescribed, with a view to preventing or delaying the progression of cancer in a subject. Thus, in another aspect, the present invention provides a method of treating a subject with cancer, the method comprising the steps of:
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and
(b) comparing the level of expression of the one or more lammin chain subunits in the biological sample to a reference value, wherein said comparison provides an indication as to whether or not the subject is at risk of cancer progression.
(c) identifying a subject that is at risk of cancer progression from step (b); and
(d) exposing the subject identified in step (c) as being at risk of cancer progression to a therapeutic regimen for preventing or delaying cancer progression.
[0007] In an embodiment, the one or more laminin chain subunits are selected from the group consisting of laminin chain subunit «3, laminin chain subunit β3, laminin chain subunit βΐ, laminin chain subunit γΐ and laminin chain subunit γ2.
[0008] In another aspect of the present disclosure, there is provided a method of determining a subject's sensitivity to a chemotherapeutic regimen for treating or preventing cancer, the method comprising:
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and
(b) comparing the level of expression of the one or more lamimn chain subunits in the biological sample to a reference value; wherein said comparison provides an indication as to subject's sensitivity to the chemotherapeutic regimen. In an embodiment, the one or more laminin chain subumts are selected from the group consisting of laminin chain subunit «3, laminin chain subunit β3, laminin chain subunit βΐ , laminin chain subunit γΐ and laminin chain subunit γ2.
[0009] In another aspect of the present disclosure, there is provided a kit comprising one or more reagents and/or devices for use in performing the method of the present invention, as disclosed herein.
Brief Description of the Drawings
[0010] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.
[0011] Figure 1A shows the disease-specific survival rates of colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits cc3 (LAMA3), βΐ (IAMBI ), β3 (IAMB3), γΐ (LAMC1) and γ2 (LAMC2) across all stages (Stages I-IV) of the disease. X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0012] Figure IB shows the disease-specific survival rates of Stage I colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits α3 (IAMA3), βΐ (IAMBI), β3 (IAMB3), γ! (LAMC1) and γ2 (IAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0013] Figure 1C shows the disease-specific survival rates of Stage 11 colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits a3 (LAMA3), βΐ (IAMBI ), β3 (LAMB3), γΐ (LAMC1) and j2 (LAMC2). X-axis: first row from top ::= number of months after resection; second row from top = number of patients in the L group; third (bottom) row ::= number of patients in the H group. [0014] Figure ID shows the disease-specific survival rates of Stage III colorectal cancer (CRC) patients with high (II; red) and low (L; green) expression of laminin chain subunits a3 (LAMA3), βΐ (LAMB1 ), β3 (LAMB3), γΐ (LAMCl) and j2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group,
[0015] Figure IE shows the disease-specific survival rates of Stage IV colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits o3 (LAMA3), βΐ (LAMM), β3 (LAMB3), γΐ (LAMCl) and y2 (LAMC2), X-axis: first row from top =;: number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0016] Figure 2A shows the disease-specific survival rates of colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits cc3 (LAMA3), β3 (LAMB3), and γ2 (LAMC2) across all stages (Stages I-IV) of the disease, X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0017] Figure 2B shows the disease-specific survival rates of Stage I colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits cc3 (LAMA3), β3 (LAMB3), and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top =;: number of patients in the L group; third (bottom) row = number of patients in the H group.
[0018] Figure 2C shows the disease-specific survival rates of Stage II colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits ix3 (LAMA3), β3 (LAMB3), and J2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top =:: number of patients in the L group; third (bottom) row := number of patients in the H group.
[0019] Figure 2D shows the disease-specific survival rates of Stage III colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits ix3 (LAM A3), β3 (LAMB3), and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top ::= number of patients in the L group; third (bottom) row = number of patients in the H group.
[0020] Figure 2E shows the disease-specific survival rates of Stage IV colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits a3 a3 (LAMA3), β3 (LAMB3), and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0021] Figure 3A shows the disease-specific survival rates of colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of lammin chain subunits oc3 (LAMA3), βΐ (LAMBl), β3 (LAMB3), and y2 (LAMC2) across all stages (Stages I-IV) of the disease. X-axis: first row from top := number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0022] Figure 3B shows the disease-specific survival rates of Stage I colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of lammin chain subunits c/3 (LAMA3), βΐ (LAMBl), β3 (LAMBS), and γ2 (LAMC2). X-axis: first row from top number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0023] Figure 3C shows the disease-specific survival rates of Stage II colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laniinm chain subunits <x3 (LAMA3), (LAMB ), β3 (LAMBS), and v2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0024] Figure 3D shows the disease-specific survival rates of Stage III colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminm chain subunits «3 (LAMA3), βΐ (LAMBl), β3 (LAMBS), and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row := number of patients in the H group. [0025] Figure 3E shows the disease-specific survival rates of Stage IV colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits a3 (LAMA3), βΐ (LAMB1), β3 (LAMB3), and γ2 (LAMC2). X-axis: first row from top number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group,
[0026] Figure 4A shows the disease-specific survival rates of colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of lammin chain subunits oc3 (LAMA3), βΐ (LAMB1), jl (LAMC1) and γ2 (LAMC2) across all stages (Stages I-IV) of the disease. X-axis: first row from top := number of months after resection; second row from top :=: number of patients in the L group; third (bottom) row := number of patients in the H group,
[0027] Figure 4B shows the disease-specific survival rates of Stage I colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of lammin chain subunits oc3 (LAMA3), βΐ (LAMB1), jl (LAMC1 ) and γ2 (LAMC2). X-axis: first row from top number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0028] Figure 4C shows the disease-specific survival rates of Stage II colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits o 3 (LAMA3), β! (LAMB1), γΐ (LAMC1) and y2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0029] Figure 4D shows the disease-specific survival rates of Stage III colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits «3 (LAMA3), βΐ (LAMB1), γΐ (LAMC1) and j2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row := number of patients in the H group.
[0030] Figure 4E shows the disease-specific survival rates of Stage IV colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits α3 (LAMA3), βΐ (LAMB1), γΐ (LAMC1) and γ2 (LAMC2). X-axis: first row from top number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group,
[0031] Figure 5A shows the disease-specific survival rates of colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits oc3 (LAMAS) and γ2 (LAMC2) across all stages (Stages I-IV) of the disease. X-axis: first row from top :=: number of months after resection; second row from top = number of patients in the L group; third (bottom) row := number of patients in the H group.
[0032] Figure 5B shows the disease-specific survival rates of Stage I colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits c 3 (LAMA3) and γ2 (LAMC2). X-axis: first row from top :=: number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0033] Figure 5C shows the disease-specific survival rates of Stage II colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits cc3 (LAMA3) and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row =;: number of patients in the H group.
[0034] Figure 5D shows the disease-specific survival rates of Stage III colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits (x3 (LAMA3) and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top := number of patients in the L group; third (bottom) row =:: number of patients in the H group.
[0035] Figure 5E shows the disease-specific survival rates of Stage IV colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits ix3 (LAMA3) and γ2 (LAMC2). X-axis: first row from top ::= number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group. [0036] Figure 6A shows the disease-specific survival rates of colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunit γ2 (LAMC2) across all stages (Stages I-IV) of the disease. X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group,
[0037] Figure 6B shows the disease-specific survival rates of Stage I colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunit y2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top :=: number of patients in the L group; third (bottom) row = number of patients in the H group.
[0038] Figure 6C shows the disease-specific survival rates of Stage II colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunit γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top :=: number of patients in the L group; third (bottom) row := number of patients in the H group.
[0039] Figure 6D shows the disease-specific survival rates of Stage III colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunit γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0040] Figure 6E shows the disease-specific survival rates of Stage IV colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminm chain subunit γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0041 ] Figure 7A shows the disease-specific survival rates of colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of lammin chain subunits a3 (LAMA3), βΐ (LAMB l), γΐ (LAMC1) and γ2 (LAMC2) across all stages (Stages I-IV) of the disease. X-axis: first row from top := number of months after resection; second row from top :=: number of patients in the L group; third (bottom) row := number of patients in the H group,
[0042] Figure 7B shows the disease-specific survival rates of Stage I colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits ix3 (LAMA3), yl (LAMCl) and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group,
[0043] Figure 7C shows the disease-specific survival rates of Stage II colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits cc3 (LAMA3), yl (LAMCl) and γ2 (LAMC2), X~axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0044] Figure 7D shows the disease-specific survival rates of Stage III colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits cc3 (LAMA3), γΐ (LAMCl) and yl (LAMC2), X-axis: first row from top = number of months after resection; second row from top ::= number of patients in the L group; third (bottom) row = number of patients in the H group.
[0045] Figure 7E shows the disease-specific survival rates of Stage IV colorectal cancer (CRC) patients with high (H; red) and low (L; green) expression of laminin chain subunits a3 (LAMA3), γΐ (LAMCl) and yl (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0046] Figure 8 shows the gene expression of laminin chain subunits a3 (LAMA3), βΐ (LAMBl), β3 (LAMB3), yl (LAMCl ) and yl (LAMC2) for each of the high risk group (H; red) and low risk group (L; green) of patients with colorectal cancer (A), liver cancer (B), pancreatic cancer (C) and invasive breast carcinoma (D).
[0047] Figure 9 shows the disease-specific survival rates of liver cancer (A), pancreatic cancer (B), bile duct cancer (C), glioma (D), glioblastoma (E), melanoma (F), prostate cancer (G), gastric cancer (H), bladder cancer (I) and invasive breast carcinoma (J) patients with high (H; red) and low (L; green) expression of laminin chain subunits «3 (LAMA3), βΐ (LAMB1), β3 (LAMB3), γΐ (LAMCl) and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0048] Figure 10 shows the disease-specific survival rates of Stage I colorectal cancer patients with (A) high (H; red) and low (L; green) expression of laminin chain subunit γ2; or (B) high (H; red) and low (L; green) expression of laminin chain subunits c¾3 (LAMA3), βΐ (LAMB1), β3 (LAMB3), γΐ (LAMCl) and v2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0049] Figure 11 shows the disease-specific survival rates of Stage II colorectal cancer patients with (A) high (H; red) and low (L; green) expression of laminin chain subunit γ2; or (B) high (H; red) and low (L; green) expression of laminin chain subunits c¾3 (LAMA3), βΐ (LAMBl), β3 (LAMB3), γΐ (LAMCl) and v2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0050] Figure 12 shows the disease-specific survival rates of Stage III colorectal cancer patients with (A) high (H; red) and low (L; green) expression of laminin chain subunit γ2; or (B) high (H; red) and low (L; green) expression of laminin chain subunits c¾3 (LAMA3), βΐ (LAMBl), β3 (LAMB3), γΐ (LAMCl) and v2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0051] Figure 13 shows the disease-specific survival rates of Stage IV colorectal cancer patients with (A) high (H; red) and low (L; green) expression of laminin chain subunit γ2; or (B) high (H; red) and low (L; green) expression of laminin chain subunits c¾3 (LAMA3), βΐ (LAMBl), β3 (LAMB3), yl (LAMCl) and γ2 (LAMC2). [0052] Figure 14 shows the disease-specific survival rates of Stage 0 (A) and Stage 1 (B) pancreatic cancer patients with high (H; red) and low (L; green) expression of lamimn chain suhumts a3 (LAMA3), βΐ (LAMB1 ), β3 (LAMB3), γΐ (LAMC1) and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0053] Figure 15 shows the disease-specific survival rates of female (A) and male (B) colorectal cancer patients from the SurvExpress colon metabase cohort with high (H; red) and low (L; green) expression of laminin chain subunits a3 (LAMA3), βΐ (LAMB1), β3 (LAMB 3), γΐ (LAMC1) and γ2 (LAMC2). X-axis: first row from top = number of months after resection; second row from top = number of patients in the L group; third (bottom) row =:: number of patients in the H group.
[0054] Figure 16 shows the disease-specific survival rates of female (A) and male (B) colorectal cancer patients from the Smith-Beauehamp cohort with high (H; red) and low (L; green) expression of lamimn chain subunits 0(3 (LAMA3), βΐ (LAMBl), β3 (LAMB3), γΐ (LAMCl ) and γ2 (I.AMC2). X-axis: first row from top := number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0055] Figure 17 shows the disease-specific survival rates of female (A) and male (B) colorectal cancer patients from the Sheffer-Domany cohort (GSE41258) with high (H; red) and low (L; green) expression of laminin chain subunits cc3 (LAMA3), βΐ (LAMBl), β3 (LAMB3), γΐ (LAMCl) and γ2 (LAMC2). X-axis: first row from top := number of months after resection; second row from top = number of patients in the L group; third (bottom) row = number of patients in the H group.
[0056] Figure 18 is a series of photomicrographs showing the gene expression profile of laminin chain subunits cd, c 2, cc3, a4, cc5, βΐ, β2, β3, γΐ, γ2 and γ3 in seven colorectal cancer cell lines - COLO205, SW480, RK0, HT-29, HT-55, T-84 and COLO320HSR. Total RNA was isolated from the ceil lines, lamimn chain subunit mRNA was reverse transcribed and the corresponding cDNA was amplified by RT-PCR using laminin chain subunit specific primers, as shown in Table 1 elsewhere herein. RT-PCR amplification products were then run through an ethidium bromide-containing agarose gel.
[0057] Figure 19 shows the % viability of six colorectal cancer cell lines following exposed to different concentrations of the epidermal growth factor receptor (EGFR) inhibitor, Gefitinib, in vitro. The six colorectal cancer cell lines can be separated into those with a high Iammin score (HT55, HT29 and SW480) and a low laminin score (Colo205, Colo320HSR and RKO),
[0058] Figure 20 shows the % viability of six colorectal cancer cell lines following exposed to different concentrations of the tyrosine kinase inhibitor, Sorafenib, in vitro. The six colorectal cancer cell lines can be separated into those with a high laminin score (HT55, HT29 and SW480) and a low laminin score (Colo205, Colo320HSR and RKO).
Detailed Description
[0059] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
[0060] The articles "a" and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "an agent" means one agent or more than one agent.
[0061] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.
Methods of diagnosis and prognosis
[0062] The present disclosure is predicated, at least in part, on the inventors' surprising findings that the expression of one or more Iammin chain subunits can (i) improve the prediction of disease-specific survival and recurrence in a patient with cancer and (ii) predict a clinical response to treatment with a chemotherapeuttc agent in a patient with cancer. The present inventors have therefore shown, for the first time, that the expression of one or more laminin chain subumts is a suitable diagnostic and prognostic indicator of cancer progression and response to cancer treatment.
[0063] In one aspect of the present disclosure, there is provided a method of determining whether a subject is at risk of cancer progression, the method comprising:
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and
(b) comparing the level of expression of the one or more laminin chain subumts in the biological sample to a reference value;
wherein said comparison provides an indication as to whether or not the subject is at risk of cancer progression.
[0064] In another aspect of the present disclosure, there is provided a method of stratifying a subject to a therapeutic regimen for treating cancer, the method comprising the steps of:
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer;
(b) comparing the level of expression of the one or more laminin chain subunits in the biological sample to a reference value, wherein said comparison provides an indication as to whether or not the subject is at risk of cancer progression; and
(c) stratifying the subject identified in step (b) as being at risk of cancer progression to a therapeutic regimen for treating the cancer.
Laminins
[0065] Laminins are a family of αβγ heterotrimeric glycoproteins usually present in the basal lamina of the basement membranes (BM) (see Domogatskaya et al, "Functional Diversity of Laminins". Annual Review of Ceil and Developmental Biology 28.1 (2012): 523-553). These macromolecules, secreted and incorporated as part of the extracellular matrix (ECM), are often found to be at least 400kDa in size. The laminin family of glycoproteins is an integral part of the structural scaffolding of tissues and is vital for their maintenance and survival. More than 15 laminin isoforms have so far been discovered in vivo and named in accordance with the combination of five a laminin cham subunits (ocl - cc5), four β laminin chain subunits (β'1-β4), and three γ laminin chain subunits (jl-jY) (see, e.g., Aumailley et al. "A Simplified Laminin Nomenclature". Matrix Biology 24.5 (2005): 326-332). As an example, iaminin-521 (LN-521) is comprised of an 5 subunit (encoded by the IAMA5 gene), a β2 subunit (encoded by the LAMB2 gene), and a γΐ subunit (encoded by the LAMC1 gene). Laminins are ubiquitously expressed, although the expression of specific laminin isoforms is largely cell- and tissue-dependent.
[0066] As disclosed herein, the present inventors have shown that the expression of the one or more laminin cham subunits have prognostic capacity with respect to cancer progression, including tumour progression following resection (also known as recurrence).
[0067] The present inventors have also shown that the expression of laminin cham γ2 in a biological sample from a patient with colorectal cancer is sufficient to predict whether that patient is at risk of cancer progression {i.e., recurrence). Thus, in an embodiment, the one or more laminin chain subunits comprises or consists of laminin chain subunit γ2.
[0068] The present inventors have also shown that the combined expression of two or more laminin chain subunits can significantly improve the predictive capacity of the expression profile for identifying whether or not a patient is at risk of cancer progression. For instance, the inventors have found that the predictive capacity of the expression profile can be improved by measuring the expression of the panel of one or more laminin chain subunits as shown in Table 2 herein.
[0069] In an embodiment, the one or more laminin chain subunits are selected from the group consisting of laminin chain subunit c 3, laminin chain subunit β3, laminin chain subunit βΐ, laminin cham subunit γΐ and laminin chain subunit γ2. In another embodiment, the one or more laminin chain subunits comprises c¾3 and "(1. In another embodiment, the one or more laminin chain subunits comprise laminin chain subunits cc3, β3 and γ2.
[0070] The present inventors have also found that laminin chain subunit βΐ or laminin chain subunit γΐ, each alone or in combination, further improves the predictive capacity of the expression profile for identifying whether or not a patient is at risk of cancer progression. Thus, in an embodiment, the one or more laminin chain subunits comprises laminin chain subunit βΐ or laminin chain subunit γΐ . In another embodiment the one or more laminin chain subunits comprise cc3, β ΐ, β3 and j2. In another embodiment, the one or more lammin chain subunits comprise cc3, γΐ and γ2. In another embodiment, the one or more lammin chain subunits comprise ίχ3, β3, yl and y2.
[0071] The present inventors have also shown that the combined expression of laminin chain subunits βΐ and γΐ can provide even greater predictive capacity in comparison to the expression of either laminin chain subunit βΐ or laminin chain subunit γΐ . Thus, in an embodiment, the one or more laminin chain subunits comprise laminin chain subunit βΐ and lammin chain subunit γΐ . In another embodiment, one or more laminin chain subunits comprise laminin chain subunits oc3, β ΐ , β3, γΐ and y2. In another embodiment, the one or more laminin chain subunits comprise cc3, β ΐ, γΐ and y2.
[0072] It is to be understood that the expression of any combination or permutation of laminin chain subunits can be used in accordance with the present invention. In an embodiment, the one or more laminin chain subunits are selected from the group consisting of laminin chain subunit o3, laminin chain subunit β3, laminin chain subunit βΐ, laminin chain subunit γΐ and laminin chain subunit y2. In another embodiment, the one or more laminin chain subunits comprises at least lammin chain subunit βΐ and/or laminin chain subunit yl .
[0073] The phrase "one or more laminin chain subunits" is to be understood an meaning at least one laminin cham subunit, at least 2 lammin chain subunits, at least 3 laminin chain subunits, at least 4 laminin cham subunits, at least 5 lammin chain subunits, at least 6 laminin chain subunits, at least 7 laminin chain subunits, at least 8 laminin chain subunits, at least 9 laminin chain subunits, at least 10 laminin chain subunits, at least I I laminin chain subunits, at least 12 laminin chain subunits, at least 1 3 laminin chain subunits, at least 14 laminin chain subunits, at least 1 5 laminin chain subunits, and so on. The present disclosure is broadly directed to the expression of one or more of the 1 5 or so known lammin chain subunits (e.g., five a lammin chain subunits (ccl -cc5), four β lammin chain subunits (β1 -β4), and three γ laminin chain subunits (γ1-γ3)), as described, for example, in Aumailley, Cell Adhesion & Migration-, 7(1): 48-55) or Aumailley ei al (2005 ; "A Simplified Laminin Nomenclature". Matrix Biology 24(5): 326-332), including homologs thereof. In an embodiment, the one or more laminin chain subunits are selected from the group consisting of cc3, β3, βΐ , yl and γ2. It is to be understood that, where a combination of two or more laminin chain subunits is selected and employed in accordance with the present invention, it is desirable that the one or lammin chain subunits that are selected for combination with one or more other laminin chain subunits in the combination will improve, or at least will not negate or reduce, the predictive capacity of the one or more other laminin chain subunits. Methods of assessing whether or not one or more lammin chain subunits improve or at least do not negate or reduce the capacity of the one or more other laminin chain subunits in a combination to predict cancer progression and/or response to treatment, as herein described, would be familiar to persons skilled in the art having regard to the present disclosure.
[0074] The term "laminin chain subunit" includes vertebrate and non-vertebrate laminin chain subunits. Suitable vertebrates that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g. , dogs), felines (e.g. , cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. In an embodiment, the laminin chain subunit is a human lammin chain subunit.
[0075] The amino acid and nucleic acid sequences of laminin chain subunits will be familiar to persons skilled in the art and accessible through public databases such as the National Center for Biotechnology Information (NCBI). In an embodiment, the human lammin chain subunit βΐ is encoded by the nucleic acid sequence as shown in GenBank accession number NM 002291. In an embodiment, the human lammin chain subunit γΐ is encoded by the nucleic acid sequence as shown in GenBank accession numbers NM 002293. In an embodiment, the human laminin chain subunit a3 is encoded by the nucleic acid sequence as shown in GenBank accession number NM_000227. In an embodiment, the human laminin chain subunit β2 is encoded by the nucleic acid sequence as shown in GenBank accession number NM 002292. In an embodiment, the human laminin chain subunit β3 is encoded by the nucleic acid sequence as shown in GenBank accession number NM_000228. In an embodiment, the human laminin chain subunit γ2 is encoded by the nucleic acid sequence as shown in GenBank accession numbers NM_005562. In an embodiment, the human laminin chain subunit oc5 is encoded by the nucleic acid sequence as shown in GenBank accession number NM 005560. In an embodiment, the human laminin chain subunit al is encoded by the nucleic acid sequence as shown in GenBank accession number NM_005559. In an embodiment, the human laminin chain subunit 0(4 is encoded by the nucleic acid sequence as shown in GenBank accession number NM 001105206. In an embodiment, the human laminin chain subunit βΐ is encoded by the nucleic acid sequence as shown in GenBank accession number NM 002291. In an embodiment, the human laminin chain subunit β4 is encoded by the nucleic acid sequence as shown in GenBank accession number NM_001318046. in an embodiment, the human laminin chain subunit γΐ is encoded by the nucleic acid sequence as shown in GenBank accession number NM 002293. In an embodiment, the human laminin chain subunit cc2 is encoded by the nucleic acid sequence as shown in GenBank accession number NM 000426. In an embodiment, the human laminin chain subunit γ3 is encoded by the nucleic acid sequence as shown in GenBank accession number \\i 006059.
[0076] As used herein, the term "laminin chain subunit" also includes homologs thereof. The term "homolog" typically refers to a peptide with similar biological activity, although differs in amino acid sequence at one or more ammo acid positions when the sequences are aligned. For example, the amino acid sequences of two homologous laminin chain subunits may differ only by one amino acid residue within the aligned amino acid sequences of five to ten amino acids. Alternatively, two homologous laminin chain subunits of ten to fifteen amino acids may differ by no more than two amino acid residues when aligned. Alternatively, two homologous laminm chain subunits of fifteen to twenty or more amino acids can differ by up to three amino acid residues when aligned. Homologous laminm chain subunits may also differ by up to approximately 5%, 10%, 20% or 25% of the amino acid residues when the amino acid sequences of the two homologs are aligned.
[0077] Homologs of laminin chain subunits may be found in the same species (i.e., between two or more individuals of the same species), in related species and/or sub-species, or in different species. For example, for a human laminm chain subunit, homologs include those found in non-human vertebrates and non-vertebrates. Suitable vertebrates that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), capnnes (e.g., goats), porcines (e.g. , pigs), equines (e.g. , horses), canines (e.g. , dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. A preferred homolog is one found in a primate (e.g., a human, ape, monkey, chimpanzee). Alternatively, a laminin chain subunit homolog may be from the same species (e.g., human).
[0078] Generally, homologs will have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a particular amino acid or nucleotide sequence, as determined, for example, by sequence alignment programs known in the art using default parameters (see, e.g., Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453). In specific embodiments, the percent identity between nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. An non-limiting set of parameters (and the one that should be used unless otherwise specified) includes a Biossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. In some embodiments, the percent identity or similarity between amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W, Miller (1989, Cahios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight, residue table, a gap length penalty of 12 and a gap penalty of 4.
[0079] In an embodiment, the laminin chain subumt is a homolog of human laminin chain subumt cc3 encoded by the nucleic acid sequence as shown in GenBank accession number NM 000227. In an embodiment, the laminin chain subunit is a homolog of human laminin chain subunit β2 encoded by the nucleic acid sequence as shown in GenBank accession number NM 002292. In an embodiment, the laminin chain subunit is a homolog of human lammin chain subunit β3 encoded by the nucleic acid sequence as shown in GenBank accession number NM 000228. In an embodiment, the laminin chain subunit is a homolog of human laminin chain subunit γ2 encoded by the nucleic acid sequence as shown in GenBank accession numbers NM 005562, In an embodiment, the laminin chain subunit is a homolog of human laminin chain subunit cc5 encoded by the nucleic acid sequence as shown in GenBank accession number NM 005560. In an embodiment, the laminin chain subunit is a homolog of human laminm chain subunit ctl encoded by the nucleic acid sequence as shown in GenBank accession number NM_005559. In an embodiment, the laminin chain subunit is a homolog of human laminin chain subunit a4 encoded by the nucleic acid sequence as shown in GenBank accession number NM_001105206. In an embodiment, the laminin chain subunit is a homolog of human laminin chain subunit βΐ encoded by the nucleic acid sequence as shown in GenBank accession number NM 002291. In an embodiment, the lammin chain subunit is a homolog of human laminin chain subunit β4 encoded by the nucleic acid sequence as shown in GenBank accession number NM_001318046. In an embodiment, the laminin chain subunit is a homolog of human laminin chain subunit yl encoded by the nucleic acid sequence as shown in GenBank. accession number NM_002293. In an embodiment, the laminm chain subunit is a homolog of human laminin chain subunit oc2 encoded by the nucleic acid sequence as shown in GenBank accession number NM 000426, In an embodiment the laminin chain subunit is a homolog of human laminm chain subumt γ3 encoded by the nucleic acid sequence as shown in GenBank accession number NM_006059.
[0080] For example, in an embodiment, a homolog of human laminin chain subunit a3 is encoded by the nucleic acid sequence as shown in GenBank accession number NM 000227.4.
Measuring the expression of laminin chain subunits
[0081] The term "expression" is used herein in its broadest context to denote a measurable presence of laminm chain subunits, including the production of RNA message (gene expression) or translation of RNA message into proteins or polypeptides (protein expression). For example, the term "laminm chain subunit expression" includes (i) the production of laminin chain subunit RNA message (i.e., laminin chain subunit gene expression), (ii) the translation of laminin chain subunit RNA message into laminin chain subunit protein and/or (lii) the transport of laminin chain subunit protein to the cell surface.
[0082] Suitable methods for measuring the expression of laminin chain subunit expression would be known to persons skilled in the art. In some embodiments, it may be desirable to measure the expression of laminin chain subunits at the protein level. It will be understood that, in some instances, it may be more desirable to measure a gene expression product, such as transcript (e.g., mRNA) levels, as described elsewhere herein.
[0083] Methods of measuring expression products such as proteins and transcripts are known to persons skilled in the art, with illustrative examples described herein. In some embodiments, measuring the expression of laminin chain subunits comprises determining the level of mRNA encoding the laminin chain subunits.
[0084] As used herein the terms "level" and "amount" are used interchangeably herein to refer to a quantitative amount (e.g., moles or number), a semi-quantitative amount, a relative amount (e.g., weight % or mole % within class or a ratio), a concentration, and the like. Thus, these terms encompasses absolute or relative amounts or concentrations of laminin chain subunits in a sample, including levels in a population of subjects represented as mean levels and standard deviations, as shown in some of the Figures herein.
[0085] Laminin chain subunits may be quantified or detected using any suitable technique, including, but not limited to, nucleic acid- and protein-based assays. In illustrative nucleic acid-based assays, nucleic acid is isolated from cells contained in a biological sample according to standard methodologies (Sambrook, et al, 1989, supra and Ausubel et al, 1994, supra). The nucleic acid is typically fractionated (e.g., poly A+ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the laminin chain subunit-encoding nucleotide sequences present in a given sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PGR), which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800, 159, Ausubel et al (supra), and in Innis et al, ("PGR Protocols", Academic Press, Inc., San Diego Calif, 1990). Briefly, in PGR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the laminin chain subunit nucleotide sequence. An excess of deoxynucleotide triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If a cognate laminin chain subunit nucleotide sequence is present in a sample, the primers will bind to the sequence and the polymerase will cause the primers to be extended along the sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the laminin chain subunit nucleotide sequence to form reaction products, excess primers will bind to the laminin chain subunit nucleotide sequence and to the reaction products and the process is repeated. A reverse transcriptase PGR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.
[0086] In an embodiment, the template-dependent amplification involves quantification of transcripts in real-time. For example, RNA or DNA may be quantified using the Real-Time PGR technique (Higuchi, 1992, et al. Biotechnology 10: 413-417). By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture, if the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. In specific embodiments, multiplexed, tandem PCR (MT-PCR) is employed, which uses a two-step process for gene expression profiling from small quantities of RNA or DNA, as described for example in US Pat. Appl. Pub. No. 20070190540. In the first step, RNA is converted into cDNA and amplified using multiplexed gene specific primers. In the second step each individual gene is quantitated by real time PCR.
[0087] In some embodiments, laminin chain subunit nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provides different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labelled) under conditions that promote denaturation and rehybridisation. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above. Following detection/quantification, one may compare the results seen in a given subject with a control reaction or a statistically significant reference group or population of control subjects as defined herein. In this way, it is possible to correlate the amount of a biomarker nucleic acid detected with the likelihood that a subject is at risk of cancer progression,
[0088] Also contemplated herein are bioehip-based technologies such as those described by Hacia et al. (1996, Nature Genetics 14; 441 -447) and Shoemaker et al. (1996, Nature Genetics 14: 450-456). Briefly, these techniques involve quantitative methods for analysing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high-density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994, Proc. Natl Acad. Sci. U.S.A. 91 : 5022-5026); Fodor et al (1991 , Science 251 : 767-773). Briefly, nucleic acid probes to laminin chain subunit nucleotide sequences are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein. The nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed laminin chain subunit nucleotide sequences, i.e., the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occur. This complementarity need not be perfect; there may be any number of base pair mismatches, which will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. In certain embodiments, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e. have some sequence in common), or separate.
[0089] In an illustrative biochip analysis, oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more biomarker polynucleotides under conditions favouring specific hybridization. Sample extracts of DNA or RNA, either single or double-stranded, may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme. Suitable DNA, which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et ah, 1994, supra, and Sam brook, et ah, et ah, 1989, supra.
[0090] Suitable RNA, which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et ah 1994, supra and Sambrook, et ah 1989, supra).
[0091] cDNA may be fragmented, for example, by som cation or by treatment with restriction endonucleases. Suitably, cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions. Alternatively, fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.
[0092] Usually the target biomarker polynucleotides are detectably labelled so that their hybridization to individual probes can be determined. The target polynucleotides are typically detectably labelled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions {e.g., Eu'*), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide. In some embodiments, in which an enzyme is used as a direct visual label, biotmylated bases are incorporated into a target polynucleotide,
[0093] The hybrid-forming step can be performed under suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA. In this regard, reference may be made, for example, to "Nucleic Acid Hybridization, A Practical Approach (Homes and Higgins, eds,, IRL press, Washington D.C., 1985), In general, whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants. Such variables also influence the time required for hybridization. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation.
[0094] After the hybrid-forming step, the probes are washed to remove any unbound nucleic acid with a hybridization buffer. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridized to a target polynucleotide. The hybridization reactions are then detected to determine which of the probes has hybridized to a corresponding target sequence. Depending on the nature of the reporter molecule associated with a target polynucleotide, a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a coloured colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light. In such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe: target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymatically generated colour spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array, in the case of a nucleic acid array, the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In certain embodiments, oligonucleotide probes specific for different biomarker polynucleotides are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5,143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labelled microbeads, the reaction may be detected using flow cytometry.
[0095] In other illustrative embodiments, laminin chain subunit protein levels can be measured using protein-based assays known in the art. For example, an antibody-based technique may be employed to determine the level of a laminin chain subunit in a sample, non-limiting examples of which include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
[0096] Protein-capture arrays that permit simultaneous detection and/or quantification of a large number of proteins may also be employed. For example, low-density protein arrays on filter membranes, such as the universal protein array system (Ge, 2000 Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector. Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-treatment. Exemplary protein capture arrays include arrays comprising spatially addressed antigen- binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins. Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibody arrays have been reported (see, e.g., Lopez et ai, 2003 J. Chromatogr. B 787: 19- 27; Cahill, 2000 Trends in Biotechnology 7:47-51; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat. App. Pub. 2003/0003599; PCT publication WO 03/062444; PCT publication WO 03/077851 ; PCT publication WO 02/59601 ; PCT publication WO 02/39120; PCT publication WO 01/79849; PCT publication WO 99/39210). Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.
[0097] Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include colour coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDots™, available from Quantum Dots), and barcoding for beads (UltraPiex™, available from Smartbeads) and multimetal microrods (Nanobarcodes™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and Bio Array Solutions). Where particles are used, individual protein-capture agents (e.g., anti-laminin chain subunit antibodies or laminin chain subunit-binding fragments thereof) are typically attached to an individual particle to provide the spatial definition or separation of the array. The particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a microtitre plate or in separate test tubes.
[0098] In an illustrative example, a protein sample, which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub. 2002/0055186), is delivered to a protein- capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array. Next, the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array, in certain embodiments, the amount of the second protein in the sample is already known or known to be invariant.
[0099] Another illustrative example of a protein-capture array is a Luminex-based multiplex assay, which is a bead-based multiplexing assay, where beads are internally dyed with fluorescent dyes to produce a specific spectral address. Biomolecules (such as an oligo or antibody) can be conjugated to the surface of beads to capture anah'tes of interest; that is, lammin chain protein or a nucleic acid molecule encoding same. Flow cytometric or other suitable imaging technologies known to persons skilled in the art can then be used for characterization of the beads, as well as for detection of analyte presence. The Lurninex technology enables are large number of proteins, genes or other gene expression products (e.g. , 100 or more, 200 or more, 300 or more, 400 or more) to be detected using very small sample volume (e.g., in a 96 or 384-well plate). In some embodiments, the protein-capture array is Bio-Plex Luminex-100 Station (Bio-Rad).
[0100] In some embodiments, the expression of the lammin chain subunits can be normalized against a housekeeping biomarker. The term "housekeeping biomarker" refers to a biomarker or group of biomarkers (e.g., polynucleotides and/or polypeptides), winch are typically found at a constant level in the cell type(s) or tissue(s) being analysed and across the conditions being assessed. In some embodiments, the housekeeping biomarker is a "housekeeping gene. " A "housekeeping gene" refers herein to a gene or group of genes which encode proteins whose activities are essential for the maintenance of cell function and which are typically found at a constant level in the cell type(s) being analysed and across the conditions being assessed.
[0101] It would be understood by those skilled in the art, as described elsewhere herein, that the method of analyzing the expression of laminin chain subunits in a biological sample can be quantitative, semi-quantitative or qualitative in nature. For example, quantitative analyses will typically provide a concentration or number of a laminin chain subunit nucleic acid molecule or protein in the sample within an appropriate error margin (e.g., mean +/- standard deviation). By contrast, semi-quantitative or qualitative analyses will typically provide an indication of the relative amount of a laminin chain subunit in a sample. This may involve a comparison of an amount of a lammin chain subunit protein in a first sample with an amount of a laminin chain subunit protein in a second sample and making a determination as to the relative amount of the laminin chain subunit protein between the first and second samples.
[0102] In some embodiments, qualitative analyses using an arbitrary number system is used to specify the expression of the one or more laminin chain subunits. By way of example only, the level of expression of each of the laminin chain subunits can be designated as Low, Medium or High and assigned a corresponding value of 1 , 5 and 10, respectively. Where the method comprises measuring the expression of a panel of two or more laminin chain subunits, the expression for each biological sample can be given as a total value or score; for example, the total value or score being the sum of values assigned to the expression of each of the lamimn chain subunits in the panel of one or more lammin chain subunits. For example, where the method comprises measuring the expression of lamimn chain subunits ct3, βΐ, β3 and γ2, and the level of expression for the laminin chain subunits in the biological sample is Low, Medium, Low and High, respectively, then the total value or score for that sample will be 17 (i.e., 1 (for Low) +5 (for Medium) +1 (for Low) +10 (for High)), in an embodiment described herein, where the method comprises measuring the expression of lammin chain subunits βΐ and γΐ, and the level of expression for the lammin chain subunits in the biological sample is Low and Medium, respectively, then the total value or score for that sample will be 6 (i.e., 1 (for Low) +5 (for Medium)). In another embodiment, the level of expression of each of the one or more laminin chain subunits are grouped into five score categories for each laminin chain subunit. For example, where measuring the expression of the one or more laminin chain subunits comprises measuring the mRNA levels of the one or more laminin chain subunits (by densitometric analysis of RT-PCR products following their separation through an ethidium bromide-containing agarose gel, as described elsewhere herein), the following scores may be given:
(i) Score 0 = no signal;
(ii) Score 1 = corrected density value between 0 and 1 ;
(iii) Score 2 = corrected density value >1 and <5; (iv) Score 3 = corrected density value >5 and <10; and
(v) Score 4 =:: corrected density value >10.
[0103] The total laminin score is then calculated as the sum of individual scores for each of the laminin chain subumts. For example, where the method comprises measuring the expression of laminin chain subumts 0(3, βΐ, β3 and γ2, and the level of corrected density for the laminin chain subunits in the biological sample is (1), (>1 and <5), (>10) and (>5 and <10), respectively, then the total laminin score for that sample will be 10 (i. e., 1 + 2 + 4 + 3),
[0104] It will be understood by persons skilled in the art that, where a comparison is made to a reference value, then the manner in which the biological sample is assessed for the expression of the one or more laminm chain subunits should be substantially identical to the manner in which the reference value is derived in order to ensure that an appropriate comparison can be made for the purposes of determining risk or sensitivity to a chemotherapeutic regimen, as herein described.
[0105] It an embodiment, the methods disclosed herein comprise measuring the expression of a laminin chain subunit gene expression product in the biological sample by quantitative or semi-quantitative PGR, methods of which will be known to persons skilled in the art, an illustrative example of which are disclosed elsewhere herein.
[0106] In an embodiment, the step of measuring the expression of the one or more laminin chain subunits in the biological sample comprises measuring the gene expression of the one or more laminin chain subunits in the biological sample.
Cancer
[0107] Whilst laminins are ubiquitously expressed, the expression of specific isoforms is both cell- and tissue-dependent, recognized by integrins and other cell-surface receptors (see Aumailley, Monique. "The Laminin Family" . Cell Adhesion & Migration 7.1 (2013): 48-55 and Aumailley, Monique, and Neil Smyth. "The Role Of Laminins In Basement Membrane Function". J Anatomy 193.1 (1998): 1 -21 ). Altered function and/or expression of laminins can result in a wide range of cellular defects and abnormal conditions, including tumour invasive and metastasis. The present inventors have demonstrated the ability of laminin chain subunit expression to predict cancer progression and sensitivity to chemotherapeutic regimens in colorectal cancer, liver cancer, and pancreatic cancer, as well as other solid tumours or cancers, such as bile duct cancer, glioma, glioblastoma, melanoma, prostate cancer, gastric cancer, bladder cancer and invasive breast carcinoma.. Thus, in an embodiment disclosed herein, the cancer is selected from the group consisting of colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, glioma, glioblastoma, melanoma, prostate cancer, gastric cancer, bladder cancer and invasive breast carcinoma.
[0108] In an embodiment, the methods disclosed herein are for determining whether the subject is at risk of colorectal cancer progression, including the risk of recurrence following resection of a primary colorectal tumour.
[0109] Colorectal cancer (CRC), also known as adenocarcinoma of the colon and rectum, is the third most common cancer, the fourth most common cause of cancer death, and the second most common cancer in terms of the number of individuals living with cancer five years after diagnosis worldwide. CRC typically begins as an uncontrolled growth of cells in the walls of the bowel (the large intestine - the colon or the rectum). If left untreated, the cancer can spread to other organs. Most CRCs arise from cells in the bowel lining, called epithelial cells. Small non-cancerous growths in the bowel lining called 'polyps' often precede CRC. Whilst most polyps do not grow any further, the cells in a polyp occasionally undergo changes that give rise to a larger tumour, which can damage or block the bowel. In more advanced stages of CRC, the cancerous cells can spread beyond the bowel and travel to other parts of the body, where they grow as metastatic or secondary tumours.
[0110] As reported by Rabeneck et al. (In: Gel band et a , editors. Cancer: Disease Control Priorities, Third Edition (Volume 3). Washington (DC): The International Bank for Reconstruction and Development/The World Bank; 2015 Nov. Chapter 6), an estimated 1 ,361,000 people are diagnosed with CRC annually; approximately 694,000 people die from CRC annually; and 3,544,000 individuals are living with CRC. Randomized controlled trials (RCT) have shown that screening is associated with a reduction in CRC mortality; in several high-income countries (HICs), organized, population-based screening programs have been introduced, starting in 2006. Some screening tests detect cancer at an early stage when treatment is less arduous and more often results in cure. Other screening tests have the ability to detect adenomas as well as cancer. Screening provides the opportunity to identify and remove adenomas and thereby to prevent the development of the disease.
[0111] CRC can be segregated into four stages of development - States I to IV - as shown in the table below:
Figure imgf000033_0001
[0112] Patients with Stage I CRC tumours have a very good prognosis. Following resection of the primary tumour, only a very small proportion of patients with Stage I CRC will develop recurrence {i.e., tumour progression). Stage II CRC tumours represent the greyest area in terms of CRC prognosis, where a significant proportion of these patients (around 50%) will show evidence of recurrence and metastatic tumours after the primary tumour has been removed via surgical resection. Patients with Stage III CRC tumours also have very poor prognosis, with around 50%-70% of these patients developing recurrence and metastatic tumours after the primary tumour has been removed via surgical resection. Stage IV CRC tumours have the worst prognosis, with a mean disease-specific survival period of less than 2 years after diagnosis.
[0113] Until the present invention, there has been no reliable prognostic test available that can identify whether a patient with CRC is at risk of recurrence. This is particularly evident in patients with Stage III CRC. [0114] In an embodiment, the subject has Stage III colorectal cancer. This is predicated on the inventors' surprisingly finding that the expression of a panel of laminin chain subunits comprising (i) cc3, jl and j2 or (u) «3 and 2 can improve the predictive capacity of the panel in determining a patient's risk of tumour progression following resection of primary Stage III CRC tumour.
[0115] In another embodiment, the method is for determining whether the subject is at risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression. In another embodiment, the subject has Stage 0 pancreatic cancer.
[0116] In an embodiment, where the method is for determining whether the subject is at risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits comprises a laminin chain subunit selected from the group consisting of laminin chain subunits a3, β3 and j2. In another embodiment, where the method is for determining whether the subject is at risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits comprises laminin chain subunits cc3, β3 and γ2. In another embodiment, where the method is for determining whether the subject is at risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits further comprises laminin chain subunit βΐ or γΐ . In yet another embodiment, where the method is for determining whether the subject is at risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits further comprises laminin chain subunit β ΐ and γΐ .
[0117] In another embodiment, where the method is for determining whether the subject is as risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits comprises laminin chain subunits cx3, βΐ , β3, γΐ and y2.
[0118] In another embodiment, where the method is for determining whether the subject is as risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits comprises laminin chain subunits cx3, yl , β3, and j2.
[0119] In another embodiment, where the method is for determining whether the subject is as risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits comprises laminin chain subunits cx3, yl and j2.
[0120] In another embodiment, where the method is for determining whether the subject is as risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subunits comprises laminin chain subunits cx3, βΐ , yl and y2,
[0121] In another embodiment, where the method is for determining whether the subject is as risk of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression or invasive breast cancer progression, the one or more laminin chain subumts comprises laminm chain sub units 0.3, β3, γΐ and j2' .
[0122] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subumts comprises laminin chain subunit β ΐ or laminm chain subunit jl .
[0123] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprise laminin chain subunit β ΐ and laminin chain subunit γΐ .
[0124] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminm chain subunits comprise laminin chain subunits o3, β ΐ, β3, jl and γ2.
[0125] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprise <x3, βΐ , β3 ¾κ1 γ2.
[0126] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprise c 3, β ΐ , jl and γ2. In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more lammin chain subumts comprise 0(3, β3, γΐ and y2.
[0127] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprise 3, yl and j2.
[0128] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprise α3 and γ2.
[0129] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprises γ2,
[0130] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprise laminin chain subunits α3, β3 and j2.
[0131] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprises laminin chain subunits a3, βΐ , β3, jl and j2,
[0132] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprises laminin chain subunits α3, γΐ, β3 and j2.
[0133] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprises laminin chain subunits a3, yl and j2.
[0134] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprises laminin chain subunits α3, βΐ, γΐ and j2.
[0135] In another embodiment, where the method is for determining whether the subject is at risk of colorectal cancer progression, the one or more laminin chain subunits comprises laminin chain subunits a3, β3, γΐ and j2.
Subject
[0136] The terms "subject," "individual" and "patient" are used interchangeably herein to refer to any subject to which the present disclosure may be applicable, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcmes (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. In some embodiments, the subject is a primate (e.g., a human, ape, monkey, chimpanzee). In a preferred embodiment, the subject is a human.
Biological sample
[0137] The biological sample can be any sample in which changes in the expression of laminin chain subunits reflect the risk of cancer progression, including recurrence after surgical resection. Suitable biological samples could be determined by persons skilled in the art. For example, the level of expression of the one or more laminin chain subunits in a biological sample obtained from a first subject who has been diagnosed as having cancer can be compared to a level of expression of the one or more laminin chain subunits in a biological sample obtained from substantially the same anatomical region of a second subject who is free of cancer and/or has had no history of cancer, wherein a higher level of expression of the one or more laminin chain subunits in the biological sample from the first subject as compared to the level of expression in the biological sample from the second subject is indicative that the biological sample is suitable for the purpose of diagnosing the nsk of cancer progression, or of a subject's disease-specific survival, in accordance with the present invention.
[0138] In another illustrative example, the level of expression of the one or more laminin chain subumts in a biological sample obtained from a first subject who has been diagnosed as having cancer can be compared to the level of expression of the one or more laminin chain subumts in a biological sample obtained from substantially the same anatomical region of a second subject who has also been diagnosed as having cancer, albeit a different grade of cancer; that is, a less or more advanced form of cancer than the first subject, wherein a higher or lower level of expression of the one or more laminin chain subunits in the biological sample from the first subject as compared to the level of expression in the biological sample from the second subject is indicative that the biological sample is suitable for the purpose of diagnosing the risk of cancer progression, or of a subject's disease- specific survival, in accordance with the present invention.
[0139] A biological sample may include a sample that has been obtained, extracted, untreated, treated, diluted or concentrated from a subject. In some embodiments, the biological sample has not been extracted from the subject, particularly where the determination steps in accordance with the present invention (e.g., the expression of the panel of laminm chain subunits) can be performed in situ.
[0140] Non-limiting examples of suitable biological samples include, but are not limited to, tissue, such as a biopsy sample or a resected tumour, bodily fluid (for example, blood, serum, plasma, saliva, urine, tears, peritoneal fluid, ascitic fluid, vaginal secretion, breast fluid, breast milk, lymph fluid, cerebrospinal fluid or mucosa secretion), umbilical cord blood, chorionic villi, amniotic fluid, an embryo, embryonic tissues, lymph fluid, cerebrospinal fluid, mucosa secretion, or other body exudate, fecal matter and one or more cells or extracts thereof that express laminin chain subunits (nucleic acid or protein), including subcellular structures obtained using protocols well established within the art. In some embodiments, the biological sample comprises blood, such as peripheral blood, or a fraction or extract thereof. The biological sample may comprise blood cells, such as mature, immature or developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, eosinophils, megakaryocytes, macrophages, dendritic cells, natural killer cells, or fraction of such cells (e.g., a nucleic acid or protein fraction).
[0141] In an embodiment disclosed herein, the biological sample is selected from the group consisting of a primary tumour sample, a circulating tumour sample and a metastasis sample. In an embodiment disclosed herein, the biological sample is a primary tumour sample.
[0142] The biological sample may be processed and analyzed for the purpose of determining the expression of the panel of one or more laminin chain subunits, in accordance with the present invention, almost immediately following collection (i.e., as a fresh sample), or it may be stored for subsequent analysis. If storage of the biological sample is desired or required, it would be understood by persons skilled in the art that it should ideally be stored under conditions that preserve the integrity of the biomarker of interest within the sample (e.g., at -80°C).
[0143] The terms "obtain", "obtaining", "obtained" and the like, as used herein, are meant to come into possession. Biological or reference samples so obtained include, for example, nucleic acid extracts or polypeptide extracts isolated or derived from a particular source. For instance, the extract may be isolated directly from a biological tissue of the subject.
Reference Value
[0144] The methods disclosed herein may comprise a comparison step (i.e., to identify whether the subject is at risk of cancer progression) in which the expression of the one or more laminin chain subumts in the biological sample of the test subject is compared to the expression of the one or more laminin chain subunits in a second biological sample that is measured prior to, concurrently or subsequent to the expression of the one or more laminin chain subunits in the first biological sample from the test subject, wherein the second biological sample is obtained either from a different subject, or from a different anatomical location of the same subject. It is to be understood, however, that the comparison step (i.e., to identify whether the subject is at risk of cancer progression) does not need to rely upon a comparison with a level of expression of the one or more laminin chain subunits in the biological sample to the level of expression in another biological sample. For example, the comparison may be carried out using a reference value; that is, a known or predetermined level of expression of the one or more laminin chain subunits that is associated with the risk of cancer progression or sensitivity to a chemotherapeutic regimen, as described elsewhere herein.
[0145] The term "reference value" is referred to interchangeably herein as a "control value". In an illustrative example, the comparison may be carried out using a reference value that is representative of a known or predetermined level of expression of the one or more laminm chain subunits in a reference sample or a plurality of reference samples, that is associated with the risk of cancer progression or sensitivity to a chemotherapeutic regimen, as described elsewhere herein. The term "reference sample" is also referred to herein as a "control sample".
[0146] The reference value is typically a predetermined level of expression of the one or more laminin chain subunits that is representative of the level of expression in a particular cohort or population of subjects (e.g., normal healthy controls, subjects with cancer, subjects with a history of cancer, subjects who had no sign of cancer at the time the reference sample was obtained but who have gone on to develop cancer, etc). The reference value may be represented as an absolute number, or as a mean value (e.g., mean +/- standard deviation), such as when the reference value is derived from (i.e. , representative of) a population of individuals.
[0147] The reference value may be equal to or not significantly different from the level of expression of the one or more laminin chain subunits in a sample population representative of patients in whom recurrence has occurred after surgical resection of the primary tumour; for example, in whom recurrence has occurred within about 1 month, within about 6 months, within about 12 months, within about 24 months, within about 36 months, within about 48 months or within about 60 months after surgical resection of the primary tumour. Thus, a level of expression of the one or more laminin chain subunits in a biological sample from a test subject that is equal to, not significantly less than or greater than the reference value is indicative of a high or higher risk of cancer progression in the test subject. Conversely, a level of expression of the one or more laminin chain subunits in a biological sample from a test subject that is significantly less than the reference value is indicative of a low or lower risk of cancer progression in the test subject.
[0148] The reference value may also be equal to or not significantly different from the level of expression of the one or more laminin chain subunits in a sample population representative of patients in whom recurrence has not occurred after surgical resection of the primary tumour; for example, in whom recurrence has not occurred within at least about 60 months after surgical resection of the primary tumour. Thus, a level of expression of the one or more laminin chain subunits in a biological sample from a test subject that is lower than, equal to, or not significantly different from, the reference value is indicative of the absence of cancer in the test subject, or a low risk of cancer progression in the test subject, including recurrence. Conversely, a level of expression of the one or more laminm chain subunits in a sample from a test subject that is significantly greater than the reference value is indicative of the presence of cancer in the test subject, or a risk of cancer progression.
[0149] Whilst persons skilled in the art would understand that using a reference value that is derived from a sample population of individuals is likely to provide a more accurate representation of the level of expression in that particular population (e.g., for the purposes of the methods and protocols disclosed herein), in some embodiments, the reference value can be a level of expression of the one or more laminin chain subunits in a single biological sample.
[0150] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a healthy subject, a subject that is otherwise free of the cancer, or a subject in which the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is greater than the reference value is indicative of a higher risk of cancer progression.
[0151] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a healthy subject, a subject that is otherwise free of the cancer, or a subject in winch the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than or equal to the reference value is indicative of a lower risk of cancer progression.
[0152] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein the prolonged period of time is at least 12 months following resection of the cancer. In another embodiment, the prolonged period of time is at least 24 months following resection of the cancer. In another embodiment, the prolonged period of time is at least 36 months following resection of the cancer. In another embodiment, the prolonged period of time is at least 48 months following resection of the cancer. In another embodiment, the prolonged period of time is at least 60 months following resection of the cancer, it is to be understood that the period of time following resection in which there has been no recurrence may depend on the stage of the cancer at the time of resection. As an illustrative example, where the subject has Stage IV CRC at the time of resection, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has not progressed after a period of 2 years following resection of the tumour. In another illustrative example, where the subject has Stage I, Stage II or Stage 3 CRC at the time of resection, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has not progressed after a period of 5 years following resection of the tumour.
[0153] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has progressed after a period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than the reference value is indicative of a lower risk of cancer progression.
[0154] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has progressed after a period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is greater than or equal to the reference value is indicative of a higher risk of cancer progression.
[0155] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has progressed after a period of time following resection of the cancer, wherein the period of time is less than about 12 months following resection of the tumour In another embodiment, the period of time is less than about 1 1 months following resection of the cancer. In another embodiment, the period of time is less than about 10 months following resection of the cancer. In another embodiment, the period of time is less than about 9 months following resection of the cancer. In another embodiment, the period of time is less than about 8 months following resection of the cancer. In another embodiment, the period of time is less than about 7 months following resection of the cancer. In another embodiment, the period of time is less than about 6 months following resection of the cancer. In another embodiment, the period of time is less than about 5 months following resection of the cancer. In another embodiment, the period of time is less than about 1 month following resection of the cancer. It is to be understood that the period of time following resection in which recurrence is evident may depend on the stage of the cancer at the time of resection. As an illustrative example, where the subject has Stage IV CRC at the time of resection, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has progressed within about one month following resection of the tumour. In another illustrative example, where the subject has Stage I, Stage II or Stage 3 CRC at the time of resection, the reference value is representative of a level of expression of the one or more laminin chain subumts in a biological sample of a subject in which the cancer has progressed within about 6 months following resection of the tumour.
[0156] In some embodiments, the reference value may be representative of the level of expression of the panel of laminin chain subunits in a population of patients who have undergone resection and have developed recurrence (also referred to interchangeably herein as a "positive reference value", "positive control value" and the like). Thus, in an embodiment disclosed herein, a subject is identified as being at risk of recurrence where the level of expression of the panel of laminin chain subunits in the biological sample from the subject is greater than or equal to the positive reference value.
Recurrence end disease-specific survival
[0157] As disclosed herein, the present inventors have surprisingly found that the level of expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer can predict whether the subject is at risk of recurrence. The present inventors have found that a higher level of expression of the one or more laminin chain subunits in cancer tissue is associated with poor prognosis, as determined by disease- specific survival. The present inventors have therefore shown, for the first time, that the expression of one or more laminin chain subunits is a suitable prognostic indicator of cancer progression.
[0158] Resection, also referred to as curative or surgical resection, remains the primary form of potentially curative treatment for solid cancers, including colorectal cancer (CRC), liver cancer and pancreatic cancer. However, despite curative resection and adjunct chemotherapy, recurrence of solid tumours cancer still persists in a large cohort of patients. For instance, for colorectal cancer, recurrence affects around 50% of patients with Stage II CRC, around 50%-70% of patients with Stage III CRC and almost 100% of patients with Stage IV CRC at the time of resection. There are many factors that are thought to influence recurrence of solid tumours, including tumour size, location, therapeutic strategy, stage classification, lymph node involvement and metastasis.
[0159] The terms "cancer progression" and "tumour progression" are used interchangeably herein the denote an increase in the size of the primary tumour size (e.g., volume), an increase in the size of the tumour after surgical resection (e.g., of a primary tumour remnant after surgical resection) and/or the appearance of one or more secondary tumours (i.e., one or more metastatic tumours). The terms "cancer progression" and "tumour progression" are also referred to herein as "recurrence" .
[0160] As disclosed herein, the greater the level of expression of the one or more laminin chain subunits in the biological sample, the shorter the disease-specific free survival period. The terms "disease-specific survival", "disease-specific survival period " and the like mean the time elapsed between treatment initiation (e.g., resection) and tumour progression or death. Patients who died from causes other than the disease being studied are typically excluded from this measurement.
[0161] Disease-specific survival is often used in clinical trials as one way of measuring how well a particular treatment works. The determination of disease-specific survival need not be absolute (i.e., a specific time point between onset of treatment and the onset of disease progression or death). In some embodiments, the disease-specific survival may be approximate {e.g., 6-12 months, 1 -2 years, etc).
[0162] As disclosed herein, a reference value can be used that is representative of the level of expression of the one or more laminin chain subunits in a biological sample obtained from a healthy subject or a subject that is otherwise free of cancer. Thus, in an embodiment, a level of expression of the one or more laminin chain subunits in the biological sample from a test subject that is greater than the reference value is indicative of a poor prognosis; that is, of poor disease-specific survival. Conversely, a level of expression of the panel of laminin chain subunits in the biological sample from a test subject that is less than the reference value would be indicative of a disease-specific survival period that is longer than the disease-specific survival period of the patient from which the reference value was derived.
[0163] In another embodiment disclosed herein, the reference value can be a value that is representative of the level of expression of the one or more laminin chain subunits in a biological sample obtained from a patient with cancer or from a patient who has undergone a resection of their tumour but has subsequently developed recurrence. Thus, a level of expression of the one or more laminin chain subunits in the biological sample from a test subject that is greater than or not significantly different from (e.g., equal to) the reference value is indicative of a poor prognosis and a disease-specific survival period that is shorter than or equal to the disease-specific survival period of the patient from which the reference value was derived. Conversely, a level of expression of the one or more laminin chain subunits in the biological sample from a test subject that is less than the reference value would be indicative of a disease-specific survival period that is longer than the disease- specific survival period of the patient from which the reference value was derived.
Risk
[0164] The term "risk", as used herein, denotes a subject's likelihood of developing cancer progression, including recurrence following resection, based on the level of expression of the one or more laminin chain subunits in a biological sample, as determined for that subject. Accordingly, the terms "risk" and "likelihood" are used interchangeably herein, unless otherwise stated. [0165] It would be apparent to persons skilled in the art that the risk of developing cancer will vary, for example, from being at low, lower or decreased risk of cancer progression to being at high, higher or increased risk of cancer progression. By "low, lower or decreased risk" is meant that the subject is less likely to develop recurrence as compared to a subject determined to be a "high, higher or increased risk" subject. Conversely, a "high, higher or increased risk" subject is a subject who is more likely to develop recurrence as compared to a subject who is not at risk or a "low, lower or decreased risk" subject.
[0166] Likelihood is suitably based on mathematical modelling. An increased likelihood, for example, may be relative or absolute and may be expressed qualitatively or quantitatively. For instance, an increased risk may be expressed as simply determining the subject's level of expression of the one or more laminm chain subunits in a biological sample and placing the subject in a high or higher risk category, based upon the level of expression of the one or more laminin chain subunits that is representative of the level of expression of the one or more laminin chain subunits that corresponds to a high or higher risk of cancer progression, as determined, for example, in biological samples of a population of patients who have developed cancer progression. Alternatively, a numerical expression of the test subject's likelihood of cancer progression may be determined based upon the level of expression of the one or more laminin chain subunits in the biological sample.
[0167] As used herein, the term "probability" refers to the probability of class membership for a sample as determined by a given mathematical model and is construed to be equivalent likelihood in this context. In some embodiments, likelihood is assessed by comparing the level of expression of the one or more laminin chain subunits to one or more preselected levels, also referred to herein as threshold or reference levels or values. Thresholds may be selected that provide an acceptable ability to predict recurrence. In illustrative examples, receiver operating characteristic (ROC) curves can be calculated by plotting the value of a variable versus its relative frequency in two populations in which a first population is considered at risk of cancer progression following resection and a second population that is not considered to be at risk, or have a low risk, of cancer progression (called arbitrarily, for example, "healthy controls"). The second population may also be represented by patients who have undergone curative resection but do not present with recurrent cancer over, for example, a 3 year period following the resection.
[0168] For any particular biomarker, such as laminin chain subunit βΐ, a distribution of levels of expression for subjects who are at risk or are at no or at low risk of developing cancer (including recurrence) may overlap. Under such conditions, a test may not absolutely distinguish a subject who is at higher risk of developing cancer (e.g., recurrence) from a subject who is not at risk or at lower risk of developing cancer with absolute (i.e., 100%) accuracy, and the area of overlap indicates where the test cannot distinguish the two subjects. A threshold can be selected, above which (or below which, depending on how a biomarker changes with risk) the test is considered to be "positive" and below which the test is considered to be "negative." The area under the ROC curve (AUC) provides the C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition (see, e.g., Hanley et al., Radiology 143: 29-36 (1982)). Such thresholds can be determined by persons skilled in the art, having regard to the teachings disclosed herein.
[0169] The level of risk, or a subject's likelihood of cancer progression (e.g., recurrence following resection) may be based on the level of expression of the one or more laminin chain subunits in a biological sample, as herein described, together with one or more additional risk factors, as determined for that subject. Suitable additional risk factors would be known to persons skilled in the art.
Therapeutic regimen
[0170] A subject who is identified as being at risk of cancer progression can be stratified into a treatment group where an appropriate therapeutic regimen can be adopted or prescribed with a view to treating or preventing cancer and/or cancer progression. Conversely, subjects identified as being at no or low risk of cancer progression can be spared an otherwise taxing therapeutic regimen or, alternatively, a less aggressive therapeutic regimen (e.g. , a lower dose of chemotherapeutic agent or radiation dose) can be adopted or prescribed. Thus, in an embodiment, the methods disclosed herein further comprise the step of exposing (i.e., subjecting) the subject identified as being at risk of cancer progression to a therapeutic regimen for treating the cancer and/or cancer progression, including a therapeutic regimen for preventing or delaying cancer progression,
[0171] In an embodiment, the therapeutic regimen comprises radiotherapy and/or the administration of a chemotherapeutic agent.
[0172] In another aspect disclosed herein, there is provided a method of treating a subject with cancer, the method comprising the steps of:
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and
(b) comparing the level of expression of the one or more laminin chain subunits in the biological sample to a reference value, wherein said comparison provides an indication as to whether or not the subject is at risk of cancer progression.
(c) identifying a subject that is at risk of cancer progression from step (b); and
(d) exposing the subject identified in step (c) as being at risk of cancer progression to a therapeutic regimen for preventing or delaying cancer progression.
[0173] In an embodiment, the one or more laminin chain subunits are selected from the group consisting of lammin chain subunit «3, laminin chain subunit β3, laminin chain subunit βΐ , laminin chain subunit γΐ and laminin chain subunit 2.
[0174] In an embodiment, step (a) comprises measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with colorectal cancer.
[0175] In another embodiment, step (a) comprises measuring the expression of one or more lammin chain subunits in a biological sample obtained from a subject with Stage III colorectal cancer.
[0176] In another embodiment, the one or more laminin chain subunits comprises laminin chain subunit βΐ or laminin chain subunit yl . In another embodiment, the one or more laminin chain subunits comprise laminin chain subunit βΐ and laminin chain subunit y] . In another embodiment, the one or more laminin chain subunits comprise lammin chain subunits 0(3, βΐ, β3, γΐ and γ2. In another embodiment, the one or more laminin chain subunits comprise tx3, βΐ , β3 and j2. In another embodiment, the one or more laminin chain subunits comprise cc3, βΐ, γΐ and y2. In another embodiment, the one or more laminin cham subunits comprise 0(3, β3, γΐ and γ2. In another embodiment, the one or more laminin chain subunits comprise cc3, jl and y2. In another embodiment, the one or more laminin chain subunits comprise a3 and y2. In another embodiment, the one or more laminin chain subunits comprises j2. In another embodiment, the one or more laminin chain subunits comprise laminin chain subunits cc3, β3 and γ2.
[0177] In an embodiment, step (a) comprises measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with liver cancer or pancreatic cancer.
[0178] In another embodiment, step (a) comprises measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with Stage 0 pancreatic cancer.
[0179] In an embodiment, the one or more laminin chain subunits comprises a laminm chain subunit selected from the group consisting of laminin chain subunits ix3, β3 and γ2. In another embodiment, the one or more laminin chain subunits comprises laminin chain subunits c¾3, β3 and γ2. In another embodiment, the one or more laminin cham subunits further comprises laminin chain subunit βΐ or γΐ . In another embodiment, the one or more laminin chain subunits further comprises laminin chain subunit βΐ and γΐ .
[0180] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a healthy subject, a subject that is otherwise free of the cancer, or a subject in which the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein a level of expression of the one or more laminm chain subunits in the biological sample that is greater than the reference value is indicative of a higher risk of cancer progression.
[0181] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a healthy subject, a subject that is otherwise free of the cancer, or a subject in winch the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than or equal to the reference value is indicative of a lower risk of cancer progression.
[0182] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has not progressed after a prolonged period of time following resection of the cancer, as described elsewhere herein.
[0183] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has progressed after a period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than the reference value is indicative of a lower risk of cancer progression.
[0184] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has progressed after a period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is greater than or equal to the reference value is indicative of a higher risk of cancer progression.
[0185] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has not progressed after a period of time following resection of the cancer, as described elsewhere herein.
[0186] In an embodiment, a subject identified as being at a higher risk of cancer progression is exposed in step (d) to a more aggressive therapeutic regimen as compared to a therapeutic regimen to which a subject identified as being at lower risk of cancer progression would be exposed. In an embodiment, the more aggressive therapeutic regimen comprises radiotherapy and/or administration of a chemotherapeutic agent.
[0187] In an embodiment, a subject identified as being at a lower risk of cancer progression is exposed in step (d) to a less aggressive therapeutic regimen as compared to a therapeutic regimen to which a subject identified as being at higher risk of cancer progression would be exposed,
[0188] In an embodiment, the less aggressive therapeutic regimen comprises radiotherapy and/or administration of a chemotherapeutic agent.
[0189] Suitable therapeutic regimen will be familiar to persons skilled in the art, the choice of which is likely to be determined by factors such as, but not limited to, the type of cancer, the seventy of the cancer (e.g., stage of progression), the age and general health status of the patient, etc. In an embodiment, the method the therapeutic regimen comprises radiotherapy and/or the administration of a chemotherapeutic agent.
[0190] Suitable chemotherapeutic agents will be known to persons skilled in the art, the choice of which is also likely to be determined by factors such as, but not limited to, the type of cancer, the seventy of the cancer (e.g., stage of progression), the age and general health status of the patient, etc. Illustrative examples of suitable chemotherapeutic agents include 5-Fluorouracil-based combination regimen (also known as 5-FU; e.g., Folfin, Folfox, Folfoxin), Trifluridine/tipiracil-based chemotherapy (Tas-102), inhibitors of vascular epidermal growth factor (VEGF) (e.g., Bevacizumab, Aflibercept), inhibitors of epidermal growth factor receptor (EGFR) (e.g., Cetuximab, Panitumumab, Gefitimb) and kinase inhibitors (e.g., Regorafenib, Sorafenib).
[0191] Another determinative factor for selecting a suitable therapeutic regimen may be the degree of risk of cancer progression, as determined, for example, by the level of the one or more laminin chain subunits, as herein described. For example, for a subject identified as being at high or higher risk of cancer progression, a more aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who is deemed at low or lower risk of cancer progression, as determined by the methods disclosed herein. Conversely, for a subject identified as being at low or lower risk of cancer progression, a less aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who is deemed at high or higher risk of cancer progression, as determined, for example, by the methods disclosed herein. Similarly, for a subject identified as having a more advance stage of cancer (as determined, e.g., by Stage assessment), a more aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who has a less advanced cancer, as determined, for example, by the methods disclosed herein. Conversely, for a subject identified as having a less advance stage of cancer (as determined, e.g., by Stage assessment), a less aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who has a more advanced stage of cancer.
[0192] As described elsewhere herein, therapeutic regimens will typically be designed by a medical practitioner or a team of medical practitioners, having regard, for example, to the age, weight, body mass index and general health of the subject, as noted elsewhere herein. Another determinative factor in the design of a suitable therapeutic regimen may be the degree of risk of cancer progression, as determined, for example, by the level of expression of the one or more laminin chain subunits. For instance, where the subject is determined to be at high risk of cancer progression, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is determined to be at no risk or low risk of cancer progression.
[0193] In some embodiments, the therapeutic regimen comprises a combination of two or more treatment modalities (e.g., 2, 3 or more, 4 or more, 5 or more, 6 or more). Treatment modalities will typically be selected with a view to treating and/or preventing cancer and/or cancer recurrence.
[0194] As used herein the terms "treat", "treatment", "treating", "prevent", "preventing" and "prevention" refer to any and all uses which remedy a condition or symptom, prevent the establishment of a condition or disease, or otherwise prevent, hinder, retard, abrogate or reverse the onset or progression of a condition or disease or other undesirable symptoms in any way whatsoever. Thus, the terms "treating" and "preventing" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery or cure. In conditions which display or a characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms. In the context of cancer recurrence, the agents, uses, methods and protocols of the present disclosure that involve treatment or prevention may prevent, reduce, ameliorate or otherwise delay cancer progression, or of a highly undesirable event associated with cancer progression or an irreversible outcome of cancer progression, but may not of itself prevent progression of the cancer or an outcome associated therewith (e.g., a symptom associated with cancer). Accordingly, treatment and/or prevention includes amelioration of the symptoms of cancer progression or preventing or otherwise reducing the risk of cancer progression.
[0195] The term "inhibiting" and variations thereof, such as "inhibition" and "inhibits", as used herein, do not necessarily imply the complete inhibition of the specified event, activity or function. Rather, the inhibition may be to an extent, and/or for a time, sufficient to produce the desired effect. Inhibition may be prevention, retardation, reduction, abrogation or otherwise hindrance of an event, activity or function. Such inhibition may be in magnitude and/or be temporal in nature. In particular contexts, the terms "inhibit" and "prevent", and variations thereof may be used interchangeably.
[0196] Many cancers have not been particularly sensitive to chemotherapeutic agents and, if used, have usually served to palliatively reduce the size of the tumour, relieve symptoms of the disease and/or increase survival time. Examples of chemotherapeutic agents that have been used to treat CRC include fluorouracil (5-FU) or its analog capecitabine, carmustme, methyl-CCNU, adriamycin (doxorubicin), mitomycin C, cisplatin and taxotere, or combinations thereof. Studies have explored the benefit of chemotherapy before surgery (e.g., to shrink tumour size) or after surgery to destroy any residual cancer cells. Combination treatment with chemotherapy and radiation therapy has demonstrated some activity in selected post-surgical settings. Radiation therapy (also referred to as radiotherapy) uses high-energy electromagnetic radiation to damage cancer cells and stop them from proliferating. When used, it is generally in combination with surgery and chemotherapy, or used only with chemotherapy in cases where the individual is unable to undergo resection. Radiation therapy may also be used to relieve pain or blockage by shrinking the tumour during palliative care.
Predicting a Response to Treatment
[0197] As noted elsewhere herein, the present inventors' have surprising found that the expression of one or more laminin chain subunits can predict the sensitivity of a cancer cell to a chernotherapeutic agent. The present inventors have therefore shown, for the first time, that the expression of one or more laminin chain subunits is a suitable diagnostic and prognostic indicator of response to cancer treatment.
[0198] Thus, in another aspect of the present disclosure, there is provided a method of determining a subject's sensitivity to a chernotherapeutic regimen for treating or preventing cancer, the method comprising;
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and
(b) comparing the level of expression of the one or more laminin chain subunits in the biological sample to a reference value;
wherein said comparison provides an indication as to subject's sensitivity to the chernotherapeutic regimen.
[0199] In another aspect of the present disclosure, there is provided a method of determining a cell's sensitivity to a chernotherapeutic agent, the method comprising:
(a) measuring the expression of one or more laminin chain subunits in a cell; and fb) comparing the level of expression of the one or more laminin chain subunits in the cell to a reference value;
wherein said comparison provides an indication as to cell's sensitivity to the chernotherapeutic agent.
[0200] In an embodiment, the one or more lammin chain subunits are selected from the group consisting of laminin chain subunit (x3, laminin chain subunit β3, lammin chain subunit βΐ, laminin chain subunit γΐ and laminin chain subunit j2. [0201] For the purpose of determining a cell's sensitivity to a chemotherapeutic agent, as herein described, the reference value is typically representative of a level of expression of the one or more laminin chain subunits in a cell or a population of cells with a known or pre-determined level of sensitivity to the chemotherapeutic regimen, also referred to interchangeably herein as a "reference cell", "reference cells", "reference sample" and the like. An illustrative example of a suitable reference sample includes a sample cell or a population of cells derived from a primary or metastatic tumour of a patient that is known to be responsive or non-responsive to said chemotherapeutic regimen, as evidenced, for example, by the absence or presence of recurrence following resection and commencement of the chemotherapeutic regimen, by a change or otherwise in tumour volume, by a change or otherwise in the number of secondary or metastatic tumours, etc. Another illustrative example of a suitable reference sample includes a cancer cell line that has a known or predetermined level of sensitivity to said chemotherapeutic regimen, as evidenced, for example, a change or otherwise in the number of viable cells following exposure of the cancer cell line to the chemotherapeutic agent or regimen in vitro. In an illustrative example, where the chemotherapeutic agent is cytotoxic to cancer cells, then a cancer cell line is said to have a degree of sensitivity to that chemotherapeutic agent .if there is a loss of viable cells when the cancer cell line is cultured in the presence of a chemotherapeutic agent for a period of time, when compared to the same cell line cultured in the absence of the chemotherapeutic agent. Conversely, a cancer cell line is said to lack or have little or no sensitivity to the chemotherapeutic agent .if there is no statistically significant loss of viable cells when the cancer cell line is cultured in the presence of the chemotherapeutic agent for a period of time, when compared to the same cell line cultured in the absence of the chemotherapeutic agent.
[0202] The level of sensitivity to a chemotherapeutic agent is typically dependent on the concentration or dose of the chemotherapeutic agent, although there will often be a suitable range of concentrations that may be tested, as can be readily determined by persons skilled in the art having regard to, for example, the type of chemotherapeutic agent, the patient being treated, the route of administration, the cell line being evaluated and the culture conditions. Illustrative examples of suitable ranges are described elsewhere herein. [0203] Suitable cancer cell lines will be known to persons skilled in the art. Typically, the cancer cell line will be derived from a primary tumour of the type of cancer to which the methods disclosed herein are directed or the cancer cell line will be a suitable surrogate for the type of cancer to which the methods disclosed herein are directed. For instance, where the method is for determining a subject's sensitivity to a chemotherapeutic regimen for treating or preventing colorectal cancer, the cancer cell line will typically be derived from or be a suitable surrogate for colorectal cancer cells. Suitable colorectal cancer cell lines will be familiar to persons skilled in the art, illustrative examples of which include HT55, HT29, SW480, SW620m RKO, Colo320, Colo205, SNUC-1, DLD-1, T-84 and HCT-116 cell lines.
[0204] In an embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in a cancer cell line selected from the group consisting of HT55, HT29, SW480, DLD-1 and T84, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is greater than or equal to the reference value is indicative that the subject has low sensitivity to the chemotherapeutic regimen.
[0205] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in cancer cell line selected from the group consisting of HT55, HT29, SW480, DLD-1 and T84, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than the reference value is indicative that the subject has high sensitivity to the chemotherapeutic regimen.
[0206] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in cancer ceil line selected from the group consisting of RKO, Colo320, SW620 and Colo205, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than or equal to the reference value is indicative that the subject has high sensitivity to the chemotherapeutic regimen.
[0207] In another embodiment, the reference value is representative of a level of expression of the one or more laminin chain subunits in cancer ceil line selected from the group consisting of RKO, Colo320, SW620 and Colo205, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is higher than the reference value is indicative that the subject has low sensitivity to the chemotherapeutic regimen.
[0208] The advantage of predicting a subject's response to a chemotherapeutic regimen, as determined by the methods disclosed herein, is that the chemotherapeutic regimen may then be modified to account for either a predicted low or high sensitivity to the chemotherapeutic regimen, as determined by the methods disclosed herein. For instance, in an embodiment disclosed herein, the method further comprising exposing a subject identified as having low sensitivity to the chemotherapeutic regimen to a more aggressive chemotherapeutic regimen as required to treat or prevent cancer progression. Suitable modifications to a chemotherapeutic regimen would be known to persons skilled in the art, as note elsewhere herein, illustrative examples of which include the use of a combination of one or more chemotherapeutic agents, combining the administration of chemotherapeutic agents with radiotherapy, increasing the dose of the chemotherapeutic agents and/or increasing the frequency of administration of the chemotherapeutic regimen. The term "chemotherapeutic regimen" is used herein the denote the administration of one or more suitable chemotherapeutic agents in a therapeutically effective amount or amounts with a view to preventing or delaying cancer progression, as herein described. Suitable chemotherapeutic agents will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein.
[0209] In an embodiment, the more aggressive therapeutic regimen comprises radiotherapy and/or administration of a higher dose of chemotherapeutic agent than would otherwise be required for a subject identified as having high sensitivity to the chemotherapeutic regimen.
[0210] The manner in which the therapeutic regimen is to be altered or otherwise modified, if necessary, having regard to whether the subject is identified as being responsive (i.e., having high, higher or greater sensitivity) or less (or non-) responsive (i.e., having low, lower or little sensitivity) to the chemotherapeutic agent may also depend on factors such as the age, weight, general health of the subject and/or the stage of the cancer (e.g., at resection). Kits
[0211] In another aspect of the present disclosure there is provided a kit comprising one or more reagents and/or devices for use in performing the methods disclosed herein. The kits may contain reagents for analyzing the expression of the panel of lammm chain subunits in a biological sample in accordance with the methods disclosed herein.
[0212] Kits for carrying out the methods of the present invention may also include, in suitable container means, (i) one or more reagents for detecting the one or more laminin chain subunits, (ii) one or more nucleic acid probes that specifically bind to the nucleic acid molecule(s) encoding each of the one or more laminin chain subunits, (iii) one or more probes that are capable of detecting and/or measuring the expression of the one or more laminin chain subunits, (iv) one or more labels for detecting the presence of the probes and/or (iv) instructions for how to measure the level of expression of the one or more laminin chain subunits. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container into which one or more reagents will be placed or suitably aliquoted. Where a second and/or third and/or additional component is provided, the kit will also generally contain a second, third and/or other additional container into which this component may be placed. Alternatively, a contamer may contain a mixture of more than one reagent, as required. The kits may also include means for containing the one or more reagents (e.g., nucleic acids) in close confinement for commercial sale. Such containers may include injection and/or blow-moulded plastic containers into which the desired vials are retained.
[0213] The kits may further comprise positive and negative controls, including a reference sample, as well as instructions for the use of kit components contained therein, in accordance with the methods disclosed herein.
[0214] Ail essential materials and reagents required for detecting and quantifying the one or more laminin chain subunits may be assembled together in a kit. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates, dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) one or more polynucleotides encoding the laminin chain subunit(s) in the panel of laminin chain subunits (which may be used as a positive control), (ii) one or more primers or probes that specifically hybridize to polynucleotides encoding the laminin chain subunits in the panel of laminin chain subunits. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Tag, Sequenase™ DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits may also comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. The kit may also feature various devices {e.g., one or more) and reagents (e.g. , one or more) for performing any one of the assays described herein; and/or printed instructions for using the kit to quantify the expression of the one or more laminin chain subunits.
[0215] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting.
[0216] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.
[0217] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
[0218] The present disclosure is further described by reference to the following non- limiting examples. Examples
Materials asid Methods
(i) SurvExpress data analysis
[0219] The correlation between laminin chain expression and disease outcome was analyzed using the datasets within the SurvExpress bioresource
Figure imgf000061_0001
Search options included averaging of duplicated probe signals, usage of Original (Quantiie-normaiized) data, and maximization of risk groups. Presented data summarizes expression levels for specified laminin chains in high or low risk patients (median, 5-95 percentile, significance of difference between the two patient groups) (see Figures 14A-14C); Kaplan-Meier analyses of disease-specific survival (Hazard ratio + confidence interval + significance; see Figures 1-13) and Kaplan-Meier analyses of disease-specific survival according to gender (Hazard ratio + confidence interval + significance). Optimized risk scores were obtained as shown in (Aguirre-Gamboa R, et a!. (2013) SurvExpress: an online biomarker validation tool and database for cancer gene expression data using survival analysis. PLoS One 8(9):e74250).
[0220] Colorectal cancer datasets were initially derived from the SurvExpress colon metabase (n = 380). Further analysis was performed on an updated SurvExpress colon metabase (n = 947), The Cancer Genome Atlas (TCGA) colon dataset (n = 350), Sheffer- Domany dataset (n = 243; Sheffer et a!., "Association of survival and disease progression with chromosomal instability: a genomic exploration of colorectal cancer". PNAS 106.17 (2009): 7131-7136) and Smith-Beauchamp dataset (n = 177; Smith et ah, "Experimentally derived metastasis gene expression profile predicts recurrence and death in patients with colon cancer". Gastroenterology 138.3 (2010): 958-968).
[0221] For bile duct cancer, analyses were performed on the TCGA cholangiocarcinoma dataset (n = 35).
[0222] For brain cancer, analyses were performed on the TCGA glioma (n = 660) and glioblastoma (n = 148) datasets. [0223] For melanoma, analyses were performed on the TCGA skin cutaneous melanoma dataset (n ::= 336).
[0224] For prostate cancer, analyse were performed on the Sboner-Rubin dataset (n =:: 281 ; Sboner et a!., "Molecular sampling of prostate cancer: a dilemma for predicting disease progression", BMC Medical Genomics 16.3 (2010): 8).
[0225] For gastric cancer, analyses were performed on the TCGA stomach adenocarcinoma dataset (n =:: 352).
[0226] For bladder cancer, analyses were performed on the TCGA bladder urothelial carcinoma dataset (n := 390).
[0227] For breast cancer, analyses were performed on the TCGA breast invasive carcinoma dataset (n =:: 962),
(u) Cell Culture
[0228] SW480 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; Lonzal2-61 F) supplemented with 10% (v/v) fetal bovine serum (FBS; Fisher BiotecFBS- AU-015), 100 units/ml Penicillin-Streptomycin (Life Technologies; 15140-122) and 2 fflM glutamine (Life Technologies; 35050-061) at 37 °C in 5% C02.
[0229] HT55 cells were grown in Eagle's Minimum Essential Medium (EMEM; Lonzal2- 125F) with the same supplements but 20% (v/v) fetal bovine serum.
[0230] HT-29, Colo205 and Colo320HSR cells were grown in RPMI-1640 (Lonza 312- 617), 10% FBS, 100 units/ml Penicillin-Streptomycin, lx Glutamax (L-alanyl -L-glutamine, TherraoFisher Scientific), ImM sodium pyruvate and 2.5g L glucose.
[0231] RKO cells were grown in MEM Eagle w/ Earle's BSS, NEAA & Sodium Pyruvate (Lonza #12-662), 10% FBS, 100 units/ml Penicillin-Streptomycin and lx Glutamax.
[0232] SW620, T84 and DLD-1 cells were grown in medium DMEM medium comprising 4.5g L glucose , 10% FBS, ix Glutamax and lx Penicillin/Streptomycin,
[0233] Cells were seeded at a density of 106 on T75 culture flasks (Corning; #430641) and passaged every three days. (Hi) Cytotoxicity assay
[0234] 5,000 cells resuspended in ΙΟΟμΙ of media were seeded in each well of 96- well culture plates (Corning; CLS3595). Cytotoxic compounds over a range of concentrations were added to the cells on the next day for an incubation of 72 hrs. Cell viability was assessed by MTT assay according to the manufacturer's instructions for the MTT powder (Sigma; 298-93-1) with minor modifications. The results were obtained from readings at 570nm subtracted by the background reading at 690nm, and plotted using the GraphPad Prism software to obtain IC50 values.
(iv) RJS'A extraction and RT-PCR
[0235] RNA was extracted from the cancer cell lines with either High Pure RNA Isolation Kit (Roche; Basel, Switzerland) using the manufacturer's protocol. The resulted RNA was reverse transcribed with oligo-dT and random hexamers using Transcriptor First Strand cDNA Synthesis Kit (Roche; Basel, Switzerland).
[0236] All PCR reactions were run for 30 cycles and were performed in 20 μΐ under standard conditions using 1U of Taq DNA Polimerase Recombinant (GIBCO Invitrogen Corporation). To reveal expression of different laminin chains and GAPDH (a known housekeeping gene) cDNAs were amplified using primers described in Table 1, below. The PCR products were analyzed on a 1.5% agarose gel containing ethidium bromide. For each RNA sample, RT-PCR without reverse transcriptase was performed to confirm that no genomic DNA was isolated.
Table 1: Laminin chain subunit primers.
Figure imgf000064_0001
Results
(A) The expression of one or more laminm chain subunits correlates with disease specific outcomes
[0237] Usmg the SurvExpress Webtool, retrospective analysis of published datasets was performed to determine whether the expression level of mRNAs encoding laminm chain subunits in primary colorectal tumors at the time of surgery (surgical resection) was correlated with specific disease outcomes (recurrence, death). Survival outcomes were analysed in correlation with the expression of multiple laminm chain subunit combinations, such as LAMA 3 /LAMB 1 /LAMB 3 /LAMC 1 /'L AMC2 (see Figure 1), LAMA3/LAMB3/ LAMC2 (see Figure 2), LAMA3/LAMB 1 /LAMB3/LAMC2 (see Figure 3), LAMA 3/ LAMB1 /LAMC1/LAMC2 (see Figure 4), LAMA3/LAMC2 (see Figure 5), LAMC2 alone (see Figure 6) and LAMA3/LAMC1/LAMC2 (see Figure 7). For each of these combinations, Kaplan-Meier survival curves were generated across all colorectal cancer patients (see Figures 1-7, panel A) or in patient cohorts grouped by tumour stage upon diagnosis (for Stage I, see Figures 1 -7, panel B; for Stage II, see Figures 1.-7, panel C; for Stage III, see Figures 1 -7, panel D; for Stage IV, see Figures 1 -7, panel E). Specific comparison between patient groups separated into High and Low risk groups in relation with their LAMC2 expression level or LAMA3/LAMB1 /LAMB3 LAMC1/LAMC2 expression level are provided for Stage I (see Figure 10), Stage II (see Figure 1 1), Stage III (see Figure 12) and Stage IV (see Figure 13) colorectal cancer. For Figures 10-13, panel A shows data for LAMC2 expression and panel B shows data for the combined expression of L AMA3/LAMB 1 /LAMB3/LAMC 1 LAMC2.
[0238] An expanded cohort of colorectal cancer patients (SurvExpress colon metahase, n = 947) and the TCGA colon dataset (n := 350) were also assessed to determine whether the survival outcomes were correlated with the expression of multiple laminin chain subunit combinations, including the combinations listed in Table 2, below. The smaller Sheffer- Domany (n = 243) and Smith-Beauchamp (n :=: 177) datasets were extracted from the larger SurvExpresss metabase cohort for independent analysis. For each combination, Kaplan- Meier survival curves were generated for each patient cohort. The p-values derived from the Kaplan-Meier survival curves are provided in Table 2, below.
[0239] Using the SurvExpress Webtool, the correlation between the expression level of mRNAs encoding specific laminin chain subunits (LAMA3/LAMB1/LAMB3/LAMC1/ LAMC2) and disease outcomes was also correlated in liver cancer (see Figure 9A), pancreatic cancer (see Figure 9B). Kaplan-Meier survival curves were also generated in pancreatic patient cohorts grouped by tumour stage upon diagnosis (for Stage NO, see Figure 14A; for Stage Nl, see Figure 14B).
[0240] Further analyses revealed also a correlation between the expression level of mRNAs encoding specific laminin chain subunits (LAMA3/LAMB1/LAMB3/LAMC1/ LAMC2) and disease outcomes in bile duct cancer (see Figure 9C), glioma (see Figure 9D), glioblastoma (see Figure 9E), melanoma (see Figure 9F), prostate cancer (see Figure 9G), gastric cancer (see Figure 9IT), bladder cancer (see Figure 91) and invasive breast carcinoma (see Figure 9J). [0241] Survival outcomes were also analysed in correlation with the expression of multiple laminin chain subunit combinations, including the combinations listed in Table 3, below. For each of these combinations, Kaplan-Meier survival curves were generated for each patient cohort. The /?- values derived from the Kaplan-Meier survival curves are provided in Table 3, below.
Table 2:
Figure imgf000066_0001
Table 3:
Bile Glioma GBM Melanoma Prostate Gastric Bladder invasive duel Breast
Carcinoma
A3B1B3C1C2 0 003 1.3exp-l4 4.0cxp-06 0.0003 S.3exp-06 0.007 9.5exp-07 2.8exp-07
A3B1B3C2 0.075 8.9exp-16 0.00023 0.0003 0.00021 0.050 0.00023 ND
A3C1C2 0.090 1.3exp-14 0.00046 0.0011 0.00013 0.012 1.6exD-05 ND
A3C2 0.125 .' vxp - l ! 0,0! 9 0.0042 0.0019 0,252 7.5exp-05 ND
C2 0.119 0.144 0.008 0.0313 0.0147 0.354 0.006 0.009
A3B1C1C2 0.003 1.8exp-14 0.00075 0.0019 2.9εχρ-06 0.011 3.6exp-05 ND
A3B3C1C2 0.032 2.7exp-14 3.3exp-06 0,00039 0.00014 0.0072 8.2exp-06 ND
A3B3C2 0 577 l.Sexp-08 0.0002 0,00089 0.00057 0.064 0.0005 ND
[0242] Figures 1-14 include Concordance Index (CI) and a Hazard ratio (H ) estimates. The CI gives a probability that patients from the high-risk groups experience the event being analysed (e.g., recurrence, death) after patients with the low-risk, with values close to 0,5 reflecting "random" occurrence between groups, while higher values reflect better prediction. The Cox model-derived Hazard ratio estimates are provided with their associated p- value and confidence intervals.
[0243] The expression level of individual laminin chain subunits in patients displaying better survival (low risk) or worse survival (high risk) was also quantified in colorectal cancer (see Figure 8A), liver cancer (see Figure 8B), pancreatic cancer (see Figure 8C) and invasive breast carcinoma (see Figure 8D) and the statistical analysis of differences in expression between high and low risk groups was determined using a t-test.
[0244] The data demonstrate that the expression of one or more laminin chain subumts has prognostic capacity with respect to cancer progression, including tumour progression following resection. The data, also show that the expression of lammm chain subunits βΐ and/or γΐ enhances the prognostic capacity when combined with one more other laminin chain subunits, such as a3, β3, γ2 and combinations thereof..
(B) The correlation between laminin chain subunit expression and disease prognosis is also associated with gender bias
[0245] Three patient cohorts were further analysed for any gender bias. Surprisingly, greater discrimination between low risk and high risk individuals was seen in males as compared to females. Figures 15, 16 and 17 shows data for the combined expression of Ι .ΛΜΛ I . V B ! ί Λ Β Ι .ΛΧ Ί Ι .ΛΜΓ2 in females (panel A) and the combined expression of Ι .ΛΜΛ3 Ι .ΛΜΒ Ι Ι .ΛΜΒ3 Ι .ΛΜΠ i .AMC?. in males (panel B). A gender bias towards males was also evident when the expression of fewer laminin chain subunits was profiled, including LAMA3/LAMB 1 /LAMB3/ LAMC2, LAMA3/LAMC1/LAMC2, LAMA3/LAMC2, L AMA3/LAMB 1 /LAMC 1 /LAMC2, LAMA3/LAMB3/LAMC1/ LAMC2 and LAMA3/LAMB3/LAMC2. (C) The expression of one or more laminin chain subunits correlates with sensitivity to chemotherapeutic regimen
[0246] Table 4, below, shows the total laminin chain score of six colorectal cancer cell lines. To generate total laminin score, laminin chain subunit RNA was amplified using RT- PCR and run on ethidmm bromide-containing agarose gels. Quantification of RNA expression was performed using densitornetric analysis of RNA bands on the agarose gels, using the Image J software.
Table 4:
Figure imgf000069_0001
[0247] To generate total laminin score, laminin chain RNAs were amplified using RT-PCR and run on ethidium bromide-containing agarose gels. Quantification of RNA expression was performed using densitornetric analysis of RNA bands on the agarose gels, using the Image J software. Density values were then corrected to withdraw non-specific background signals. The densitometry values were then grouped into five score categories for each laminin chain subunit, as follows;
Score 0 = = no signal;
Score 1 = ::: corrected density value between 0 and 1 :
Score 2 = = corrected density value >1 and <5;
Score 3 = ::: corrected density value >5 and <10; and
Score 4 = = corrected density- value >10. [0248] The total Iaminin score was calculated as the sum of individual scores for each of the five Iaminin chain subunits.
[0249] The cell lines can be separated into those with a high total Iaminin score (HT55, HT29 and SW480) and a low total Iaminin score (Coio2Q5, Colo320HSR and RKO), as shown in Table 4,
[0250] Figure 18 provides a series of photomicrographs showing RT-PCR-amplified Iaminin chain subunit cDNA separated on ethidium bromide-stained 1.5% agarose gels, following RNA extraction from the Colo320HSR, Colo205, RKO, HT-55, HT-29, SW480 and T84 colorectal cancer cell lines.
[0251] As summarised in the dose-response curves in Figure 19, colorectal cancer cell lines HT55, HT29 and SW480 showed lower sensitivity to Gefitinib as compared to colorectal cancer cell lines Colo205, Colo320HSR. and RKO. Similarly, colorectal cancer cell lines HT55, FIT29 and SW480 showed lower sensitivity to Sorafenib as compared to colorectal cancer cell lines Colo205, Colo320HSR and RKO (see Figure 20). These data show that cancer cells with relatively high expression of Iaminin chain subunits have a relatively low degree of sensitivity to chemotherapeutic agents when compared to cancer cells with relatively low expression of Iaminin chain subunits.
[0252] Table 5, below, shows that the colorectal cancer cells that have a higher total Iaminin score (FIT55, HT29 and SW480) have a lower sensitivity to Gefitinib and Sorafenib in comparison to the colorectal cancer cells that have a lower total Iaminin score (Colo205, Coio320HSR and RKO). Sensitivity was determined by exposing cells to increasing concentrations of Gefitinib and Sorafenib and determining the IC5 (concentration necessary to kill 50% of cells), as shown in Figure 19 and 20. T le 5ι
Figure imgf000071_0001
[0253] Table 6, below, shows that the colorectal cancer cells that have a higher total laminin score (HT55, HT29 and SW480) also have higher sensitivity to the Folfox in comparison to the colorectal cancer cells that have a lower total laminin score (Colo205, Colo320HSR and RKO). Sensitivity was determined by exposing ceils to increasing concentrations of FOLFOX and determining the IC50 (concentration necessary to kill 50% of cells).
Table 6:
Figure imgf000071_0002
[0254] This analysis was repeated using additional ceil lines SW620, T84 and DLD-1 and additional chemotherapeutics Folfiri, Regorafenib and Cnzotmib.
[0255] Ceils were grown in medium + 10% FBS at 37C in 5% C02 until 80% confluent. Upon reaching 80% confluence cells were lysed and RNAs were purified. Real Time (RT) quantitative polymerase ehara reaction (qPCR) was performed to detect 5 laminin chains, as indicated in Table 7 below, as well as the housekeeping gene RPLO. Expression values for individual laminin chains were calculated using the DeltaCp (DCp) method (Cp value for the target laminin RNA - Cp value for the housekeeping gene), in this method, lower Cp values reflect higher expression, which can be counter- intuitive. The 1/DCp value for each of these RNAs was therefore calculated, which makes the correlation between output value and effective expression proportional, rather than inversely proportional. The Laminin score represents the sum of 1/DCp values for each of the 5 chains (see Combined I /DCp in Table 7). The 1/DCp values were then multiplied by 100 (see New LAM score).
[0256] As shown in Table 7, colorectal cancer cells that have a higher total laminin score (HT55, HT29, SW480, T84 and DLD-1) have a significantly lower sensitivity to Sorafenib and Regorafenib in comparison to the colorectal cancer cells that have a lower total laminin score (Coio205, Colo320HSR, RKO and SW620). Furthermore, colorectal cancer cells that have a higher total laminin score (HT55, HT29, SW480, T84 and DLD-1 ) also have a significantly higher sensitivity to Folfox in comparison to the colorectal cancer cells that have a lower total laminin score (Colo205, Colo320HSR, RKO and SW620). Sensitivity was determined by exposing ceils to increasing concentrations of Folfox, Foifiri, Sorafenib, Regorafenib and Crizotinib and determining the IC50 (concentration necessary to kill 50% of cells).

Claims

Claims
1. A method of determining whether a subject is at risk of cancer progression, the method comprising;
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and
(b) comparing the level of expression of the one or more laminin chain subunits in the biological sample to a reference value;
wherem said comparison provides an indication as to whether or not the subject is at risk of cancer progression.
2. The method of Claim 1 , wherein the one or more laminin chain subunits are selected from the group consisting of laminin chain subunit ix3, laminin chain subunit β3, laminin chain subunit β ΐ, laminin chain subunit jl and laminin chain subunit y2.
3. The method of Claim 1 or Claim 2, wherein the method is for determining whether the subject is at risk of colorectal cancer progression.
4. The method of any one of Claims 1 to 3, wherein the subject has Stage III colorectal cancer.
5. The method of any one of Claims 1 to 4, wherein the one or more laminin chain subunits comprises laminin chain subunit βΐ or laminin chain subunit γΐ .
6. The method of any one of Claims 1 to 5, wherein the one or more laminin chain subunits comprise laminin chain subunit βΐ and lamimn chain subunit γΐ .
7. The method of any one of Claims 1 to 6, wherein the one or more laminin chain subunits comprise laminin chain subunits cc3, βΐ, β3, jl and jl.
8. The method of any one of Claims 1 to 5, wherein the one or more laminin chain subunits comprise oc3, βΐ, β3 and " 2.
9. The method of any one of Claims 1 to 6, wherein the one or more laminin chain subunits comprise oc3, βΐ, γΐ and γ2.
10. The method of any one of Claims 1 to 5, wherein the one or more laminin chain subunits comprise oc3, γΐ and γ2.
11. The method of any one of Claims 1 to 4, wherein the one or more laminin chain subunits comprise oc3 and γ2.
12. The method of any one of Claims 1 to 4, wherein the one or more laminin chain subunits comprises γ2.
13. The method of any one of Claims 1 to 4, wherein the one or more laminin chain subunits comprise laminin chain subunits oc3, β3 and j2,
14. The method of Claim 1, wherein the method is for determining whether the subject is at risk of liver cancer progression or pancreatic cancer progression.
15. The method of Claim 14, wherein the subject has Stage 0 pancreatic cancer.
16. The method of Claim 14 or Claim 15, wherein the one or more laminin chain subunits comprises a laminin chain subunit selected from the group consisting of laminin chain subunits α3, β3 and j2.
17. The method of any one of Claims 14 to 16, wherein the one or more laminin chain subunits comprises laminin chain subunits oc3, β3 and " 2.
18. The method of any one of Claims 14 to 17, wherein the one or more laminin chain subunits further comprises laminin chain subunit βΐ or γΐ .
19. The method of Claim 1 8, wherein the one or more laminin chain subunits further comprises laminin chain subunit βΐ and γΐ .
20. The method of any one of Claims 1 to 19, further comprising the step of exposing the subject identified as being at risk of cancer progression to a therapeutic regimen for preventing or delaying cancer progression.
21. The method of Claim 20, wherein the therapeutic regimen comprises radiotherapy and/or the administration of a chemotherapeutic agent.
22. A method of treating a subject with cancer, the method comprising the steps of: (a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and
(b) comparing the level of expression of the one or more laminin chain subunits in the biological sample to a reference value, wherein said comparison provides an indication as to whether or not the subject is at risk of cancer progression.
(c) identifying a subject that is at risk of cancer progression from step (b); and
(d) exposing the subject identified in step (c) as being at risk of cancer progression to a therapeutic regimen for preventing or delaying cancer progression.
23. The method of Claim 22, wherein the one or more laminm chain subunits are selected from the group consisting of laminin chain subunit ix3, laminin chain subunit β3, laminin chain subunit β ΐ, laminin chain subunit jl and laminin chain subunit y2.
24. The method of Claim 22, wherein step (a) comprises measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with colorectal cancer.
25. The method of Claim 22, wherein step (a) comprises measuring the expression of one or more laminm chain subunits in a biological sample obtained from a subject with Stage III colorectal cancer.
26. The method of any one of Claims 22 to 25, wherein the one or more laminin chain subunits comprises laminin chain subunit βΐ or laminm chain subunit γΐ .
27. The method of any one of Claims 22 to 26, wherein the one or more laminin chain subunits comprise laminin chain subunit βΐ and lammm chain subunit γΐ .
28. The method of Claim 27, wherein the one or more laminm chain subunits comprise laminin chain subunits a3, βΐ, β3, γΐ and γ2.
29. The method of any one of Claims 22 to 26, wherein the one or more laminin chain subunits comprise oc3, βΐ, β3 and " 2.
30. The method of any one of Claims 22 to 26, wherein the one or more laminin chain subunits comprise oc3, βΐ, γΐ and γ2.
31. The method of any one of Claims 22 to 26, wherein the one or more laminm chain subumts comprise oc3, γΐ and γ2.
32. The method of any one of Claims 22 to 25, wherein the one or more laminin chain subunits comprise oc3 and γ2.
33. The method of any one of Claims 22 to 25, wherein the one or more laminin chain subunits comprises γ2.
34. The method of any one of Claims 22 to 25, wherein the one or more laminin chain subunits comprise laminin chain subunits oc3 , β3 and yl,
35. The method of Claim 22, wherein step (a) comprises measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with liver cancer or pancreatic cancer.
36. The method of Claim 35, wherein step (a) comprises measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with Stage 0 pancreatic cancer.
37. The method of Claim 35 or Claim 36, wherein the one or more laminin chain subunits comprises a laminin chain subunit selected from the group consisting of laminm chain subunits «3, β3 and γ2.
38. The method of Claim 35 or Claim 36, wherein the one or more laminin chain subunits comprises laminin cham subunits ix3, β3 and γ2.
39. The method of any one of Claims 35 to 38, wherein the one or more laminm chain subunits further comprises laminin chain subunit β 1 or yl .
40. The method of Claim 39, wherein the one or more laminin chain subunits further comprises laminin chain subunit β ΐ and yl.
41. The method of any one of Claims 22 to 40, wherein the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a healthy subject, a subject that is otherwise free of the cancer, or a subject in which the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein a level of expression of the one or more lamimn chain subunits in the biological sample that is greater than the reference value is indicative of a higher risk of cancer progression.
42. The method of any one of Claims 20 to 40, wherein the reference value is representative of a level of expression of the one or more lamimn chain subunits in a biological sample of a healthy subject, a subject that is otherwise free of the cancer, or a subject in which the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein a level of expression of the one or more lanunin chain subunits in the biological sample that is less than or equal to the reference value is indicative of a lower risk of cancer progression.
43. The method of Claim 41 or Claim 42, wherein the reference value is representative of a level of expression of the one or more la unin chain subunits in a biological sample of a subject in which the cancer has not progressed after a prolonged period of time following resection of the cancer, wherein the prolonged period of time is at least 60 months following resection.
44. The method of any one of Claims 22 to 40, wherein the reference value is representative of a level of expression of the one or more lamimn chain subunits in a biological sample of a subject in which the cancer has progressed after a period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than the reference value is indicative of a lower risk of cancer progression.
45. The method of any one of Claims 22 to 40, wherein the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has progressed after a period of time following resection of the cancer, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is greater than or equal to the reference value is indicative of a higher risk of cancer progression.
46. The method of Claim 44 or Claim 45, wherein the reference value is representative of a level of expression of the one or more laminin chain subunits in a biological sample of a subject in which the cancer has not progressed after a period of time following resection of the cancer, wherein the period of time is less than about 6 months following resection.
47. The method of any one of Claims 41 to 46, wherem a subject identified as being at a higher risk of cancer progression is exposed in step (d) to a more aggressive therapeutic regimen as compared to a therapeutic regimen to which a subject identified as being at lower risk of cancer progression would be exposed.
48. The method of Claim 47, wherein the more aggressive therapeutic regimen comprises radiotherapy and/or administration of a chemotherapeutic agent.
49. The method of any one of Claims 41 to 48, wherem a subject identified as being at a lower risk of cancer progression is exposed in step (d) to a less aggressive therapeutic regimen as compared to a therapeutic regimen to which a subject identified as being at higher risk of cancer progression would be exposed.
50. The method of Claim 49, wherein the less aggressive therapeutic regimen comprises radiotherapy and/or administration of a chemotherapeutic agent.
51. The method of any one of Claims 1 to 50, wherein the biological sample is selected from the group consisting of a primary tumour sample, a circulating tumour sample and a metastasis sample.
52. The method of Claim 51 , wherein the biological sample is a primary tumour sample obtained from a resected tumour.
53. The method of any one of Claims 1 to 52, wherein the step of measuring the expression of the one or more laminin chain subunits in the biological sample comprises measuring the gene expression of the one or more laminin chain subunits in the biological sample.
54. A method of determining a subject's sensitivity to a chemotherapeutic regimen for treating or preventing cancer, the method comprising:
(a) measuring the expression of one or more laminin chain subunits in a biological sample obtained from a subject with cancer; and (b) comparing the level of expression of the one or more lamimn chain subunits in the biological sample to a reference value;
wherein said comparison provides an indication as to subject's sensitivity to the chemotherapeutic regimen.
55. The method of Claim 54, wherein the one or more laminin chain subunits are selected from the group consisting of laminin chain subunit (x3, laminin chain subunit β3, laminin chain subunit β ΐ, laminin chain subunit jl and laminin chain subunit y2.
56. The method of Claim 54, wherem the subject has colorectal cancer.
57. The method of Claim 55, wherein the subject has Stage III colorectal cancer.
58. The method of any one of Claims 54 to 57, wherein the one or more laminin chain subunits comprises laminin chain subunit β3 or laminin chain subunit γ2.
59. The method of any one of Clamis 54 to 58, wherein the one or more laminm chain subunits comprise laminin chain subunit β3 and laminin chain subunit γ2.
60. The method of any one of Claims 54 to 59, wherem the one or more laminin cham subunits comprise laminin cham subunits cc3, βΐ, β3, γΐ and j2.
61. The method of any one of Claims 54 to 60, wherein the reference value is representative of a level of expression of the one or more laminm chain subunits in cancer cell line selected from the group consisting of HT55, HT29 and SW480, wherem a level of expression of the one or more laminin chain subunits in the biological sample that is greater than or equal to the reference value is indicative that the subject has low sensitivity to the chemotherapeutic regimen.
62. The method of any one of Claims 54 to 60, wherein the reference value is representative of a level of expression of the one or more laminm chain subunits in cancer cell line selected from the group consisting of HT55, HT29 and SW480, wherem a level of expression of the one or more laminin chain subunits in the biological sample that is less than the reference value is indicative that the subject has high sensitivity to the chemotherapeutic regimen.
63. The method of any one of Claims 54 to 60, wherein the reference value is representative of a level of expression of the one or more laminin chain subunits in cancer cell line selected from the group consisting of RKO, Colo320 and Colo205, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is less than or equal to the reference value is indicative that the subject has high sensitivity to the chemotherapeutic regimen.
64. The method of any one of Claims 54 to 60, wherein the reference value is representative of a level of expression of the one or more laminin chain subunits in cancer cell line selected from the group consisting of RKO, Colo320 and Colo205, wherein a level of expression of the one or more laminin chain subunits in the biological sample that is higher than the reference value is indicative that the subject has low sensitivity to the chemotherapeutic regimen.
65. The method of any one of Claims 54 to 64, further comprising exposing a subject identified as having low sensitivity to the chemotherapeutic regimen to a more aggressive chemotherapeutic regimen as required to treat or prevent the cancer.
66. The method of Claim 65, wherein the more aggressive therapeutic regimen comprises radiotherapy and/or administration of a higher dose of chemotherapeutic agent than would otherwise be required for a subject identified as having high sensitivity to the chemotherapeutic regimen.
67. The method of any one of Claims 1 to 66, wherein the biological sample is selected from the group consisting of a primary tumour sample, a circulating tumour sample and a metastasis sample.
68. The method of Claim 67, wherem the biological sample is a primary tumour sample obtained from a resected tumour.
69. The method of any one of Claims 1 to 68, wherein the step of measuring the expression of the one or more laminin chain subunits in the biological sample comprises measuring the gene expression of the one or more laminin chain subunits in the biological sample,
70. A kit comprising one or more reagents and/or devices for use in performing the method of any one of Claims 1 to 69.
71 . The method of any one of Claims 1 , 22 and 54, where the cancer is selected from the group consisting of liver cancer progression, pancreatic cancer progression, bile duct cancer progression, glioma progression, glioblastoma progression, melanoma progression, prostate cancer progression, gastric cancer progression, bladder cancer progression and invasive breast cancer progression.
72. The method of Claim 71, wherein the one or more la unin chain subunits comprises laminm chain subunits a3, βΐ , β3, γΐ and j2.
73. The method of Claim 71, wherein the one or more lanunin chain subunits comprises laminm chain subunits a3, γΐ , β3, and j2.
74. The method of Claim 71, wherein the one or more lanunin chain subunits comprises lanunin chain subunits α3, γΐ and j2.
75. The method of Claim 71, wherein the one or more laminin chain subunits comprises laminm chain subunits a3, βΐ , γΐ and j2,
76. The method of Claim 71, wherein the one or more lanunin chain subunits comprises laminin chain subunits α3, β3, j\ and j2.
PCT/AU2017/050245 2016-03-18 2017-03-17 Use of laminins as biomarkers for cancer diagnosis and prognosis WO2017156594A1 (en)

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