EP1071808A1 - Dosage de telomerase preleve dans des liquides organiques utilise pour le depistage du cancer, l'evaluation du stade d'evolution de la maladie et l'etablissement du pronostic - Google Patents

Dosage de telomerase preleve dans des liquides organiques utilise pour le depistage du cancer, l'evaluation du stade d'evolution de la maladie et l'etablissement du pronostic

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Publication number
EP1071808A1
EP1071808A1 EP99907069A EP99907069A EP1071808A1 EP 1071808 A1 EP1071808 A1 EP 1071808A1 EP 99907069 A EP99907069 A EP 99907069A EP 99907069 A EP99907069 A EP 99907069A EP 1071808 A1 EP1071808 A1 EP 1071808A1
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EP
European Patent Office
Prior art keywords
telomerase
cancer
telomerase activity
blood
body fluid
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99907069A
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German (de)
English (en)
Inventor
Jeffrey W. Strovel
Judith Stamberg
Edward Highsmith
Lynne V. Abruzzo
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University of Maryland at Baltimore
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University of Maryland at Baltimore
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Publication date
Application filed by University of Maryland at Baltimore filed Critical University of Maryland at Baltimore
Publication of EP1071808A1 publication Critical patent/EP1071808A1/fr
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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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the invention is in the field of minimally invasive cancer detection and assessment.
  • the telomerase assay disclosed and claimed herein has both diagnostic and therapeutic utility.
  • telomere DNA Most somatic cells lose telomere DNA steadily as they divide and do not possess detectable telomerase activity [Kim et al., Science, 266: 2011-2014 (1994)]. Because telomerase prevents degradation of the chromosomal DNA, it has essential functions for ceil immortalization.
  • the current view of carcinogenesis is that uncontrolled cell proliferation is part of the development of malignant disease and uncontrolled cell populations that do progress to the malignant state attain the ability to replicate indefinitely.
  • the enzyme telomerase is vital for unlimited cell proliferation. Thus, with few exceptions, telomerase is highly expressed in and specific to malignant cells.
  • Tumor growth is characterized by distinct phases, from dysplasia to carcinoma to angiogenesis, that
  • tumor cells have a life cycle. Although malignant tumors are "immortal", cells within tumors become necrotic due to lack of oxygen and nutrients and are then shed into the bloodstream at a constant log rate. After angiogenesis occurs, cells on the exterior of tumors are also constantly shed into the bloodstream and this release of cells increases in rapidly growing tumors [Kohn et al., Cancer Res, 55: 1856-1861 (1995)]. While not wishing to be bound by theory, we believe that, due to the natural life cycle of tumor cells shedding into the bloodstream, evidence of malignant cells can be detected in
  • tumor marker levels may be too low to be detected in the circulation by known methods; (b) the tumor marker may not be specific to malignant cells, resulting in a high false positive rate; (c) the tumor marker may be found in only a percentage of the tumors tested, resulting in a high false negative rate [Canney et al, Br J Cancer, 50: 765-769 (1984); Bates et al, Cancer Treat Rev, 12: 163-207(1985); Scott et al., / Cell Biochem, l ⁇ : 175-183 (1993)].
  • the development of a non-invasive assay that quantitatively detects a tumor marker specific to all malignancies leading to early diagnosis of cancer would represent a major breakthrough in the field of cancer detection.
  • telomere activity in tumor tissues with prognosis in various cancers such as neuroblastoma, acute myeloid leukemia, breast, and gastrointestinal cancers [Hiyama et al., Nature Med, 1: 249- 255 (1995); Counter et al, Blood, 85: 2315-2320 (1995); Zhang et al, Clin Cancer Res, 2: 799-803 (1996); Hiyama et al., / Natl Cancer Inst, 88 (1996); Tahara et al., Semin Oncol, 23: 307-315 (1995)]. All of these studies have been carried out on the tumors themselves. However, in order for measurements of a tumor marker such as telomerase to be useful in screening for cancer, one must address several critical questions including: is the marker differentially expressed in normal and high-risk tissue; at what stage of progression does the marker appear; is there a
  • telomerase activity is a valid marker in the
  • telomere activity in cancer has been done using samples directly from the tumor itself. For example, detection of telomerase activity in lung cancer patients is done by examining biopsied or surgically resected lung tumor; in brain tumor patients, by examining the brain tumor, etc. In the case of blood tumors (leukemia), a sample of white blood cells is examined.
  • Our invention is fundamentally different and superior. We have discovered that it is possible to detect evidence of malignant cells shed by organs other than blood in the non-cellular portion of the blood of cancer patients. For
  • telomere activity we have detected elevated telomerase activity levlels in the non- cellular portion of the blood of lung cancer patients whose tumor is not a blood tumor.
  • TRAP telomeric repeat amplification protocol
  • the level of telomerase expression can be used to monitor cancer therapy effectiveness. The failure of telomerase activity to regress to within the normal barely detectable range in blood after chemotherapy will be predictive of
  • telomerase activity derived from a
  • telomere activity levels in blood during follow-up visits An increase in telomerase activity will indicate relapse earlier when treatment is more effective.
  • telomere end integrity is maintained by telomerase [Shay et al., (1996)].
  • a minuscule blood sample can be analyzed for telomerase levels and reveal the presence of a new or metastatic tumor, be it of the lung, liver, breast or any organ, in time to eradicate it. The sensitivity of this non-invasive test could change our conception of cancer. Rather than becoming a diagnosis leading to limited treatment options, early stage tumors will be caught and eradicated.
  • telomerase and telomerase activity measurements are used as a tumor marker for diagnostic purposes. It is a further object of the invention to use these telomerase and telomerase activity measurements to monitor in a minimally invasive manner the status of cancer patients and assess prognosis of the disease. It is a further object of the invention to develop a cancer screening kit for routine use on healthy individuals to detect cancer in the early stages using easily obtainable body fluids. It is a further object of the invention to correlate measured telomerase and telomerase activity with the tumor bulk and grading and /or staging of the disease. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 The end-replication problem (prior art)
  • Figure 2 Time windows for intervention vs. treatment, (prior art)
  • Figure 3 Telomerase repeat amplification protocol (TRAP) assay of body
  • FIG. 4 Plasma telomerase activity (expressed as a percentage of the activity
  • Figure 5 Plasma telomerase activity in cancer patients.
  • FIG. 6 Plasma telomerase activity in cancer patients in remission versus
  • lung cancer patients As the primary study because the lungs contain a very dense capillary bed and receive the complete cardiac output every minute [West, Wilkins and Wilkins (1982)]. Therefore, lung tumors have easy access to the body's circulation system which increases the likelihood of detecting a tumor marker in blood.
  • lung cancer patients In general, lung
  • NSCLC non-small cell lung cancer
  • Lung cancer is one of the most common fatal malignancies in the world, accounting for more than 28% of all cancer deaths each year [Miller et al., Natl Cancer Inst, 93 . : 2789-2795 (1993)].
  • overall lung cancer survival has not improved during the past two decades, with 5-year survival remaining about 13% [Ries et al., Natl Cancer Inst, 94. 263-276 (1994)].
  • telomerase In order for a tumor marker such as telomerase to be utilized in the assessment of malignancy in blood samples, it must be expressed by the primary tumor.
  • telomere activity was detected in SCLC which displayed levels comparable to immortalized lung cancer cell lines used as positive controls [Hiyama et al.,/ Natl Cancer Inst (1995)].
  • SCLC Highest telomerase activity was detected in SCLC which displayed levels comparable to immortalized lung cancer cell lines used as positive controls [Hiyama et al.,/ Natl Cancer Inst (1995)].
  • Small cell lung cancer is a very aggressive malignancy that progresses quickly to widespread disease. At presentation, more than 90% of SCLC patients have advanced and /or
  • telomerase activity in blood can provide prognostic data.
  • tumor volume is assessed by a clinical staging evaluation. This evaluation confirms histologic data, determines whether metastasis is present, and assesses the patient for operability. If the tumor is relatively confined-stage 1 or 2-surgical resection is generally recommended. However, after surgery as much as 35% of patients are found to be understaged [Ihde, Curr Prob Cancer, 15: 65-72 (1991)]. In these patients surgical removal of the tumor does not improve prognosis and is thus an unnecessary invasive procedure. Measurements of telomerase activity levels, however, can provide data helpful in the accurate clinical staging of cancer.
  • telomere activity levels correlates to patients with extensive disease who are not good surgical candidates.
  • the ascertainment of how quickly a tumor will grow or spread is a critical component in determining post-surgical treatment, either radiation or
  • telomerase Since telomerase is down-regulated in the GO phase, tumors with high telomerase activity will generally contain a low number of cells in GO [Holt et al.,
  • the detection of occult neoplastic cells in surgical resection margins is a
  • telomerase activity can be detected in tissues composed of only .01% telomerase positive cells [Wright et al., Trends Cell Biol, 5: 293-297 (1995)]. Current histological techniques, on the other hand, may fail to detect metastasis in sections containing less than 2% neoplastic cells [Brennan et al.,(1995)]. In addition, quantitative measurements of telomerase activity in serial blood samples taken from the same patient may reveal evidence of residual disease outside of tumor resection margins. After tumor resection, telomerase activity
  • tumor resection margins and blood can provide a sensitive means to assess
  • telomerase activity was tested for their effect on measurable telomerase activity in blood samples: 1) type of blood drawing tube (heparinized, EDTA, sodium citrate, or no additives) 2) time elapsed between blood draw and protein extraction (0, 2, 4, and 24 hours), 3) temperature at which sample was held until protein extraction (room temperature vs. refrigerated), and 4) addition of RNase inhibitors to protein lysis buffer.
  • Each blood sample was "spiked" with K562 cell extract, subjected to the aforementioned variables, and assayed for telomerase activity.
  • K562 is a human leukemia cell line that has been shown to exhibit very high telomerase activity [Kim (1994)].
  • telomerase activity in cells is standardly measured using the TRAP (telomeric repeat amplification protocol) assay developed by Kim and Shay.
  • the TRAP assay measures telomerase activity in protein extracts isolated directly from tumor tissues and tumor cells [Kim et al., (1994)].
  • telomerase has not been measured in blood fluids for two reasons. First, telomerase is a cellular
  • telomerase activity in tissues containing blood components would be expected to present a technical challenge for the following reasons: (1) heme products found in blood have a major inhibitory effect on the PCR amplification, an integral part of the TRAP assay; and (2) ribonucleases (RNases), which are released in the blood by the destruction of lysosomes, should inactivate telomerase by degrading its ribonucleic acid (RNA) template, rendering telomerase activity undetectable by the TRAP assay [Akane et al.
  • a high fidelity Taq polymerase replaces the recommended Taq polymerase from Gibco, BRL.
  • TS oligonudeotide 5 -AAT CCG TCG AGC AGA GTT-3'
  • thin-walled reaction tubes obtained from Perkin Elmer
  • 20 mM TRIS-HCl pH 8.3
  • 1.5 mM MgCl 1.5 mM
  • 63 mM KC1 0.05% Tween 20 (Sigma)
  • telomerase adds TTAGGG repeats to the TS oligonudeotide.
  • the PCR tubes were heated to 94 degrees C for 2 minutes to inactivate telomerase and decrease formation of primer/dimers.
  • telomerase assay standard were added to bring the volume to 50 microliters.
  • the ITAS is a 150 base pair DNA fragment with binding sites for the forward TS
  • the products were amplified for 30 cycles at 94 degrees C for 30 seconds, 50 degrees C for 30 seconds, and 72 degrees C for 45 seconds.
  • a 10 microliter aliquot of the amplified products were resolved on a 12.5% non-denaturing polyacrylamide gel in 45 mM TRIS base/45 mM boric acid/1 mM EDTA at 200 volts for 30 minutes, followed by 2.5 hours at 275 volts.
  • the gels were dried and exposed for 16 hours to Phosphorlmager screens (Molecular Dynamics). Positive and negative controls were run in parallel with each experiment.
  • the level of specific telomerase activity in 50 K562 cells was set to 100% in each assay.
  • RNA was isolated from 140 ⁇ l of plasma protein (preparation of plasma protein lysate done according to the protocol listed above) using the QIAamp viral RNA isolation kit (QIAGEN). The RNA is stored at 70 °C.
  • hTR telomerase RNA template
  • RNA is incubated at 94 °C for 4 minutes and placed immediately on ice. The incubation temperature allows for the elimination of RNA secondary structure, thereby allowing RNA to be reverse transcribed into cDNA.
  • the tubes are transferred to the PCR block and thermocycled using the hTR PCR program described below.
  • the program includes the following incubation steps, wherein steps 3 to 5 are repeated 34 times:
  • PCR products are analyzed by agarose gel electrophoresis. First, 5 ⁇ l of 10X loading buffer is added to each reaction tube. Next, 20 ⁇ l of PCR product is loaded on a 2% Nusieve gel (FMC Bioproducts) stained with 5 ⁇ l Ethidium Bromide (5mg/ml). A lane with a 25 base pair ladder (1 ⁇ g) is included. Products are electrophoresed at 125 volts for 45 minutes in 0.5X TBE. Gel is placed on the transilluminator and photographed. The expected PCR product is 126 base pairs.
  • Example 1 Telomerase activity in a control population: Blood samples were collected from 20 control subjects and tested repeatedly utilizing the modified TRAP assay disclosed herein. In all cases, telomerase activity was less than 5% of the positive control cell line ( Figure 4).
  • Example 2 Telomerase activity in a cancer population: Blood samples were collected from 20 lung cancer patients and tested in a double blind fashion utilizing the modified TRAP assay disclosed herein. Specifically, we were not informed of the patients disease status until after the tests; likewise, the clinician caring for the patient was not told of our results until after the patient was staged and prognosed, after the completion of the pilot study. In 14 cases, telomerase activity was less than or equal to 5% of the positive control cell line. In these 14 cases, it was subsequently learned that the patient was in complete or near complete remission at the time of testing. In the 6 cases with high telomerase activity (24% to 160% of the positive control), all had
  • telomerase activity levels were well above 5%. Because not every tumor tissue expresses elevated telomerase levels [found in
  • Example 3 Telomerase activity as a means to assess cancer type and prognosis: Blood samples were collected from 20 lung cancer patients and tested utilizing the modified TRAP assay disclosed herein. In 14 case cases, telomerase activity was less than or equal to 5% of the control cell line. In these 14 cases, the patient was in complete or near complete remission at the time of testing. In the 6 cases with high telomerase activity (24% to 160%), all had actively advancing and/or metastatic disease (Figure 5).
  • telomerase assay is specifically discussed for testing protein extracts from blood fluid, it is clear that other body fluids such as lymph, pleural fluid, spinal
  • body fluid selected for telomerase detection would depend on the patient, the type of cancer to be screened or assessed as well as the manifestation of the
  • telomerase activity is not limited to the disclosed protocol. Rather, any mechanism for measuring telomerase levels can be used.
  • reagents or chemical entities that specifically bind to or recognize telomerase such as telomerase antibodies, may be used.
  • serologic assays such as complement fixation, ELISA, immunoblots and equivalents thereof , may be used to measure telomerase activity levels.

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Abstract

Les taux élevés de l'activité de la télomérase dans des extraits de tissus et de cellules ont été utilisés pour le dépistage du cancer dans un échantillon tissulaire. Cette invention concerne un dosage amélioré d'un protocole d'amplification télomérique répétée (TRAP) pour quantifier l'ARN télomérase ou les taux d'activité de la télomérase, en utilisant des liquides organiques pouvant être obtenus facilement, tels que le sang, le plasma, la lymphe, le liquide pleural, le liquide céphalorachidien spinal, la salive, l'expectoration, l'urine et le sperme pour à la fois dépister le cancer, et évaluer le stade d'évolution de la maladie chez un patient et en établir le pronostic. Par ailleurs, on peut utiliser le dosage TRAP amélioré comme marqueur diagnostique tumoral et comme moyen de surveillance de l'évolution et de l'efficacité des cancérothérapies.
EP99907069A 1998-02-16 1999-02-16 Dosage de telomerase preleve dans des liquides organiques utilise pour le depistage du cancer, l'evaluation du stade d'evolution de la maladie et l'etablissement du pronostic Withdrawn EP1071808A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US7479398P 1998-02-16 1998-02-16
PCT/US1999/003302 WO1999041406A1 (fr) 1998-02-16 1999-02-16 Dosage de telomerase preleve dans des liquides organiques utilise pour le depistage du cancer, l'evaluation du stade d'evolution de la maladie et l'etablissement du pronostic
US74793P 2008-06-23

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EP1071808A1 true EP1071808A1 (fr) 2001-01-31

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EP (1) EP1071808A1 (fr)
JP (1) JP2002503480A (fr)
AU (1) AU2682299A (fr)
CA (1) CA2321259A1 (fr)
WO (1) WO1999041406A1 (fr)

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EP1158055A1 (fr) * 2000-05-26 2001-11-28 Xu Qi University of Teaxs Laboratoire de Leucémie Chen Méthode pour le diagnostic de cancers
ITMI20022743A1 (it) * 2002-12-23 2004-06-24 Ist Naz Stud Cura Dei Tumori Metodo per la determinazione del rischio di comparsa, della presenza o del decorso di malattie tumorali.
PL1712639T3 (pl) 2005-04-06 2009-02-27 Maurice Stroun Sposób diagnozowania nowotworu przez wykrywanie krążącego DNA i RNA
CN106940372A (zh) * 2017-03-06 2017-07-11 南京中医药大学 检测端粒酶的亚甲基蓝、上转换纳米粒修饰纤维化纸传感器及其制备方法

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US5830644A (en) * 1992-05-13 1998-11-03 Geron Corporation Method for screening for agents which increase telomerase activity in a cell
US5840490A (en) * 1995-06-07 1998-11-24 Mcmaster University Telomerase activity associated with hematological and colorectal malignancies

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JP2002503480A (ja) 2002-02-05
AU2682299A (en) 1999-08-30
CA2321259A1 (fr) 1999-08-19

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