WO2015041788A1 - Mutations du promoteur tert dans la néoplasie urothéliale - Google Patents

Mutations du promoteur tert dans la néoplasie urothéliale Download PDF

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Publication number
WO2015041788A1
WO2015041788A1 PCT/US2014/051808 US2014051808W WO2015041788A1 WO 2015041788 A1 WO2015041788 A1 WO 2015041788A1 US 2014051808 W US2014051808 W US 2014051808W WO 2015041788 A1 WO2015041788 A1 WO 2015041788A1
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WO
WIPO (PCT)
Prior art keywords
tert promoter
bladder cancer
seq
genetic alteration
urine
Prior art date
Application number
PCT/US2014/051808
Other languages
English (en)
Inventor
Bert Vogelstein
Kenneth W. Kinzler
Luis Diaz
Nickolas Papadopoulos
George J. NETTO
Ralph Hruban
Isaac A. KINDE
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The Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Johns Hopkins University filed Critical The Johns Hopkins University
Publication of WO2015041788A1 publication Critical patent/WO2015041788A1/fr
Priority to US15/077,284 priority Critical patent/US10870890B2/en
Priority to US17/097,510 priority patent/US11667976B2/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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention is related to the area of cancer. In particular, it relates to urothelial cancers.
  • Urothelial carcinoma of the bladder is the most common malignancy of the urinary tract with 73,000 new cases and 15,000 deaths expected in 2013 in the US alone (1). These invasive carcinomas arise from histologically well-defined papillary and flat precursor lesions, providing a potential opportunity for early detection and treatment (2). Although urine cytology enjoys a reasonable sensitivity and specificity for detecting high-grade neoplasms, its performance in detecting low-grade tumors is poor, with a sensitivity and specificity of 4% and 30%, respectively (3).
  • a number of urine-based markers have been developed to improve the accuracy of noninvasive screening and surveillance in bladder cancer.
  • Food and Drug Administration (FDA) approved tests the Immunocy test (Scimedx Corp, Danville, NJ), nuclear matrix protein 22 (NMP22) immunoassay test (Matritech, Cambridge, MA) and multitarget fluorescence in situ hybridization (FISH) (UroVysion; Abbott Park, IL) (4) have demonstrated an overall sensitivity of 70% and a specificity range of up to 89%. Performance inconsistencies, as a result of variability in pre-analytical and analytical specimen factors, have impeded their wide-spread clinical use.
  • TERT telomerase reverse transcriptase
  • Muscle-invasive urothelial carcinoma is responsible for the vast majority of bladder cancer related deaths and many of these deaths could be prevented if precursor lesions were detected and surgically excised prior to their invasion into the muscle (10-13). New strategies for the early detection of such lesions are therefore urgently needed (14). There is a continuing need in the art to find ways of detecting early, curable, bladder disease and for monitoring recurrences of bladder cancer after tumor resection.
  • a method is provided. Nucleic acids obtained from a urine sample of a human are tested for a genetic alteration in telomerase reverse transcriptase promoter at one or more nucleotides between 1295205 and 1295297 on the minus strand of chromosome 5 in version hgl9 of human genome sequence. The human has not been diagnosed with bladder cancer.
  • a method is provided. Nucleic acids obtained from a urine sample of a patient are tested for a genetic modification in telomerase reverse transcriptase promoter. One or more nucleotides between 1295205 and 1295297 on the minus strand of chromosome 5 in version hgl9 of human genome sequence are interrogated. The patient has had a surgical excision or other treatment for a bladder cancer.
  • a first primer comprises a first segment of TERT promoter region gcggaaaggaaggggag (SEQ ID NO: 5), and a second primer comprises a second segment of TERT promoter region CCGTCCCGACCCCT (SEQ ID NO: 6).
  • a first primer comprises a first segment of TERT promoter region ggccgcggaaaggaag (SEQ ID NO: 7), and a second primer comprises a second segment of TERT promoter region CGTCCTGCCCCTTCACC (SEQ ID NO: 8).
  • Fig. 1 shows a schematic of the TERT locus and positioning of the Safe-SeqS amplification primers.
  • the yellow marks indicate the positions (offset by -1,295,000 base pairs) of the most common TERT promoter mutations previously reported and identified in this study.
  • UID Unique identifier
  • UPS Universal primer binding site.
  • Fig. 2 Table 1 : Clinicopathologic characteristics of patients analyzed in this study
  • Fig. 3 Table 2: TERT promoter mutations
  • Fig. 4 Table 3: Correlation between TERT promoter mutation status and tumor recurrence.
  • Fig. 5 Table 4: Correlation of TERT promoter mutation status and tumor progression
  • Fig. 6 Table 5: Correlation of TERT mutation status in original diagnostic transurethral resection biopsy (TURB) tissue and TERT mutation status in urine collected at follow-up.
  • Fig. 7 Table 6: Correlation of TERT promoter mutation status in follow-up urine samples with recurrence
  • Fig. 8 Table SI . TERT promoter mutation status in 59 pTa and 17 carcinoma in situ (CIS) patients. DETAILED DESCRIPTION OF THE INVENTION
  • TERT promoter mutations are the most common genetic alteration in noninvasive bladder cancer identified to date, occurring in the majority (74%) of such precursor lesions. They occur in cancers developing through both the papillary and flat routes to tumor progression (15), and occur in low-grade as well as high-grade tumors. These mutations can be detected in the urine of patients with bladder cancer using sensitive techniques we have developed. TERT promoter mutations provide a useful biomarker for the early detection of bladder cancers, and patients at high risk for this disease can be screened in this noninvasive way.
  • TERT promoter mutations occur early, are specific for neoplasia, and can be identified in the urine with techniques such as those described below.
  • Testing nucleic acids can be accomplished by any means known in the art for determining a nucleotide identity at one or more positions. Testing typically involves isolation of nucleic acids from a clinical sample and doing at least one transformative reaction on the nucleic acids. Alternatively, a sample can be treated to make its nucleic acids accessible to probes, to perform an in situ assay. For example, the nucleic acids can be denatured and hybridized to a probe. The nucleic acids can be amplified. The nucleic acids can be hybridized to a primer and the primer extended by one or more bases. The nucleic acids can be modified by an enzyme that uses DNA as a substrate. Each of these methods involves a transformation of nucleic acids. The nucleic acids are a physical substance and are not a representation. One or both strands can be tested or assayed.
  • Therapies which can be prescribed are any that are known in the art for treating bladder cancer. These may include Bacillus Calmette-Guerin (BCG) and intravesical chemotherapy. Adjuvant therapies may include chemotherapy agents such as cisplatin and gemcitabine. Other chemotherapy agents or biological agents can be used in combination or separately. Similarly, radiation can be used alone or together with other therapies. Prescribing a therapy typically involves a medical professional making a determination based on fact or surmise that a particular therapy will be or might be efficacious. The medical professional typically permanently records this determination in a medical chart or file. In addition, the drug and dosage are reduced to a verbal communication, such as a writing, a voice recording, or an electronic message that is delivered to a dispensing pharmacy.
  • a verbal communication such as a writing, a voice recording, or an electronic message that is delivered to a dispensing pharmacy.
  • Any confirmatory test can be performed as is known in the art if a bladder cancer is detected by means of the urine test.
  • One such test is a cytoscopy. Any test that can sensitively detect a bladder cancer can be used.
  • Elevated risk of a bladder cancer can be ascertained based on known exposure to a carcinogen. Such exposures may be, for example, environmental, nutritional, or pharmaceutical. Cigarette or other tobacco smoking or ingesting, which may be considered an environmental exposure, is a major risk factor. Alternatively or in addition, the elevated risk may be due to a family history of bladder cancer.
  • Primers according to the invention are complementary to portions of the TERT promoter region.
  • the primer will comprises a sequence according to any of SEQ ID NOs: 5, 6, 7, or 8.
  • Primers will typically be at least 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides in length and typically will be less than 50, 45, 40, 35, 30, 25 nucleotides in length.
  • the primers will also comprise sequences that are not complementary to portions of the TERT promoter.
  • any of the specified TERT promoter sequences will be linked to other non-TERT promoter sequences.
  • the TERT promoter sequences may be linked to a universal priming site (UPS) and/or a unique identifier (UID).
  • UPS universal priming site
  • UID unique identifier
  • the UID may, for example be comprised of a number of degenerate N bases (equal likelihood of being an A, C, T, or G).
  • the degenerate bases may range from 4-20, typically.
  • the sequence of the primers is not naturally occurring.
  • the primer may be linked to a non- nucleotide moiety, such as to a fluor, a chromophor, or a radioactive moiety.
  • FFPE formalin-fixed paraffin-embedded
  • the forward and reverse amplification primers contained the rE ⁇ -specific sequences at their 3 ' ends and a universal priming site (UPS) at their 5' end.
  • the reverse primer additionally contained a 14-base unique identifier (UID) comprised of 14 degenerate N bases (equal likelihood of being an A, C, T, or G) between the UPS and gene-specific sequences.
  • USD 14-base unique identifier
  • the sequences of the forward and reverse primers were either 5'-
  • PCR products were amplified in 25 ⁇ ⁇ reactions containing IX Phusion Flash High-Fidelity PCR Master Mix and 0.5 ⁇ amplification primers that each contained the first-stage UPS at their 3' ends and the grafting sequences required to hybridize to the sequencing instrument flow cell at their 5' ends (8, 9).
  • the reverse amplification primer additionally included a 6 bp index sequence, unique to each sample, inserted between the UPS and grafting sequences.
  • 17 cycles of PCR were performed in the following manner: 98°C for 10 seconds, 63°C for 120 seconds, and 72°C for 120 seconds.
  • the PCR products were purified with AMPure and sequenced on a MiSeq instrument.
  • TERT promoter region of reads containing UIDs was matched to a reference sequence using a custom script. TERT promoter sequences with five or fewer mismatches were retained for further analysis. Tumor samples were considered positive if the fraction of mutations exceeded 1% of alleles (which was a frequency at least lOx higher than found in control DNA templates from FFPE tissues). Urine samples were considered positive when the frequency of mutation exceeded 0.1% of alleles (a frequency at least lOx higher than found in control DNA templates from urine samples of patients without TERT mutations in their primary tumors). All sequencing assays scored as positive were confirmed in at least one additional, independent PCR and sequence assay.
  • Pearson's chi-squared test was used for analysis of association of categorical variables. A two-tailed probability ⁇ 0.05 was required for statistical significance.
  • TERT promoter mutations were identified in 56/76 (74%) of these urothelial carcinomas (Table 2). In contrast, none of the eight samples of adjacent normal urothelium harbored TERT promoter mutations. Additionally, we did not detect TERT promoter mutations in 15 samples of peripheral blood from the same patients. Twelve of the blood samples and five of the normal urothelial samples were from patients whose tumors harbored TERT promoter mutations. These data demonstrate that the TERT promoter mutations in these patients were unequivocally somatic and limited to the neoplastic urothelium in the bladder. The predominant alterations were g.
  • TERT promoter mutations occur early in bladder cancers and did not correlate with grade or type. Such early mutations would not be likely associated with recurrence or progression, but to evaluate this possibility, our series of samples included cases both with and without recurrence during follow up.
  • Tables 3 and 4 the relationship between TERT promoter mutation status and tumor recurrence or progression, respectively, are displayed: TERT promoter mutation status was not associated with likelihood of recurrence or progression in any subgroup.
  • TERT promoter mutations could be identified in cells in the urine.
  • urine samples are routinely taken at follow-up visits following TURB procedures to help determine whether residual tumor cells are present (via cytology or other methods).
  • SurePath® Preservative (Becton Dickinson) is an alcohol-based, preservative fluid.
  • the preservation solution serves as a transport, preservative and antibacterial medium for gynecologic (and urine cytology) specimens.
  • the rate of TERT positivity was higher in patients who had a positive diagnosis by cytology, compared to those who had atypical diagnosis by cytology.
  • the rate of TERT promoter mutation positivity was in turn higher in patients with a diagnosis of atypical cytology compared to those that were negative on cytologic exam.

Abstract

Cette invention concerne des mutations du promoteur TERT qui se produisent à la fois dans les cancers de la vessie de type papillaire et de type lésion plate, les altérations les plus fréquentes identifiées à ce jour dans les lésions précurseurs non invasives de la vessie, lesdites mutations étant détectables dans les urines, et semblant être fortement associées à une récidive du cancer de la vessie. Les mutations du promoteur TERT sont un biomarqueur urinaire utile à la fois pour le dépistage précoce et la surveillance de la néoplasie de la vessie.
PCT/US2014/051808 2013-02-18 2014-08-20 Mutations du promoteur tert dans la néoplasie urothéliale WO2015041788A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US15/077,284 US10870890B2 (en) 2013-02-18 2016-03-22 TERT promoter mutations in urothelial neoplasia
US17/097,510 US11667976B2 (en) 2013-02-18 2020-11-13 TERT promoter mutations in urothelial neoplasia

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361881113P 2013-09-23 2013-09-23
US61/881,113 2013-09-23

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US14/765,692 Continuation-In-Part US10711310B2 (en) 2013-02-18 2014-02-18 Tert promoter mutations in gliomas and a subset of tumors
PCT/US2014/016906 A-371-Of-International WO2014127359A1 (fr) 2012-02-18 2014-02-18 Mutations du promoteur de tert dans les gliomes et un sous-ensemble de tumeurs
PCT/US2014/016906 Continuation-In-Part WO2014127359A1 (fr) 2012-02-18 2014-02-18 Mutations du promoteur de tert dans les gliomes et un sous-ensemble de tumeurs

Related Child Applications (3)

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US201514765692A Continuation 2013-02-18 2015-08-04
US15/077,284 Continuation US10870890B2 (en) 2013-02-18 2016-03-22 TERT promoter mutations in urothelial neoplasia
US17/097,510 Continuation US11667976B2 (en) 2013-02-18 2020-11-13 TERT promoter mutations in urothelial neoplasia

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020044046A3 (fr) * 2018-08-29 2020-04-09 Oncologica UK Limited Test et thérapie
CN116987787A (zh) * 2023-06-09 2023-11-03 北京泛生子基因科技有限公司 检测膀胱癌是否复发的装置和计算机可读存储介质

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FRIEDRICH ET AL.: "Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients", CLINICAL CANCER RESEARCH, vol. 10, no. 22, 2004, pages 7457 - 7465 *
HORN ET AL.: "TERT promoter mutations in familial and sporadic melanoma", SCIENCE, vol. 339, no. 6122, 22 February 2013 (2013-02-22), pages 959 - 961 *
HUANG ET AL.: "Highly recurrent TERT promoter mutations in human melanoma", SCIENCE, vol. 339, no. 6122, 22 February 2013 (2013-02-22), pages 957 - 959 *
KILLELA ET AL.: "TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal", PNAS, vol. 110, no. 15, 9 April 2013 (2013-04-09), pages 6021 - 6026 *
KINDE ET AL.: "TERT promoter mutations occur early in urothelial neoplasia and are biomarkers of early disease and disease recurrence in urine", CANCER RESEARCH, vol. 73, no. 24, 11 October 2013 (2013-10-11), pages 7162 - 7167 *
LIU ET AL.: "Highly prevalent TERT promoter mutations in bladder cancer and glioblastoma", CELL CYCLE, vol. 12, no. 10, 15 May 2013 (2013-05-15), pages 1637 - 1638 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020044046A3 (fr) * 2018-08-29 2020-04-09 Oncologica UK Limited Test et thérapie
CN116987787A (zh) * 2023-06-09 2023-11-03 北京泛生子基因科技有限公司 检测膀胱癌是否复发的装置和计算机可读存储介质

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