Original Study
Multicohort Retrospective Validation of a Predictive Biomarker for Topoisomerase I Inhibitors

https://doi.org/10.1016/j.clcc.2020.11.005Get rights and content

Abstract

Purpose

The camptothecin (CPT) analogs topotecan and irinotecan specifically target topoisomerase I (topoI) and are used to treat colorectal, gastric, and pancreatic cancer. Response rate for this class of drug varies from 10% to 30%, and there is no predictive biomarker for patient stratification by response. On the basis of our understanding of CPT drug resistance mechanisms, we developed an immunohistochemistry-based predictive test, P-topoI-Dx, to stratify the patient population into those who did and did not experience a response.

Patients and Methods

The retrospective validation studies included a training set (n = 79) and a validation cohort (n = 27) of gastric cancer (GC) patients, and 8 cohorts of colorectal cancer (CRC) patient tissue (n = 176). Progression-free survival for 6 months was considered a positive response to CPT-based therapy. Formalin-fixed, paraffin-embedded slides were immunohistochemically stained with anti–phospho-specific topoI-Serine10 (topoI-pS10), quantitated, and analyzed statistically.

Results

We determined a threshold of 35% positive staining to offer optimal test characteristics in GC. The GC (n = 79) training set demonstrated 76.6% (95% confidence interval, 64-86) sensitivity; 68.8% (41-88) specificity; positive predictive value (PPV) 92.5% (81-98); and negative predictive value (NPV) 42.3% (24-62). The GC validation set (n = 27) demonstrated 82.4% (56-95) sensitivity and 70.0% (35-92) specificity. Estimated PPV and NPV were 82.4% (56-95) and 70.0% (35-92) respectively. In the CRC validation set (n = 176), the 40% threshold demonstrated 87.5% (78-94) sensitivity; 70.0% (59-79) specificity; PPV 70.7% (61-79); and NPV 87.0 % (77-93).

Conclusion

The analysis of retrospective data from patients (n = 282) provides clinical validity to our P-topoI-Dx immunohistochemical test to identify patients with disease that is most likely to respond to topoI inhibitors.

Introduction

Colorectal cancer (CRC) is the third most common form of cancer, and despite progress in effective screening, one fifth of patients present with metastatic disease (mCRC), and another fifth develop metastasis during clinical courses.1 The standard first-line treatment has been developed on the backbone of 5-fluorouracil (5-FU) since the 1950s.2 Currently, 5-FU and leucovorin are combined with either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI). These doublets, depending on the status of the RAS gene, are combined with bevacizumab (a VEGF inhibitor), cetuximab, or panitumumab (a EGFR inhibitor).3 Patients carrying RAS mutations (approximately 45%-55% of mCRC patients) has disease that does not respond to EGFR-targeted therapy, so they receive antiangiogenic bevacizumab. Other biomarkers that predict the therapy outcomes in mCRC patients are BRAF mutations (8%-12% of patients), HER2 amplifications (5%), and microsatellite instability (MSI) (4%-5%). BRAF mutants do not respond to EGFR inhibitors. However, a combination of 3 kinase inhibitors, BRAF inhibitor encorafenib, MEK inhibitor binimetinib, and EGFR inhibitor cetuximab in BRAF V600E–positive patients, has shown significantly higher progression-free survival (PFS) compared to historical PFS (8 vs 2 months).4 Although cytotoxic agents like 5-FU, oxaliplatin, and irinotecan remain a part of mainstay therapy in combination, there is no predictive biomarker for any of these chemotherapeutic agents. More importantly, 3 clinical trials, FIRE-3, PEAK, and CALB/SWOG, were set up to determine the efficacy of combining targeted therapy with FOLFOX or FOLFIRI to determine a comparative response rate as first-line therapy. None of these studies met their primary endpoint (response rate, PFS, and overall survival [OS]). Therefore, a critical and optimal combination of first-line chemotherapy and targeted therapy in mCRC has not yet been found.5

Gastric cancer (GC) is the fourth most common cancer, with a poor 5-year survival rate. GC is biologically and genetically heterogeneous, with a poorly understood carcinogenesis at the molecular level.6 Although various combinations of platinum compounds and 5-FU derivatives improve patient outcome, no accepted global standard exists for the treatment of GC.7 More recently, a FLOT (5-FU, leucovorin, oxaliplatin, docetaxel) study showed significantly improved survival compared with ECF/ECX (epirubicin and cisplatin plus either 5-FU or capecitabine), with median OS of 50 versus 35 months.8 A review of 60 randomized controlled trials (11,698 participants) of chemotherapy for advanced GC concluded the following: (1) chemotherapy extends OS by approximately 6.7 months more than the best supportive care; (2) combination chemotherapy extends OS by an additional month versus single-agent chemotherapy; (3) irinotecan extends OS slightly (by an additional 1.6 months) versus non–irinotecan-containing regimens; (4) the efficacy of the 3-drug combination of cisplatin, 5-FU, and epirubicin compared to the same combination without epirubicin is not significantly different; and (5) in this 3-drug regimen, irinotecan performs better without any additional cytotoxicity. For this reason, irinotecan/5-FU–containing combinations are an attractive option for first-line treatment.9 Importantly, trastuzumab deruxtecan (Enhertu), an antibody–drug conjugate (ADC) with trastuzumab conjugated to topoisomerase I (topoI) inhibitor, was approved for HER2-positive GC patients.

Camptothecin and its analogs (CPTs), like topotecan and irinotecan, specifically inhibit topoI and are used extensively in clinical oncology to treat various solid tumors. However, response rate is low, and the mechanism of drug resistance is only partly understood.10, 11, 12, 13, 14 One of the most remarkable cellular phenomena observed in response to CPT is the ubiquitin proteasomal pathway (UPP)-mediated degradation of topoI. Importantly, cells that degrade topoI rapidly are resistant to CPT.15 Though the mechanism of UPP-mediated topoI degradation is not understood, our work has identified the molecular determinants of topoI degradation by UPP and its correlation with CPT response. We have recently published that a DNA-dependent protein kinase catalytic subunit–dependent higher basal level of phosphorylated topoI serine 10 (topoI-pS10), ensures rapid degradation of topoI in response to CPT and CPT resistance.16 On the basis of this understanding, we have developed and validated an immunohistochemistry (IHC)-based test, P-topoI-Dx, which will identify the patients with disease most likely to respond to CPT-based therapy, including FOLFIRI.17

We report here the results of retrospective clinical validation data of our predictive biomarker (P-topoI-Dx) in the GC and CRC patient populations. Formalin-fixed, paraffin-embedded (FFPE) slides from irinotecan-treated patients were immunostained, quantitatively analyzed, and statistically validated. The intended use of this predictive biomarker was to identify patients with disease likely to respond to irinotecan-based therapy.

Section snippets

Patients and Methods

Eight cohorts of CRC tissue were collected. Table 1 lists block IDs and collection centers.

GC Training Set

Patient characteristics for the training set of GCs (n = 79) are shown in Table 2. Of the 79 patients, 67% (n = 53) were male and 33% (n = 26) female. The mean ± standard deviation age was 64.3 ± 12.4, ranging from 29 to 90 years. Forty-six percent of patients (n = 36) had intestinal type disease and 54% (n = 43) diffused type. Tumor stages were distributed as follows: stage I, 22% (n = 17); stage II, 15% (n = 12); stage III, 21% (n = 17); and stage IV, 42% (n = 33).

Table 3 shows the

Discussion

The inclusion of irinotecan with 5-FU, leucovorin (FOLFIRI), and oxaliplatin (FOLFOX) resulted in significantly higher PFS, higher OS, and higher rate of confirmed response.21 A clinical trial to determine the relative efficacy of FOLFOX versus FOLFIRI demonstrated the similar efficacy of both regimens. At present, the selection of combination therapy in the first or second line is not based on any predictive biomarker. However, the fact that a substantial proportion of patients did not receive

Disclosure

A.B. has equity interest and is a paid consultant for Predictus Biosciences Inc. The other authors have stated that they have no conflict of interest.

Acknowledgments

The Department of Medicine, Boston University Medical School, and Boston University Office of Technology Development supported the publication. The authors also acknowledge the help of Neelabh Bharti for editorial work and for formatting the tables.

References (38)

  • E. Elez et al.

    First-line treatment of metastatic colorectal cancer: interpreting FIRE-3, PEAK, and CALGB/SWOG 80405

    Curr Treat Options Oncol

    (2015)
  • T. Matsuoka et al.

    Biomarkers of gastric cancer: current topics and future perspective

    World J Gastroenterol

    (2018)
  • A.D. Wagner et al.

    Chemotherapy for advanced gastric cancer

    Cochrane Database Syst Rev

    (2017)
  • L.F. Liu et al.

    Mechanism of action of camptothecin

    Ann N Y Acad Sci

    (2000)
  • Y. Pommier

    Topoisomerase I inhibitors: camptothecins and beyond

    Nat Rev Cancer

    (2006)
  • J.C. Wang

    Cellular roles of DNA topoisomerases: a molecular perspective

    Nat Rev Mol Cell Biol

    (2002)
  • R.B. Ewesuedo et al.

    Topoisomerase I inhibitors

    Oncologist

    (1997)
  • S.D. Desai et al.

    Ubiquitin/26S proteasome–mediated degradation of topoisomerase I as a resistance mechanism to camptothecin in tumor cells

    Cancer Res

    (2001)
  • K. Ando et al.

    Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway

    Oncotarget

    (2017)
  • View full text