WO2014092647A1

WO2014092647A1 - A method for diagnosis of hpv-related non-genital cancers using pcr

Info

Publication number: WO2014092647A1
Application number: PCT/SG2013/000522
Authority: WO
Inventors: Kenji Oba; Misako YAJIMA; Naoki Yamamoto; Masaru Nikaido; Nobuhiro Gemma
Original assignee: National University Of Singapore; Toshiba Corporation
Priority date: 2012-12-10
Filing date: 2013-12-10
Publication date: 2014-06-19

Abstract

The disclosure concerns a diagnostic and prognostic method of cancer progression in patients with breast and other non-genital cancers in human subjects by the detection of high-risk Human papilloma viruses (HR-HPV) and the monitoring expression level of one or more of estrogen receptor (ER), progesterone receptor (PR), cytidine deaminase family genes and gamma Histone2A member X protein ( H2AX: phosphorylated H2AX).

Description

A METHOD FOR DIAGNOSIS OF HPV-RELATED NON-GENITAL CANCERS USING PCR Field of the Invention The disclosure concerns a diagnostic and prognostic method of cancer progression in patients with breast and other non-genital cancers in human subjects by the detection of high-risk Human papilloma viruses (HR-HPV) employing DNA microarray analysis and short fragment (sf) -PCR, and the monitoring expression level of one or more of estrogen receptor (ER), progesterone receptor (PR), cytidine deaminase family genes (for example APOBEC/AID family) and gamma Histone 2A member X protein (γΗ2ΑΧ: phosphorylated H2AX).

Background to the Invention Cancer is an abnormal disease state in which uncontrolled proliferation of one or more cell populations interferes with normal biological function. The proliferative changes are usually accompanied by other changes in cellular properties, including reversion to a less differentiated state. Cancer cells are typically referred to as "transformed". Transformed cells generally display several of the following properties: spherical morphology, expression of foetal antigens, growth-factor independence, lack of contact inhibition, anchorage-independence, and growth to high density. Cancer cells form tumours and are referred to as "primary" or "secondary" tumours. A primary tumour results in cancer cell growth in an organ in which the original transformed cell develops. A secondary tumour results from the escape of a cancer cell from a primary tumour and the establishment of a secondary tumour in another organ. The process is referred to as metastasis and this process may be aggressive, for example as in the case of hepatoma or lung cancer; or non-aggressive, for example early prostate cancer. The transformation of a normal cell to a cancer cell involves alterations in gene expression that results in the altered phenotype of the cancer cell. In some examples the genes expressed by cancer cells are unique to a particular cancer.

Breast cancer is the leading female cancer in the world and approximately 1 in 8 women will be diagnosed with breast cancer during their lifetime. The National Cancer Institute US predicts for 2012 that 226,870 women will be diagnosed and around 39,510 women will die of breast cancer. As most cancers, the severity of breast cancer is categorized in different stages allowing more targeted treatment plans and a certain level of prognostics. The staging system is based on the extent of the tumour and whether cancerous cells have spread to nearby lymph nodes or to more distant areas of the body and therefore presenting metastasis. This classification system ranges from stage 0 to IV, with 0 indicating a carcinoma in situ, whilst stage IV presents cancer which has spread to other organ(s).

Although a number of risk factors including age, weight, use of hormone replacement therapy or family history increase the prevalence of breast cancer, in 50-80% of patients diagnosed with breast cancer, these risk factors are absent. Recent studies suggested that breast cancer may be linked to viral infections such as Epstein-Barr virus, mouse mammary tumour virus and human papilloma virus (HPV). HPV infections are mostly asymptomatic but however persist in a small minority and are known to be the major cause of cervico-uterine cancer and have been associated with oropharyngeal carcinomas or anogenital tract malignancies. The recognition that HPV is a major cause of cervical cancer led to the recent development of vaccines, which are currently monitored worldwide for their effectiveness.

Whether HPV plays a role in breast carcinogenesis is controversial. HPV DNA has been detected in numerous breast cancer specimens. However, some reports suggest the occurrence of HPV in breast cancer tissue whereas others failed to identify HPV in breast carcinomas. Common methods used to identify the occurrence of HPV in breast tissue are Polymerase Chain Reactions (PCR), Southern blotting or in situ hybridisation. The success of these techniques is strongly dependent on the primer or probe specificity which are required to prevent false-positives and false-negative results. Moreover, these methods may not be sensitive enough to detect the virus once integrated into the host genome as the copy number sharply declines during this process. Improved, highly sensitive and specific methods should therefore facilitate a more conclusive detection of the HPV and subtype in breast and other cancerous tissue.

Several patent applications disclose methods to improve detection of HPV and subtypes. For example, US2011201516 discloses a real time Taq-Man PCR assay employing various sets of degenerate and specific primers to detect closely-related serotypes of HPV. WO2012/116220 discloses a method for the detection of different risk type HPV nucleic acids from a biological sample by hybridisation of RNA to immobilised DNA of interest and detecting this complex by antibodies comprising a detectable marker. WO2011/116797 discloses the use of highly specific PCR primer to detect HPV-33.

The present application discloses a diagnostic and prognostic method of cancer progression in patients with breast and non-genital cancers in normal individuals by detecting HR-HPV genomic DNA using microarray analysis and a highly sensitive and specific PCR method facilitating the amplification of, for example, HPV-16 and HPV-18 short genomic nucleic acid fragments. Results are then confirmed by Southern blot analysis using specific probes thus eliminating false positive results.

Statements of Invention

According to an aspect of the invention there is provided a method to diagnose a non- genital cancer in a human subject comprising:

i) providing an isolated biopsy sample from a human subject to be tested; ii) forming a preparation comprising said sample and at least one oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising human papilloma viral [HPV] genomic DNA; a thermo-stable DNA polymerase, deoxynucleotide triphosphates and co-factors required for the specific polymerase chain amplification of HPV DNA; iii) providing polymerase chain reaction conditions sufficient to amplify said nucleic acid molecule[s];

iv) analyzing the amplified products of said polymerase chain reaction for the presence or absence of a nucleic acid moleculefs] comprising one or more HPV nucleotide sequencefs]; and optionally

v) comparing the amplified produces] with a normal matched control to determine the identity of one or more HPV types.

According to a further aspect of the invention there is provided a method to determine the prognosis of a non-genital cancer in a human subject that has been diagnosed with a non-genital cancer comprising:

i) providing an isolated biopsy sample from a human subject to be tested; ii) forming a preparation comprising said sample and at least one oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising HPV genomic DNA; a thermo-stable DNA polymerase, deoxynuc!eotide triphosphates and co-factors required for the specific polymerase chain amplification of HPV genomic DNA;

iii) providing polymerase chain reaction conditions sufficient to amplify said nucleic acid molecule[s];

iv) analyzing the amplified products of said polymerase chain reaction for the presence or absence of a nucleic acid molecule[s] comprising one or more HPV nucleotide sequencefs];

v) comparing the amplified produces] with a normal matched control to determine the identity of one or more HPV types; and

vi) determining the likely prognosis of the cancer in said human subject.

According to a still further aspect of the invention there is provided a method to diagnose and treat a non-genital cancer in a human subject comprising:

i) providing an isolated biopsy sample from a human subject to be tested; ii) forming a preparation comprising said sample and at least one oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising human papilloma viral [HPV] genomic DNA; a thermo-stable DNA polymerase, deoxynucleotide triphosphates and co-factors required for the specific polymerase chain amplification of HPV DNA; iii) providing polymerase chain reaction conditions sufficient to amplify said nucleic acid moleculefs];

iv) analyzing the amplified products of said polymerase chain reaction for the presence or absence of a nucleic acid molecule[s] comprising one or more HPV nucleotide sequence[s];

vi) designing a treatment regimen for the prevention or treatment of cancer as determined by the result of said diagnostic test. In a preferred method of the invention human papilloma virus is selected from the group consisting of. HPV-16, HPV-18, HPV-31 , HPV-33, HPV-35, HPV-39, HPV-45, HPV-51 , HVP-52, HVP-HPV-56, HPV-58, HPV- 59 and HPV-68.

In a preferred method of the invention said human papilloma virus is selected from the group consisting of: HPV-16, HPV-18, HPV-35 and HPV-59. In a preferred method of the invention said human papilloma virus is HVP-18 and HPV- 35.

In a preferred method of the invention said human papilloma virus is HVP-18 and HPV- 59.

In a preferred method of the invention said human papilloma virus is HVP-18, HPV-35 and HPV-59. In a preferred embodiment of the invention said human papilloma virus is HPV-16 and/or HPV- 8.

In a preferred method of the invention said human papilloma virus is HPV-31, HPV-35, HPV-56 and HPV-59.

Human papilloma viruses vary in their pathological effects. For example, in humans so called low risk HPVs such as HPV-6 and HPV- 1 cause benign hyperplasia such as genital warts while high risk HPVs, for example HPV-16, HPV-18, HPV-31 , HPV-33, HPV-52, HPV-54 and HPV-56 can cause cancers such as cervical and penile carcinoma. HPV-5 and HPV-8 cause malignant squamous cell carcinomas of the skin. HPV-2 is found in malignant and non-malignant lesions in cutaneous and squamous epithelium.

In a preferred method of the invention said polymerase chain amplified nucleic acid molecules are hybridized to a DNA array comprising HPV genomic DNA isolated from one or more HPV type.

In a preferred method of the invention said DNA array comprises at least HPV-16 and/or HPV-18.

In a preferred method of the invention said DNA array comprises at least HPV-31 , HPV- 35, HPV-56 and HPV-59.

In an alternative preferred method of the invention said polymerase chain amplified nucleic acid molecules are sized fractionated. In a preferred method of the invention said size fractionation is by electrophoresis. Preferably said electrophoretic separation is gel electrophoresis.

In a preferred method of the invention said polymerase chain reaction is a short fragment length polymerase chain reaction.

The detection of HPV is problematic. HPV cannot be cultured in vitro and detection of HPV in sera is not possible and therefore the direct detection of HPV genomic DNA is the only available assay for HPV. The existence of multiple HPV genotypes with heterogeneous nucleotide sequences makes the detection of specific HPV type difficult. The approaches either involve the identification of type specific PCR primers or the use of broad specific detection which will not give the required specificity. "Short fragment length PCR" is a method that utilizes sequence specific oligonucleotide primers for the detection of short [for example typically between 100 to 300bp e.g. 108-275bp] HPV genomic fragments that can be analysed either by sequencing or Southern blot hybridization. The inventors have surprisingly found that standard PCR followed by DNA array hybridization is unreliable as a means to detect HPV in clinical samples and samples that at first appear negative or variable using DNA array technology is clarified if combined with short fragment length PCR.

In a preferred method of the invention said method combines detection of HPV by hybridization to a DNA array and by short fragment length polymerase chain reaction.

The combination of detection methods can be simultaneous or sequential.

In a still further alternative method of the invention said polymerase chain amplified nucleic acid molecule is subject to DNA sequencing and sequence comparison to confirm the nucleotide sequence identity. In a preferred method of the invention the amplified human papilloma virus nucleic acid moleculefs] are at least 100-300 base pairs [bp].

In a preferred method of then invention said amplified human papilloma virus nucleic acid molecule[s] are between about 108-275 bp. Preferably, said amplified nucleic acid molecules are about 147bp, 196bp, 204bp, 215bp and 275bp. As used herein, the term "non-genital cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth which are not cancers typically associated with genital tissue, for example, tissue of the penis, vulvar/vagina, anus, testes, prostate and cervix. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal tract as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, breast carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation. Of particular relevance to the present invention are non-genital cancers such as breast, lung, and colon and bladder cancer.

In a preferred method of the invention the non-genital cancer is a carcinoma. In a preferred method of the invention said carcinoma is selected from the group consisting of: breast, tonsil, colorectal, oesophageal, bladder and lung.

In an alternative preferred method of the said cancer is a sarcoma. In a preferred method of the invention said amplified nucleic acid molecule comprises or consists essentially of HPV genomic DNA that encodes all or part of the L1 capsid protein.

In a preferred method of the invention said amplified nucleic acid molecule comprises or consists of the nucleotide sequence as set forth in SEQ ID NO: 1. In a preferred method of the invention said amplified nucleic acid molecule consists essentially of one or more nucleotide sequence[s] selected from the group: SEQ ID NO: 2, 3 or 4. In a preferred method of the invention said amplified nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 5

In a preferred method of the invention said amplified nucleic acid molecule consists essentially of one or more nucleotide sequence[s] selected from the group: SEQ ID NO: 6, 7 or 8.

In a preferred method of the invention said primer pairs are selected from the group consisting of:

forward: 5'- CAGATGTCTCTTTGGCTGCCTAG-3' [SEQ ID NO: 9]

reverse: 5'- GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 10]

forward: 5'- GGTGTCAGAACCATATGGCGAC-3' [SEQ ID NO: 11]

reverse: 5'- GTTGGTTACCCCAACAAATGCC-3' [SEQ ID NO: 12]

forward: 5'- CCAGCACCTAAAGAAGATGATCCC-3' [SEQ ID NO: 3]

reverse: 5'- GCAGTTGTAGAGGTAGATGAGGTGG-3' [SEQ ID NO: 14]

forward: 5'- GCCTGTATACACGGGTCCTG-3' [SEQ ID NO: 15]

reverse: 5'-GGCCGCCACAAAGCCATCTG-3' [SEQ ID NO: 16]

forward: 5'-GATACTGGATATGGTGCCATGGAC-3' [SEQ ID NO: 17]

reverse: 5 -GTCACCCATAGTACCTGCTCTATTCC-3 [SEQ ID NO: 18]

forward: 5'- GTTCAGGCTGGATTGCGTCG-3' [SEQ ID NO: 19]

reverse: 5'- CCTGGCACGTACACGCACAC-3 [SEQ ID NO: 20],

forward: 5'-CAGATGTCTCTTTGGCTGCCTAG-3', [SEQ ID NO: 21],

reverse: 5'-GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 22],

forward: 5'-GGTGTCAGAACCATATGGCGAC-3', [SEQ ID NO: 23],

reverse: 5'-GTTGGTTACCCCAACAAATGCC-3\ [SEQ ID NO: 24],

forward: 5'-CCAGCACCTAAAGAAGATGATCCC-3\ [SEQ ID NO: 25],

reverse: 5 -GCAGTTGTAGAGGTAGATGAGGTGG-3'. [SEQ ID NO: 26], or oligonucleotide primer nucleotide sequences that have at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequences set forth in SEQ ID NO: 9-26 and which amplify a L1 capsid nucleic acid molecule from genomic DNA.

forward: 5'- CAGATGTCTCTTTGGCTGCCTAG-3' [SEQ ID NO: 9]

reverse: 5'- GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 10]

forward: 5'- GGTGTCAGAACCATATGGCGAC-3' [SEQ ID NO: 11]

reverse: 5'- GTTGGTTACCCCAACAAATGCC-3' [SEQ ID NO: 12]

forward: 5'- CCAGCACCTAAAGAAGATGATCCC-3' [SEQ ID NO: 13]

reverse: 5'- GCAGTTGTAGAGGTAGATGAGGTGG-3' [SEQ ID NO: 14].

forward: 5 -CAGATGTCTCTTTGGCTGCCTAG-3', [SEQ ID NO: 21],

reverse: 5'-GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 22],

forward: 5'-GGTGTCAG AACCATATGGCGAC-3' , [SEQ ID NO: 23],

reverse: 5'-GTTGGTTACCCCAACAAATGCC-3', [SEQ ID NO: 24],

forward: 5'-CCAGCACCTAAAGAAGATGATCCC-3', [SEQ ID NO: 25],

reverse: 5'-GCAGTTGTAGAGGTAGATGAGGTGG-3\ [SEQ ID NO: 26].

In an alternative preferred method of the invention said primer pairs are selected from the group consisting of:

forward: 5'- GCCTGTATACACGGGTCCTG-3' [SEQ ID NO: 15]

reverse: 5'-GGCCGCCACAAAGCCATCTG-3' [SEQ ID NO: 16]

forward: 5'-GATACTGGATATGGTGCCATGGAC-3' [SEQ ID NO: 17]

reverse: 5'-GTCACCCATAGTACCTGCTCTATTCC-3 [SEQ ID NO: 18]

forward: 5'- GTTCAGGCTGGATTGCGTCG-3' [SEQ ID NO: 19]

reverse: 5'- CCTGGCACGTACACGCACAC-3 [SEQ ID NO: 20].

In a preferred method of then invention said primer pairs amplify HPV-16 genomic DNA. In an alternative preferred method of the invention said primer pairs amplify HPV- 8 genomic DNA.

In a preferred method of the invention said method compares the detection of said human papilloma viral genomic DNA in a tumour biopsy and a non-tumour biopsy from the same human subject. In a preferred method of the invention said method is combined with detection of one or more further non-HPV biomarkers which is diagnostic or prognostic of said cancer. In a preferred method of the invention said biomarker is selected from the group consisting of: Human Epidermal Growth Factor Receptor 2 [HER2], Eestrogen Receptor [ER], Progesterone Receptor [PR], cytidine deaminase [A3B], for example APOBEC3B, and gamma Histone2A member X [yH2AX].

In a preferred method of the invention the treatment comprises the administration of an anti-cancer agent in an effective amount sufficient to prevent or slow the progression of cancer in said subject. In a preferred method of the invention said treatment is the administration of a chemotherapeutic agent.

A general definition of "chemotherapeutic agent" is an agent that typically is a small chemical compound that preferably kills cells in particular diseased cells or is at least cytostatic. Agents can be divided with respect to their structure or mode of action. For example, chemotherapeutic agents include alkylating agents, anti-metabolites, anthracyclines, alkaloids, plant terpenoids and topoisomerase inhibitors. Chemotherapeutic agents typically produce their effects on cell division or DNA synthesis. Examples of alkylating agents are is cisplatin, carboplatin or oxaliplatin. Examples of anti-metabolites include purine or pyrimidine analogues. Purine analogues are known in the art. For example thioguanine is used to treat acute leukaemia. Fludarabine inhibits the function of DNA polymerases, DNA primases and DNA ligases and is specific for cell-cycle S-phase. Pentostatin and cladribine are adenosine analogues and are effective against hairy cell leukaemias. A further example is mecrcaptopurine which is an adenine analogue. Pyrimidine analogues are similarly known in the art. For example, 5-fluorouracil (5-FU), floxuridine and cytosine arabinoside. 5-FU has been used for many years in the treatment of breast, colorectal cancer, pancreatic and other cancers. 5-FU can also been formed from the pro-drug capecitabine which is converted to 5-FU in the tumour. Leucovorin, also known as folinic acid, is administered as an adjuvant in cancer chemotherapy and which enhances the inhibitory effects of 5-FU on thymidylate synthase. Alkylating agents are also known in the art and include vinca alkaloids, for example vincristine or vinblastine. Terpenoids have been used for many years and include the taxanes, for example, palitaxel.

Treatment of breast cancer depends on the stage and grade and includes surgery, chemotherapy, hormone treatment, antibody therapy and radiation. Commonly used chemotherapeutic drugs for the treatment of cancerous breast tissue are amongst others methotrexate, paclitaxel, cyclophosphamide or fluorouracil. Antibody therapies include Trastuzumab, a monoclonal antibody that blocks the effects of HER2, which sends growth signals to breast cancer cells, or Lapatinib a tyrosine kinase inhibitor that blocks the effects of the HER2 protein and other proteins inside tumour cells. Poly (ADP- ribose) polymerase (PARP) inhibitors are used to block DNA repair and causing cancer cells to die. Hormone therapy includes estrogen inhibitors as Tamoxifen or aromatase inhibitors. In an alternative preferred method of the invention said treatment is the administration of a vaccine.

In a still further alternative method of the invention said treatment is administration of a therapeutic antibody.

According to a further aspect of the invention there is provided a kit comprising one or more pairs of oligonucleotide primers selected from the group consisting of.

forward: 5'- CAGATGTCTCTTTGGCTGCCTAG-3' [SEQ ID NO: 9]

reverse: 5'- GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 10]

forward: 5'- GGTGTCAGAACCATATGGCGAC-3' [SEQ ID NO: 11]

reverse: 5'- GTTGGTTACCCCAACAAATGCC-3' [SEQ ID NO: 12]

forward: 5'- CCAGCACCTAAAGAAGATGATCCC-3' [SEQ ID NO: 13]

reverse: 5'- GCAGTTGTAGAGGTAGATGAGGTGG-3' [SEQ ID NO: 14]

forward: 5'- GCCTGTATACACGGGTCCTG-3' [SEQ ID NO: 15]

reverse. 5'-GGCCGCCACAAAGCCATCTG-3' [SEQ ID NO. 16]

forward: 5'-GATACTGGATATGGTGCCATGGAC-3' [SEQ ID NO: 17]

reverse: 5'-GTCACCCATAGTACCTGCTCTATTCC-3 [SEQ ID NO: 18]

forward: 5'- GTTCAGGCTGGATTGCGTCG-3' [SEQ ID NO: 19] reverse:.5 - CCTGGCACGTACACGCACAC-3 [SEQ ID NO: 20],

P1 : 5 -CAGATGTCTCTTTGGCTGCCTAG-3', [SEQ ID NO: 21],

P1 : 5'-GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 22],

P2: 5'-GGTGTCAGAACCATATGGCGAC-3', [SEQ ID NO: 23],

P2: 5'-GTTGGTTACCCCAACAAATGCC-3', [SEQ ID NO: 24],

P3: 5'-CCAGCACCTAAAGAAGATGATCCC-3' , [SEQ ID NO: 25],

P3: 5'-GCAGTTGTAGAGGTAGATGAGGTGG-3'. [SEQ ID NO: 26], or primer oligonucleotide nucleotide sequences that have at least 90%, 95%, 96%, 97%, 98% or 99%o sequence identity to the nucleotide sequences set forth in SEQ ID NO: 9-26 and which amplify a L1 capsid nucleic acid molecule from genomic DNA.

In a preferred embodiment of the invention said kit also comprises polymerase chain reaction components including a thermo-stable DNA polymerase, deoxynucleotide triphosphates and co-factors required for the specific polymerase chain amplification of HPV genomic DNA and optionally control HPV DNA.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. "Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 : illustrates isolated DNA samples from breast cancer (BC) tissues and the cells from 10 cell lines and 24 cell lines transplantable in nude mice. Samples were analyzed with DNA chip as well as sf-PCR methods. The result showed that 5, 8 and 1 samples were additionally recorded from patients with BC, cell line and xeno-graft samples, respectively as positive for HPV DNA by sf-PCR; Figure 2: illustrates HPV genotyping using a Toshiba DNA chip;

Figure 3: A) is a schematic representation of the design of small-fragment PCR (sf-PCR) to detect fragmented HPV genome; B) illustrates the detection of the HPV16 genome in HCC1599 BC cells by sf-PCR; C) is a nucleotide sequence analysis of the PCR amplified small fragment shown in B); and D) illustrates Southern blot of human genomic DNA to show integration of HPV-16 into the human genome using a 147bp small fragment probe;

Figure 4: illustrates the detection of HPV using both DNA chip and small fragment PCR on clinical samples; and

Figure 5: A) is the full length HPV16 L1 capsid genomic nucleotide sequence (Accession No; K02718; B) is the full length HPV18 L1 capsid genomic nucleotide sequence (Accession No; AY262282); C, D, and E are amplified genomic fragments from HPV-16; and F, G, H are amplified genomic fragments from HPV-18.

Figure 6; Relationship between HPV status and patholological features. Numbers in each bar represent number of HPV positives/ total sample numbers. Difference of HPV prevalence between invasive ductal carcinoma (IDC) and invasive LC (ILC) was statistically significant (p=0.0003). IS; in situ.

Figure 7A: Significant difference of duration until recurrence between HPV-positive and - negative tumours. Fifty BC samples of the patients from whom information were available were compared between HPV-positive and -negative groups. Numbers in brackets indicate total number of samples for HPV-positive and -negative cases, respectively. Student's t-test was performed, with p-values indicated on the graph. Figure 7B: Difference of duration until recurrence between HPV-positive and -negative tumours among ER-positive and -negative cases. Recurrent samples were extracted, and then disease-free interval was compared across HPV-positive or -negative tumours, among ER-positive cases. Black bars on graph and numbers at bottom of graph indicate average disease-free interval for each category. Numbers in brackets indicate total number of samples for each category. Student's t-test was performed, and p-values were indicated on graph.

Figure 8: Induction of APOBEC3B by HPV in the normal breast, MCF10-A cells.

(A) APOBEC3B (A3B) promoter activity. Indicated plasmids were digested by restriction enzymes followed by transfection with A3B-Promoter-luciferase and TK-RLuc plasmids into MCF10A cells. Fold A3B-luciferase activity was normalized by TK-Rluc value. (B) A3B or A3G mRNA level in stably HPV18 infected cells. Cells were lysed to extract total RNA, and its RNA was subjected to quantitative PCR (qPCR). The fold induction of A3B or A3G was calculated after normalization by GAPDH mRNA level. (C) RT-PCR for E6, E7, E1 E2 and E5 gene. Cells were lysed to extract total RNA, and its RNA was subjected to conventional RT-PCR. (D) E7 expression in MCF 0A-HPV18 cells. Cells were fixed by paraformaldehyde followed by staining of E7 protein and nuclear using anti-HPV18 E7 Ab and DAPI respectively.

Figure 9: APOBEC3B (A3B) activation by HPV infection increase genome instability. (A) γΗ2ΑΧ immunofluorescence. Cells were fixed by paraformaldehyde followed by staining of γΗ2ΑΧ protein and nuclear using anti-yH2AX Ab and DAPI respectively. (B) γΗ2ΑΧ western blot. Cells were lysed and subjected to western blot. The γΗ2ΑΧ and β- actin were detected using anti-yH2AX and β-actin Ab. (C) Commet assay. Cells were seeded onto agarose-coated slide glass after mixing with 1.5% agarose followed by lysis. Slides were subjected to electrophoresis and then genomic DNA was stained by PI. (D) The possible pathways to induce DNA breaks and γΗ2ΑΧ through HPV-A3B or just HPV. Figure 10: Abrogation of HPV-induced cancer phenotypes by shRNA against HPV E6, E7 and A3B (A) yH2AX level in A3B stably knocked down cells. A3B stably knocked down cells were established using shRNA retroviral vector. Cells were lysed after selection using puromycin and then subjected to western blot. (B) γΗ2ΑΧ level in transient knockdown of A3B, HPV18E6 or E7 cells. Cells were transfected with indicated shRNA plasmids. Cells were lysed and then subjected to western blot. The γΗ2ΑΧ and β-actin were detected using anti-yH2AX and β-actin Ab. The number at bottom of panel shows band intensity ration after normalized by β-actin level.

Figure 11 : Up-regulation of APOBEC3B mRNA level in HPV-infected BC. Total RNA from BC patients were obtained from NUHS TR. Samples were subjected to quantitative PCR (qPCR). The fold induction of A3B or A3G was calculated after normalization by GAPDH mRNA level.

Figure 2 illustrates a possible mechanism of HPV-induced initiation of BC;

Figure 13 is a the proportion of HPV types in HPV positive samples; and

Figure 14A is the organization of genes in the HPV genome; Figure 14B is detection of HPV16 L1 target regions P1 , P2 and P3 in HPV positive samples.

Materials and Methods

DNA samples

Most specimens including coupled samples of matched normal and tumour tissues were collected by surgical operations in the University Hospital and banked in the Tissue Repository of National University of Singapore. Ten breast cancer cell lines were obtained from American Type Culture Collection (ATCC). Transplantable breast tumour samples (Xenograft cells), which were inoculated with breast cancer tissues into nude mice, were obtained from the Central Institute for Experimental Animals (CIEA).

Genomic DNAs were extracted by QIAGEN DNeasy Bood & Tissue kit (Hilden, Germany) and then kept at -20^°C before use. After extraction, all specimens were subjected to DNA microarray (TOSHIBA, Tokyo, Japan) for detection of 13 high-risk HPV genomes according to the manufacturer's instructions as well as sf-PCR and Southern blot analysis to detect small fragments of HPV.

HPV detection using TOSHIBA microarray

HPV detection using TOSHIBA DNA chip was performed according to manufacture's instructions. Briefly, 200-500ng of genomic DNAs were added into each 6 LAMP reaction tubes after denaturing at 95°C for 5 min. Loop mediated isothermal amplification (LAMP) reaction, which targets L1 region of 13 high-risk HPVs (16, 18, 31 , 33, 35, 39, 45, 51 , 52, 56, 58, 59 and 68) was performed with following condition: 65^°C for 90 min and 80°C for 5 min. Samples were collected into one tube followed by addition of hybridization buffer and then applied into electrochemical DNA chip which contain specific DNA probe for L1 region of 13 high-risk HPVs. Hybridization and detection of HPV was performed using a Genelyzer (TOSHIBA, Tokyo, Japan).

HPV detection using combination with small-fragment PCR and Southern hybridization

To identify the HPV16 and 18 genome in genomic DNA samples derived from fresh- frozen tissues, transplantable breast tumour samples and breast cancer cell lines, specific primers which can amplify 147bp, 275bp and 215bp of HPV16 sequence, and 196bp, 204bp and 108bp of HPV18 sequence in L gene were newly designed as mentioned above. Small-fragment PCR was performed using Blend taq plus (Tokyo, Japan), specific primers and 50ng of genomic DNAs with following condition: Step ; 96°C 10min, Step2; 96^°C 30sec, 55°C 30sec, 72^°C 45sec, Step2 were repeated 30 cycles, Step3: 72^°C 5min, keep at 16^°C. Samples were applied for agarose gel electrophoresis. Gels were soaked into acidic buffer for 5min, alkaline buffer for 30 min and neutralizing buffer for 30 min to denature DNAs. Small fragment DNAs were transferred to membrane followed by hybridization to detect HPV genome.

HPV 16 or 18 specific hybridization probe were generated by PCR using HPV16 or HPV18 plasmids and primers as same with sf-PCR with following condition; Stepl ; 96^°C 10min, Step2; 96°C 30sec, 55°C 30sec, 72^°C 45sec, Step2 were repeated 35 cycles, Step3: 72°C 5min, keep at 16°C. PCR products were labeled with alkaline phosphate using Alkphos Direct labeling kit (GE, Buckinghamshire, UK) according to manufacturer's instruction.

Membrane containing small-fragments DNAs were reacted with hybridization buffer (GE) at 72°C for 15 min followed by addition of probes and incubation at 72°C for overnight. After the reaction of probe, membranes were washed with primary wash buffer and secondary wash buffer. Detection was performed using CDP-star detection reagent according to manufacturer's instructions. Quantification of viral DNA

Total DNA was isolated from fresh frozen tissues using the DNeasy Tissue & Blood Kit (QIAGEN) according to manufacturer's instructions. Viral DNA was quantified by realtime quantitative polymerase chain reaction (qPCR) using the TaqMan Gene Expression Master Mix and 7500 Real-Time PCR system (Applied Biosystems) with primers and TaqMan probe as described previously^{58, 59}. Viral DNA was calculated based on the standard curve of control DNA. HPV16 L1 (forward; 5'- TTGTTGGGGTAACCAACTATTTGTTACTGTT-3' [SEQ ID 27], reverse; 5'- CCTCCCCATGTCTG AGGTACTCCTTAAAG-3 '[SEQ ID 28], probe; 6FMA-5'- GTCATTATGTGCTGCCATATCTACTTC-3'-TAMRA) [SEQ ID 29], HPV18 L1 (forward; 5'-GCATAATCAATTATTTGTTACTGTGGTAGATACCACT-3'[SEQ ID 30], reverse; 5 - GCTATACTGCTTAAATTTGGTAGCATCATATTGC-3'[SEQ ID 31], probe; 6FAM-5'- AACAATATGTGCTTCTACACAGTCTCCTGT-3'-TAMRA [SEQ ID 32] and hGAPDH (forward; 5'-TGTGCTCCCACTCCTGATTTC-3'[SEQ ID 33], reverse; 5'- CCTAGTCCCAGGGCTTTGATT-3'[SEQ ID 34], probe; 6FAM-5'- AAAAGAGCTAGGAAGGACAGGCAACTTGGC-3'-TAMRA[SEQ ID 35]). This is illustrated in Table 3.

Genomic DNA extraction from paraffin-embedded BC samples and HPV16 detection

Twenty-five BC samples preserved in paraffin were gifted obtained from the University of Ryukyu, Okinawa, Japan. The gDNA from paraffin-embedded BC samples was extracted using the Macherey-Nagel NucleoSpin FFPE DNA Isolation Kit (Duren, Germany) and then stored at -20°C before use. After extraction, gDNAs were subjected to sf-PCR (and Southern blot analysis) to detect small fragments of the HPV genome.

Three types of primers targeting the HPV16 L1 gene were designed (P1 , P2 and P3) as follows: P1 ; 5'-cagatgtctctttggctgcctag-3', 5'-gggatgtccaactgcaagtagtc-3' P2; 5'- ggtgtcagaaccatatggcgac-3', 5'-gttggttaccccaacaaatgcc-3', P3; 5'- ccagcacctaaagaagatgatccc-3', 5'-gcagttgtagaggtagatgaggtgg-3'. The sf-PCR was performed using Blend Taq-plus (Toyobo), specific primers and 50ng of gDNA with the following conditions: 96°C for 10min (step 1), 96°C for 30s (step 2), 55^°C for 30s (step 3), 72°C for 45s (step 4), repeat step 2 for 30 cycles, 72^°C for 5min (step 5), hold at 16^°C. Samples were applied to agarose gel electrophoresis. This is quantified in Figure 14B. Example 1

TOSHIBA PNA microarray TOSHIBA DNA microarray can sensitively detect 13 high-risk HPVs (16, 18, 31 , 33, 35, 39, 45, 51 , 52, 56, 58, 59 and 68) by one assay. Therefore, it's suitable for HPV genotyping. Representative data are shown in Figure 2.

HPV18 was detected in matched normal tissue as well as tumour tissues on patient 0115. However, HPV-35 genome was detected only in matched normal tissue. We could observe HPV16 in matched normal sample of the patient 0122. Table 1 shows the summary of data. Genotypes of HPV detected seem to be similar in matched normal and tumour region. Intriguingly, matched normal samples tend to show high HPV positive ratio compared to tumour samples. Since matched normal samples are considered to reflect the infection condition before malignancy, these data suggest that HPV infection actually took place in normal breast cells initially and resulted in induction of tumour in breast. However, HPV tends to be eliminated by unknown mechanisms along the tumour progression. Irrespective of the reasons, this implies that HPV infection and tumour progression might show an inverse correlation which will be useful in monitoring fate and risk of cancer. Therefore, proper monitoring of HPV DNAs in both matched normal and tumour tissues should help estimating prognosis of breast cancer patients.

Example 2

Sf-PCR and Southern hybridization

TOSHIBA microarray data on BC indicate that tumour samples seem to lose HPV genome with time. According to the model shown in cervical cancer system, HPV is integrated into human genome to exert its carcinogenic activity of E6/E7 oncoprotein.

E7 gene can induce genome instability to introduce oncogenic mutation on human genome. Therefore, we speculated that fragmentation of HPV genome in BC might take place due to genome instability, so that HPV genome could not be detected when we use TOSHIBA microarray. TOSHIBA microarray are targeting to middle part of the L1 gene. HPV DNA is normally integrated into human genome (with a part of L2 or L1 gene to middle of E2 gene). Because of this fact, we hypothesize that HPV genome fragmentation would occur in BC, so that small-fragments of HPV genome might remain in BC tissues as well as in transplantable tumour samples and cell lines, which were all negative for HPV by TOSHIBA microarray.

To reveal this hypothesis, we designed the small-fragment PCR (sf-PCR) to detect fragmented HPV genome (Figure 3 panel A). As compared to positive control cell, Siha, smaller amount of HPV16 genome which the sized is 147bp was detected in HCC1599 BC cells by sf-PCR (panel B) although we could not detect any HPV16 DNA by TOSHIBA microarray. Sequencing analysis revealed that HCC1599 genomic DNA actually had 147bp of smaller fragment of HPV16 DNA (panel C). In addition, Southern blot analysis using identified sequence as a probe clearly showed that small fragment of HPV16 genome was integrated into human genome (panel D). These data seem to support our hypothesis that HPV genome fragmentation would occur during the step of breast carcinogenesis.

All these data together with microarray results suggest that HPV genome DNAs are eliminated from BC tissues more preferentially as compared to matched normal tissues. Especially, we assumed that tumour region might still maintain small-fragment of viral DNA in cases when samples were negative with DNA microarray method but their matched normal counterparts were positive for HPV. Thus, we performed combination analysis using sf-PCR and Southern blot to detect fragmented HPV DNAs in tumour region. Fragmented HPV16 positive samples by Southern blot tend to be corresponding to HPV16 positive samples by TOSHIBA microarray. Interestingly, HPV16 L1 P1 probe could detect fragmented HPV16 genome in some of BC tissue samples even though these samples were completely negative for HPV by TOSHIBA microarray. These data strongly support our contention that HPV genome fragmentation might occur during multi-step carcinogenesis of BC. Detection of 147bp of HPV short fragments in the patients and BC-derived cells in vitro and in mice (Figurel), is highly suggestive of its active role in BC carcinogenesis.

Herein we showed that 147bp of HPV16 small fragment DNA could be efficiently detected by sf-PCR as representative examples. Likewise, other sequences such as 275 and 215bp of HPV16 LI, and 196, 204 and 108bp of HPV18 L1 sequences seem to be able to be used for detection of fragmented HPV genome according to our data.

All these findings strongly suggest the unique association between HPV and progression of breast cancer. Hence, it is possible to establish a unique diagnosis system through combining TOSHIBA microarray and sf-PCR methods. Prognosis and the risk of breast cancer could be better monitored through testing HPV DNAs in matched normal and tumour tissues with these methods comparatively. Example 3

Molecular epidemiological studies in vivo; BC tissues showed high prevalence of HPV infection

We first examined the relationship between HPV status and pathological features using 209 female BC cases using the DNA chip system and genomic DNA extracted from frozen samples in Singapore (Figure 6, Table 2). High-risk HPV DNA was detected in 31 % in average of all BC samples. Majority of HPV types found in BC were HPV-16 (47%) and HPV-18 (36%) followed by HPV-35, -56, -59 and -31 subtype distribution (data not shown). The prevalence of HPV varied greatly depending on sample populations ranging from 25 to 100% (Figure 6). Especially, almost all lobular carcinoma (LC) type of BC were positive for HPV even though the numbers were small for both LC in situ (IS) (LCIS) and invasive LC (ILC) (p=0.0003). The incidence of HPV infection in pre-malignant (IS) state of carcinomas seemed to be higher than that of invasive carcinomas though it was not statistically significant (p=0.77) (Figure 6).

Example 4

Matched normal (MN) samples showed higher HPV infection rate than tumour (T) in coupled BC samples and HPV subtypes in both regions were closely related We then compared the prevalence of HPV infection in coupled samples from 40 BC patients (all IDC) from whom not only tumour (T) but also matched normal (MN) samples were available. Amongst 40 patients, 11 cases were positive for HPV in T (27.5%) (data not shown). However, HPV prevalence was increased to 55% (22/40) if calculation was performed per patient because MN showed higher infection rate than T (p=0.0597). In these 40 patients, 9 patients showed evidence of multiple infections with different HPV subtypes which might reflect numbers of infection events. Importantly, the same viral subtypes were found 12 times in both MN and T of these 9 cases (Table 1). These data suggest that HPVs found in T and MN are closely linked. Example 5

The BC with estrogen receptor (ER) showed significantly higher HPV prevalence than ER- negative tumors

Table 2 also shows the prevalence of the HPV infection according to various demographic and pathological factors in BC. The HPV positivity was lower when the patients were younger but it seemed to increase with age (p for trend= 0.1155) (not shown). Irrespective of ethnicity, all 3 major groups (Chinese, Malay and Indian) showed essentially a similar HPV prevalence including others (not shown). The patients with a history of BC in the family and the women with BC at both sides seemed to show higher HPV rate suggesting an infectious etiology of BC. Importantly, HPV positive ratio was quite different between positive and negative groups for ER and PR status. Especially, the BC with ER showed significantly higher HPV prevalence than ER-negative tumors (p=0.0378) (Table 2). Example 6

HPV-positive BC showed better prognosis than virus-negative tumors

HPV-positive tumours showed significantly longer duration period for recurrence after BC diagnosis as compared to virus-negative tumors (p=0.047) (Figure 7A). When the same analysis was performed among ER-positive cases exclusively, again HPV-positive tumours showed a longer duration until recurrence as compared to HPV-negative tumours although this was not statistically significant (p=0.135) (Figure 7B).

Example 7

BC tissues harboured viral copy numbers at the level far below than 1 copy/cell

We also checked the copy number of HPV in our BC samples using HPV-16 or -18 L1 gene specific quantitative-PCR. Most of the cervical cancers used as controls had more than 1 copy/cell of HPV-16 or HPV-18 genome, whereas all samples at various stages of BC tissues harboured viral copy numbers at much lower level than 1 copy/cell (data not shown).

Example 8 In vitro virological/molecular biological studies; HPV induces A3B expression in normal breast cells

Above results suggest that BC and pre-BC tissues are highly infected with HPV but the viral load in BC is quite low. Thus, it is unlikely that integrated HPV plays an active role in the growth of established breast tumour cells. However, another possibility is still open; that is, a viral involvement in early stages of breast carcinogenesis through induction of host genetic change, possibly by its oncogenes. Actually, recent evidence has implicated that a family of cytidine deaminase A3B is a source of mutations in various cancers, especially BC. Thus, it was interesting to determine whether viral infections could trigger A3B induction and involve in mutagenesis of host genomes. For this purpose, normal breast derived cells, MCF10-A, were transfected with A3B reporter gene, A3B-Pro+TK- RLuc and either HPV16 BamHI or HPV18 EcoRI DNA fragments generated by restriction enzymes to see whether HPV-infected cells show that transcription factor(s) binding to A3B promoter is activated. Significantly higher luciferase activity (3-5-fold) was recorded upon transfection of viral DNA fragments.

We then established the MCF10-A cells persistently infected with HPV-18. These cells express mRNA of E1 (middle to C-terminal of E1 is deleted), E6 and E7 of HPV18 as well as E7 protein, abundantly (Fig.8C and D). When these cells were compared for the expression of A3B, HPV-positive cells expressed mRNA for A3B about 2.5 times more than parental cells calculated based on A3B/GAPDH. Interestingly, expression of another cytidine deaminase, A3G was strongly inhibited in HPV-infected cells (Fig. 8B).

Next, we asked which oncogene of HPV is responsible for induction of A3B. 293T cells were transfected by control empty vector, E6 and E7 oncogenes of HPV and A3B- Pro/TK-RLuc activity was monitored. Although each E6 or E7 stimulated A3B promoter significantly, maximum activity was achieved when both E6 and E7 genes were simultaneously transfected suggesting that A3B induction requires both oncogenes (data not shown). Example 9

Cancer phenotypes triggered by HPV infection in MCF 10-A cells

Then, we addressed whether HPV infection and resulting A3B induction trigger other cancer hallmarks using HPV-18-infected cells (Fig.9). When acinar formation was performed using Matrigel 3-D culture, acinar size of HPV-infected MCF10-A cells showed larger though there was no statistical significance. Also, these acinar structures included polarized epithelial cells and hollow lumen so that the cells seemed to still possess normal cell phenotype. In the culture, HPV-infected cells exhibited small growth advantage over uninfected cells (data not shown). Thus, HPV infection did not transform the cell which is consistent with previous observation. However, effect of HPV infection was more clearly shown when other parameters such induction of phosphorylated γΗ2ΑΧ (a marker for DNA damage for cancer phenotypes) and comet formation (a marker for DNA fragmentation) were employed. HPV infection apparently caused γΗ2ΑΧ induction (Fig.9A and B), and DNA fragmentation, as evidenced by visible comets (Fig. 9C) which are important for the induction of these cancer phenotypes.

Example 10

Abrogation of HPV-induced cancer phenotypes by shRNA against HPV E6, E7 and A3B

We showed that HPV infection could induce A3B expression resulting in increase of γΗ2ΑΧ level and genomic DNA breaks in breast cells. To further confirm the role of A3B and HPV on DNA damage in human genome, we next employed short-hairpin RNA (shRNA) specific to A3B, HPV 8 E6 or E7 to knock down those gene expression. Silencing of HPV E6, E7 and A3B abrogated HPV-induced malignant phenotypes in MCF10A-HPV18 cells which are either stably or transiently expressing shRNA (Fig.10A and B). In addition, γΗ2ΑΧ reduction level in HPV18 E6 or E7 shRNA treated cells seems to be slightly greater than its level in A3B shRNA treated cells. These results suggest that DNA damage and yH2AX would be induced through possible two pathways; 1) HPV18 E6/E7-A3B (A3B-dependent) pathway and 2) HPVE6/E7 direct (A3B- independent) pathway. Example 11

A3B RNA level based on HPV and ER status in BC samples

Induction of A3B by HPV expression in vitro prompted us to address whether HPV- positive tumours express higher levels of A3B than negatives in patients' samples very preliminarily with small number of samples. Mean relative mRNA levels for A3B for HPV- positive samples was much higher than that of HPV-negative ones (Fig.11). In conclusion, we showed that high-risk HPVs are definitely but uniquely associated with BC patients. Based on our patients' data as well as in vitro studies, we propose here a provocative carcinogenic mechanism of BC that aberrant A3B expression caused by HPV infection leading to γΗ2ΑΧ induction and single/double strand DNA breaks might be a mechanism of mutation accumulation in the breast epithelium during HPV-associated breast carcinogenesis which possibly occurs in the early stage of multi-step, multifactorial carcinogenesis (Fig. 12). If this model turns out to be at all feasible, reevaluation of several other HPV-associated non-genital cancer systems such as those in lung, colon, bladder, and so on is warranted since these tumours represent a significant reminiscence to BC in terms of high infection rate but low copy numbers (though not monitored in most cases) of high-risk HPV in cancer tissue. Recent results with genome- wide sequencing of cancer DNA suggesting that A3B (and possibly A3A) is a possible enzymatic source of mutation in these cancers also support our view and the relevance of such approach. Moreover, the same idea could be applied even to the other types of human cancers induced by other oncogenic DNA viruses such as EBV and human polyomaviruses (e.g. nasopharyngeal cancer). Eventually, the role of HPV in development of BC and other HPV-associated non-genital cancers will be ascertained by monitoring the effect on the disease prevalence in women by cervical cancer vaccinations against high-risk HPV types. Table 1 : Actual data for the HPV prevalence in T and MN specimens from 40 BC patients as determined by DNA chip (data represent only a part).

Table 2: Prevalence of the HPV infection according to various demographic and pathological factors in breast cancers

HPV-positive HPV-negative Total

P-value

N(%) N(%) N(%)

Age (Invasive samples )

<40 1 (6.7) 14(93.3) 15

40-49 12 (23.5) 39 (76.5 51

50-59 17 25.8) 74.2 66 ^♦*0.11SS

60-69 14(31.8) 68.2' 44 ** Trend test

>70 8 (30.8) 69.2' 26

Total 52 (25.7) 150 (74.3) 202 (100)

Ethnicity (All)

Chinese 32(30.8) 72 69.2 104

Malay 11(22.4) 38 77.6 49

Indian 6(26.1) 17 73.9 23 ***0.445

Others 4 (16.7) 20 83.3 24 *** Fisher Cvs I

Total 53 (26.5) 147(73.5) 200(100)

Family history (Tumor - MN)

Yes 7(29.2) 17 (70.8) 24

No 35 (25.9) 100(74.1) 135 *0.8026

Unknown 1(67) 14 (93.3) 15 * Fisher: Yes vs No

Total 43(24.7) 131(75.3) 174 (100)

BC at both sides (Tumor - MN)

Yes 4 36.4) 7(63.6) 11

No 42 (25.9) 120(74.1) 162 ^♦0.4857

Unknown 0 6.7) 1 (93.3) 1 * Fisher: Yes vs No

Total 46(26.4) 128(73.6) 174 (100)

Estrogen receptor (Tumor - MN)

Positive 35,(29.7) 83 (70.3)

Negative 8 (14.5) 47(85.5

Unknown 0(0.0) Ψ .5 *0.0378

lflOO) 1 * Ftsher: Positive vs negative

Total 43(24.7) 131(75.3) 174 (100)

Progesterone receptor (Tumor - MN)

Positive 30(26.8) 82(73.2) 112

Negative 13(21.3) 61 78.7) 61 ^♦0.466

Unknown 0 (0.0) 1 (100) 1 * Fisher: Positive vs negative

Total 43 (24.7) 131(75.3) 174 (1O0)

HER2 (Tumor -MN)

Positive 7(19.4) 29(80.6) 36

Negative .23 (28.8) 57 71.2 80 "0.3626

Unknown or N/A 13(22.4) 45 (77.6) 58 * Fisher: Positive vs negative

Total 43(24.7) 131(75.3) 174 (100)

Vascular invasion (Tumor - MN)

Present 19 28.8 47 (71.2) 66

Absent 24 24.0) 76 (76.0) 100 ^♦0.5875

ND 0(0.0) 8 (lOOj ^♦ Fisher: positive vs negative

Total , , 43 24.7) 131 (75.3) 174 (100)

Lymph vessel invasion (Tumor - MN)

Present 19 29.2) 40 (60.7) 65

Absent 24 24.0) 76(76.0) 100 ^♦0.4727

ND 0(0.0) 9 (100) 9 * Fisher: positive vs negative

Total , , 43(26.4) 131(69.7) 174 (100)

Lymph node metastasis (invasive)

Low (1-29%) 15 (28.3) 38(71.7 53 ^ ^¾she : Meta Low vs No meta

Mid (30-59%) 5 26.3 14 73.7^' 19 ^♦0.5199

High >60%) 7 28.0) 18 72.o; 25 * Fisher: Meta Mid vs No meta

Total (1-100%) 27(27.8) 70 72.2 97 ^♦0.3906

No metastasis 13(18.3 58(81.7 71 * Fisher: Meta high vs No meta

Unknown or N/A 1(33.3) 2 (fe₆.7)' 3 ^♦0.1993

Total 41(24.0) 130 (76.0) 171 (100) * Fisher: Meta total vs No meta

HPV16 HPV16 HPV18 HPV18 % tumor cells

Sample ID

DNAChip (copfe$/10¼ell) DNAChip (copies/KHcell) in sample

Γ

BC284T Jj ! » ί ^" ]j 701DCIS)

BC284N + 8 -

BC319T i ^♦ 1 . 118 - - 50 (IDC)

BC 319N + 34 - -

BC 20T I - i I - j SO (IDC)

BC 20N + 40 + 2

BC437T - - 1 - j 90 (IDC)

BC 37N + 58

BC441T + I · - 50 (IDC)

BC441N + 28 -

CC053T 1

i ⁺ 31746

CC150T + 445466 -

!

CC 128B ! + j 1100 ! +

CC 3S2T + 18218

T: Tumor sample N: Non-tumor sample (adjacent to tumor) -: undetectable

Table 3

Claims

1 A method to diagnose a non-genital cancer in a subject comprising:

i) providing an isolated biopsy sample from a human subject to be tested;

ii) forming a preparation comprising said sample and at least one oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising human papilloma viral [HPV] genomic DNA; a thermo-stable DNA polymerase, deoxynucleotide triphosphates and co-factors required for the specific polymerase chain amplification of HPV DNA;

iv) analyzing the amplified products of said polymerase chain reaction for the presence or absence of a nucleic acid molecule[s] comprising one or more HPV nucleotide sequence[s]; and optionally

2. A method to determine the prognosis of a non-genital cancer in a subject that has been diagnosed with a non-genital cancer comprising:

i) providing an isolated biopsy sample from a human subject to be tested;

ii) forming a preparation comprising said sample and at least one oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising HPV genomic DNA; a thermo-stable DNA polymerase, deoxynucleotide triphosphates and co-factors required for the specific polymerase chain amplification of HPV genomic DNA;

iv) analyzing the amplified products of said polymerase chain reaction for the presence or absence of a nucleic acid molecule[s] comprising one or more HPV nucleotide sequence[s]; v) comparing the amplified produces] with a normal matched control to determine the identity of one or more HPV types; and

vi) determining the likely prognosis of the cancer in said human subject.

3. A method to diagnose and treat a non-genital cancer in a subject comprising:

i) providing an isolated biopsy sample from a human subject to be tested;

vi) designing a treatment regimen for the prevention or treatment of cancer as determined by the result of said diagnostic test.

4. The method according to any one of claims 1 to 3, wherein said human papilloma virus is selected from the group consisting of: HPV-16, HPV-18, HPV-31 , HPV-33, HPV- 35, HPV-39, HPV-45, HPV-51 , HVP-52, HVP-HPV-56, HPV-58, HPV- 59 and HPV-68.

5. The method according to claim 4 wherein said human papilloma virus is selected from the group consisting of: HPV-16, HPV-18, HPV-35 and HPV-59.

6. The method according to claim 5 wherein said human papilloma virus is HVP-18 and HPV-35.

7. The method according to claim 5 wherein said human papilloma virus is HVP-18 and HPV-59.

8. The method according to claim 5 wherein said human papilloma virus is HVP-18, HPV-35 and HPV-59.

9. The method according to claim 4, wherein said HPV is HPV-16 and/or HPV-18.

10. The method according to claim 4 wherein said human papilloma virus is HPV-31 , HPV-35, HPV-56 and HPV-59.

11. The method according to any one of claims 1 to 5, wherein said polymerase chain amplified nucleic acid molecules are hybridized to a DNA array comprising HPV genomic DNA isolated from one or more HPV sub-types.

12. The method according to claim 1 1 wherein said DNA array comprises at least HPV-16 and/or HPV-18.

13. The method according to claim 1 1 wherein said DNA array comprises at least virus is HPV-31 , HPV-35, HPV-56 and HPV-59.

14. The method according to any one of claims 1 to 13 wherein said polymerase chain amplified nucleic acid molecules are sized fractionated.

15. The method according to claim 14 wherein said size fractionation is by electrophoresis.

16. The method according to any one of claims 1 to 15 wherein said polymerase chain reaction is a short fragment length polymerase chain reaction.

17. The method according to any one of claims 11 to 16 wherein said method combines detection of HPV by hybridization to a DNA array and by short fragment length polymerase chain reaction.

18. The method according to any one of claims 11 to 17 wherein the amplified human papilloma virus nucleic acid molecule[s] are at least 100 base pairs [bp].

19. The method according to claim 18 wherein said amplified human papilloma virus nucleic acid moleculefs] are between 100 and 300 bp, preferably between about 108 bp and 275 bp.

20. The method according to claim 18 or 19 wherein said amplified nucleic acid molecules are about 147bp, 196bp, 204bp, 215bp and 275bp.

21. The method according to any one of claims 1 to 10 wherein said polymerase chain amplified nucleic acid molecule is subject to DNA sequencing and sequence comparison to confirm the nucleotide sequence identity.

22. The method according to any one of claims 1 to 21 wherein the non-genital cancer is a carcinoma.

23. The method according to claim 22 wherein said carcinoma is selected from the group consisting of: breast, tonsil, colorectal, oesophageal, bladder and lung.

24. The method according to claim 23 wherein said carcinoma is breast cancer.

25. The method according to any one of claims 1 to 21 wherein said cancer is a sarcoma.

26. The method according to any one of claims 1 to 25 wherein said amplified nucleic acid molecule comprises or consists essentially of HPV genomic DNA that encodes all or part of the L1 capsid protein.

27. The method according to claim 26 wherein said amplified nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO: .

28. The method according to claim 27 wherein said amplified nucleic acid molecule consists essentially of one or more nucleotide sequence[s] selected from the group: SEQ ID NO: 2, 3 or 4.

29. The method according to claim 26 wherein said amplified nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 5

30. The method according to claim 29 wherein said amplified nucleic acid molecule consists essentially of one or more nucleotide sequence[s] selected from the group: SEQ ID NO: 6, 7 or 8.

31. The method according to any one of claims 1 to 30 wherein said primer pairs are selected from the group consisting of:

forward: 5'- CAGATGTCTCTTTGGCTGCCTAG-3' [SEQ ID NO: 9]

reverse: 5'- GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 10]

forward: 5'- GGTGTCAGAACCATATGGCGAC-3' [SEQ ID NO: 1 ]

reverse: 5'- GTTGGTTACCCCAACAAATGCC-3' [SEQ ID NO: 12]

forward: 5'- CCAGCACCTAAAGAAGATGATCCC-3' [SEQ ID NO: 13]

reverse: 5'- GCAGTTGTAGAGGTAGATGAGGTGG-3' [SEQ ID NO: 14]

forward: 5'- GCCTGTATACACGGGTCCTG-3' [SEQ ID NO: 15]

reverse: 5'-GGCCGCCACAAAGCCATCTG-3' [SEQ ID NO: 16]

forward: 5'-GATACTGGATATGGTGCCATGGAC-3' [SEQ ID NO: 17]

reverse: 5'-GTCACCCATAGTACCTGCTCTATTCC-3 [SEQ ID NO: 18]

forward: 5'- GTTCAGGCTGGATTGCGTCG-3' [SEQ ID NO: 19]

reverse: 5'- CCTGGCACGTACACGCACAC-3 [SEQ ID NO: 20]

forward: 5'-CAGATGTCTCTTTGGCTGCCTAG-3\ [SEQ ID NO: 21],

reverse: 5'-GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 22],

forward: 5'-GGTGTCAGAACCATATGGCGAC-3', [SEQ ID NO: 23],

reverse: 5'-GTTGGTTACCCCAACAAATGCC-3', [SEQ ID NO: 24],

forward: 5'-CCAGCACCTAAAGAAGATGATCCC-3', [SEQ ID NO: 25],

reverse: 5'-GCAGTTGTAGAGGTAGATGAGGTGG-3'. [SEQ ID NO: 26],

or oligonucleotide primer nucleotide sequences that have at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequences set forth in SEQ ID NO: 9-26 and which amplify a L1 capsid nucleic acid molecule from genomic DNA.

32. The method according to claim 31 wherein said primer pairs are selected from the group consisting of:

forward: 5'- CAGATGTCTCTTTGGCTGCCTAG-3' [SEQ ID NO: 9]

reverse: 5'- GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 10]

forward: 5'- GGTGTCAGAACCATATGGCGAC-3' [SEQ ID NO: 11]

reverse: 5'- GTTGGTTACCCCAACAAATGCC-3' [SEQ ID NO: 12]

forward: 5'- CCAGCACCTAAAGAAGATGATCCC-3' [SEQ ID NO: 13]

reverse: 5'- GCAGTTGTAGAGGTAGATGAGGTGG-3' [SEQ ID NO: 14]

forward: 5'-CAGATGTCTCTTTGGCTGCCTAG-3', [SEQ ID NO: 21],

reverse: 5'-GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 22],

forward: 5'-GGTGTCAGAACCATATGGCGAC-3\ [SEQ ID NO: 23],

reverse: 5'-GTTGGTTACCCCAACAAATGCC-3', [SEQ ID NO: 24],

forward: 5'-CCAGCACCTAAAGAAGATGATCCC-3', [SEQ ID NO: 25],

reverse: 5'-GCAGTTGTAGAGGTAGATGAGGTGG-3'. [SEQ ID NO: 26].

33. The method according to claim 31 wherein said primer pairs are selected from the group consisting of:

forward: 5'- GCCTGTATACACGGGTCCTG-3' [SEQ ID NO: 15]

reverse: 5'-GGCCGCCACAAAGCCATCTG-3' [SEQ ID NO: 16]

forward: 5'-GATACTGGATATGGTGCCATGGAC-3' [SEQ ID NO: 17]

reverse. 5'-GTCACCCATAGTACCTGCTCTATTCC-3 [SEQ ID NO: 18]

forward: 5'- GTTCAGGCTGGATTGCGTCG-3' [SEQ ID NO: 19]

reverse: 5'- CCTGGCACGTACACGCACAC-3 [SEQ ID NO: 20]

34. The method according to claim 31 or 32 wherein said primer pairs amplify HPV- 16 genomic DNA.

35. The method according to claim 31 or 32 wherein said primer pairs amplify HPV- 18 genomic DNA.

36. The method according to any one of claims 1 to 35 wherein said method compares the detection of said human papilloma viral genomic DNA in a tumour biopsy and a non-tumour biopsy from the same human subject.

37. The method according to any one of claims 1 to 36 wherein said method is combined with detection of one or more further non-HPV biomarkers which is diagnostic or prognostic of cancer.

38. The method according to claim 37 wherein said biomarker is selected from the group consisting of: Human Epidermal Growth Factor Receptor 2 [HER2], Oestrogen

Receptor [ER], Progesterone Receptor [PR], cytidine deaminase [A3B] for example APOBEC3B, and gamma Histone 2A member X [γΗ2ΑΧ].

39. The method according to any one of claims 3 to 38 wherein the treatment comprises the administration of an anti-cancer agent in an effective amount sufficient to prevent or slow the progression of cancer in said subject.

40. The method according to claim 39 wherein said treatment is the administration of a chemotherapeutic agent.

41.. The method according to claim 39 wherein said treatment is the administration of a vaccine.

42. The method according to claim 39 wherein said treatment is administration of a therapeutic antibody.

43. A kit comprising one or more pairs of oligonucleotide primers selected from the group consisting of:

forward: 5'- CAGATGTCTCTTTGGCTGCCTAG-3' [SEQ ID NO: 9]

reverse: 5'- GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 10]

forward: 5'- GGTGTCAGAACCATATGGCGAC-3' [SEQ ID NO: 11]

reverse: 5'- GTTGGTTACCCCAACAAATGCC-3' [SEQ ID NO: 2]

forward: 5'- CCAGCACCTAAAGAAGATGATCCC-3' [SEQ ID NO: 13]

reverse: 5'- GCAGTTGTAGAGGTAGATGAGGTGG-3' [SEQ ID NO: 14] forward: 5'- GCCTGTATACACGGGTCCTG-3' [SEQ ID NO: 15]

reverse: 5'-GGCCGCCACAAAGCCATCTG-3' [SEQ ID NO: 16]

forward: 5 -GATACTGGATATGGTGCCATGGAC-3' [SEQ ID NO: 17]

reverse: 5 -GTCACCCATAGTACCTGCTCTATTCC-3 [SEQ ID NO: 18]

forward: 5'- GTTCAGGCTGGATTGCGTCG-3' [SEQ ID NO. 19]

reverse: 5'- CCTGGCACGTACACGCACAC-3 [SEQ ID NO: 20]

forward: 5 -CAGATGTCTCTTTGGCTGCCTAG-3', [SEQ ID NO: 21],

reverse: 5'-GGGATGTCCAACTGCAAGTAGTC-3' [SEQ ID NO: 22],

forward: 5'-GGTGTCAGAACCATATGGCGAC-3', [SEQ ID NO: 23],

reverse: 5 -GTTGGTTACCCCAACAAATGCC-3', [SEQ ID NO: 24],

forward: 5'-CCAGCACCTAAAGAAGATGATCCC-3', [SEQ ID NO: 25],

reverse: 5'-GCAGTTGTAGAGGTAGATGAGGTGG-3'. [SEQ ID NO: 26],

or primer oligonucleotide nucleotide sequences that have at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequences set forth in SEQ ID NO: 9-26 and which amplify a L1 capsid nucleic acid molecule from genomic DNA.

44. The kit according to claim 43 wherein said kit also comprises polymerase chain reaction components including a thermo-stable DNA polymerase, deoxynucleotide triphosphates and co-factors required for the specific polymerase chain amplification of HPV genomic DNA and optionally control HPV DNA.