Resistance to Selective FGFR Inhibitors in FGFR-Driven Urothelial Cancer.

Several fibroblast growth factor receptor (FGFR) inhibitors are approved or in clinical development for the treatment of FGFR-driven urothelial cancer, and molecular mechanisms of resistance leading to patient relapses have not been fully explored. We identified 21 patients with FGFR-driven urothelial cancer treated with selective FGFR inhibitors and analyzed postprogression tissue and/or circulating tumor DNA (ctDNA). We detected single mutations in the FGFR tyrosine kinase domain in seven (33%) patients (FGFR3 N540K, V553L/M, V555L/M, E587Q; FGFR2 L551F) and multiple mutations in one (5%) case (FGFR3 N540K, V555L, and L608V). Using Ba/F3 cells, we defined their spectrum of resistance/sensitivity to multiple selective FGFR inhibitors. Eleven (52%) patients harbored alterations in the PI3K-mTOR pathway (n = 4 TSC1/2, n = 4 PIK3CA, n = 1 TSC1 and PIK3CA, n = 1 NF2, n = 1 PTEN). In patient-derived models, erdafitinib was synergistic with pictilisib in the presence of PIK3CA E545K, whereas erdafitinib-gefitinib combination was able to overcome bypass resistance mediated by EGFR activation. SIGNIFICANCE: In the largest study on the topic thus far, we detected a high frequency of FGFR kinase domain mutations responsible for resistance to FGFR inhibitors in urothelial cancer. Off-target resistance mechanisms involved primarily the PI3K-mTOR pathway. Our findings provide preclinical evidence sustaining combinatorial treatment strategies to overcome bypass resistance. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.

Feasibility and first reports of the MATCH-R repeated biopsy trial at Gustave Roussy.

Unravelling the biological processes driving tumour resistance is necessary to support the development of innovative treatment strategies. We report the design and feasibility of the MATCH-R prospective trial led by Gustave Roussy with the primary objective of characterizing the molecular mechanisms of resistance to cancer treatments. The primary clinical endpoints consist of analyzing the type and frequency of molecular alterations in resistant tumours and compare these to samples prior to treatment. Patients experiencing disease progression after an initial partial response or stable disease for at least 24 weeks underwent a tumour biopsy guided by CT or ultrasound. Molecular profiling of tumours was performed using whole exome sequencing, RNA sequencing and panel sequencing. At data cut-off for feasibility analysis, out of 333 inclusions, tumour biopsies were obtained in 303 cases (91%). From these biopsies, 278 (83%) had sufficient quality for analysis by high-throughput next generation sequencing (NGS). All 278 samples underwent targeted NGS, 215 (70.9%) RNA sequencing and 222 (73.2%) whole exome sequencing. In total, 163 tumours were implanted in NOD scid gamma (NSG) or nude mice and 54 patient-derived xenograft (PDX) models were established, with a success rate of 33%. Adverse events secondary to invasive tumour sampling occurred in 24 patients (7.6%). Study recruitment is still ongoing. Systematic molecular profiling of tumours and the development of patient-derived models of acquired resistance to targeted agents and immunotherapy is feasible and can drive the selection of the next therapeutic strategy.

Molecular mechanisms of resistance to BRAF and MEK inhibitors in BRAFV600E non-small cell lung cancer.

INTRODUCTION: BRAF is a confirmed therapeutic target in non-small cell lung cancer (NSCLC), as the BRAF inhibitor dabrafenib, in combination with the MEK inhibitor trametinib, is approved for the treatment of NSCLC harbouring BRAF V600E mutation. Scant evidence is available concerning the mechanisms of resistance to BRAF/MEK inhibitors in BRAFV600E NSCLC. PATIENTS AND METHODS: Patients with BRAFV600E NSCLC with acquired resistance to BRAF/MEK inhibitors were included in the institutional, prospective MATCH-R (from "Matching Resistance") trial and underwent tumour and liquid biopsies at the moment of radiological progression. Extensive molecular analyses were performed, including targeted next-generation sequencing (NGS), whole-exome sequencing (WES), RNA sequencing and comparative genomic hybridisation (CGH) array. RESULTS: Of the 11 patients included, eight had progressed on dabrafenib-trametinib combination, two on dabrafenib monotherapy and one on vemurafenib (BRAF inhibitor). Complete molecular analyses were available for seven patients, whereas an additional case had only targeted NGS and CGH array data. Among these eight patients, acquired molecular events potentially responsible for resistance were detected in three who progressed on dabrafenib-trametinib combination, that is, MEK1 K57N, RAS viral (v-ras) oncogene homolog (NRAS) Q61R and rat sarcoma viral oncogene homolog (KRAS) Q61R mutations. One patient progressing on dabrafenib monotherapy developed a PTEN frameshift mutation. No molecular hints addressing resistance emerged in the remaining four patients with analyses performed. Tumour mutational burden, evaluated by WES in seven patients, was low (median = 2.06 mutations/megabase, range = 1.57-3.75 mut/Mb). CONCLUSIONS: Novel resistance mechanisms to BRAF/MEK inhibitors in BRAFV600E NSCLC were identified, pointing out the recurring involvement of the MAPK pathway and guiding the development of new treatment strategies.

Facts and New Hopes on Selective FGFR Inhibitors in Solid Tumors.

Precision oncology relies on the identification of molecular alterations, responsible for tumor initiation and growth, which are suitable targets of specific inhibitors. The development of FGFR inhibitors represents an edifying example of the rapid evolution in the field of targeted oncology, with 10 different FGFR tyrosine kinase inhibitors actually under clinical investigation. In parallel, the discovery of FGFR activating molecular alterations (mainly FGFR3 mutations and FGFR2 fusions) across many tumor types, especially urothelial carcinomas and intrahepatic cholangiocarcinomas, widens the selection of patients that might benefit from selective FGFR inhibitors. The ongoing concomitant clinical evaluation of selective FGFR inhibitors in molecularly selected solid tumors brings new hopes for patients with metastatic cancer, for tumors so far excluded from molecularly guided treatments. Matching molecularly selected tumors with selective FGFR inhibitors has indeed led to promising results in phase I and II trials, justifying their registration to be expected in a near future, such as the recent accelerated approval of erdafitinib granted by the FDA for urothelial cancer. Widening our knowledge of the activity, efficacy, and toxicities relative to the selective FGFR tyrosine kinase inhibitors under clinical investigation, according to the exact FGFR molecular alteration, will be crucial to determine the optimal therapeutic strategy for patients suffering from FGFR-driven tumors. Similarly, identifying with appropriate molecular diagnostic, every single tumor harboring targetable FGFR alterations will be of utmost importance to attain the best outcomes for patients with FGFR-driven cancer.

Diverse Resistance Mechanisms to the Third-Generation ALK Inhibitor Lorlatinib in ALK-Rearranged Lung Cancer.

PURPOSE: Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK-rearranged lung cancer and design new therapeutic strategies in this setting. EXPERIMENTAL DESIGN: Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance. RESULTS: Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial-mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors. CONCLUSIONS: This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression.

Making the first move in EGFR-driven or ALK-driven NSCLC: first-generation or next-generation TKI?

The traditional approach to the treatment of patients with advanced-stage non-small-cell lung carcinoma (NSCLC) harbouring ALK rearrangements or EGFR mutations has been the sequential administration of therapies (sequential treatment approach), in which patients first receive first-generation tyrosine-kinase inhibitors (TKIs), which are eventually replaced by next-generation TKIs and/or chemotherapy upon disease progression, in a decision optionally guided by tumour molecular profiling. In the past few years, this strategy has been challenged by clinical evidence showing improved progression-free survival, improved intracranial disease control and a generally favourable toxicity profile when next-generation EGFR and ALK TKIs are used in the first-line setting. In this Review, we describe the existing preclinical and clinical evidence supporting both treatment strategies - the 'historical' sequential treatment strategy and the use of next-generation TKIs - as frontline therapies and discuss the suitability of both strategies for patients with EGFR-driven or ALK-driven NSCLC.

Tumor Mutation Burden as a Biomarker in Resected Non-Small-Cell Lung Cancer.

PURPOSE: The survival benefit with adjuvant chemotherapy for patients with resected stage II-III non-small-cell lung cancer (NSCLC) is modest. Efforts to develop prognostic or predictive biomarkers in these patients have not yielded clinically useful tests. We report findings from the Lung Adjuvant Cisplatin Evaluation (LACE)-Bio-II study, in which we analyzed next-generation sequencing and long-term outcomes data from > 900 patients with early-stage NSCLC treated prospectively in adjuvant landmark clinical trials. We used a targeted gene panel to assess the prognostic and predictive effect of mutations in individual genes, DNA repair pathways, and tumor mutation burden (TMB). METHODS: A total of 908 unmatched, formalin-fixed, paraffin-embedded, resected lung cancer tumor specimens were sequenced using a targeted panel of 1,538 genes. Stringent filtering criteria were applied to exclude germline variants and artifacts related to formalin fixation. Disease-free survival, overall survival, and lung cancer-specific survival (LCSS) were assessed in Cox models stratified by trial and adjusted for treatment, age, sex, performance score, histology, type of surgery, and stage. RESULTS: Nonsynonymous mutations were identified in 1,515 genes in 908 tumor samples. High nonsynonymous TMB (> 8 mutations/Mb) was prognostic for favorable outcomes (ie, overall survival, disease-free survival, and LCSS) in patients with resected NSCLC. LCSS benefit with adjuvant chemotherapy was more pronounced in patients with low nonsynonymous TMBs (≤ 4 mutations/Mb). Presence of mutations in DNA repair pathways, tumor-infiltrating lymphocytes, TP53 alteration subtype, and intratumor heterogeneity was neither prognostic nor predictive. Statistically significant effect of mutations in individual genes was difficult to determine due to high false-discovery rates. CONCLUSION: High nonsynonymous TMB was associated with a better prognosis in patients with resected NSCLC. In addition, the benefit of adjuvant chemotherapy on LCSS was more pronounced in patients with low nonsynonymous TMBs. Studies are warranted to confirm these findings.

A novel antibody-based approach to detect the functional ERCC1-202 isoform.

ERCC1/XPF endonuclease plays an important role in multiple DNA repair pathways and stands as a potential prognostic and predictive biomarker for cisplatin-based chemotherapy. Four distinct ERCC1 isoforms arising from alternative splicing have been described (201, 202, 203 and 204) but only the 202 isoform is functional in DNA excision repair, when interacting with its obligate partner XPF. Currently, there is no tool to assess specifically the expression of ERCC1-202 due to high sequence homology between the four isoforms. Here, we generated monoclonal antibodies directed against the heterodimer of ERCC1 and its obligate interacting partner XPF by genetic immunization. We obtained three monoclonal antibodies (2C11, 7C3 and 10D10) recognizing specifically the heterodimer ERCC1-202/XPF as well as the ERCC1-204/XPF with no affinity to ERCC1 or XPF monomers. By combining one of these three heterodimer-specific antibodies with a commercial anti-ERCC1 antibody (clone 4F9) unable to recognize the 204 isoform in a proximity ligation assay (PLA), we managed to specifically detect the functional ERCC1-202 isoform. This methodological breakthrough can constitute a basis for the development of clinical tests to evaluate ERCC1 functional proficiency.

DNA repair deficiency sensitizes lung cancer cells to NAD+ biosynthesis blockade.

Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non-small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house-generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro - ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells - and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.

TPF induction chemotherapy increases PD-L1 expression in tumour cells and immune cells in head and neck squamous cell carcinoma.

BACKGROUND: Antiprogrammed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) therapies have demonstrated promising activity in advanced head and neck squamous cell carcinoma (HNSCC), with overall response rates of approximately 20% in unselected populations and survival benefit. Whether induction docetaxel, platinum and fluorouracil (TPF) modifies PD-L1 expression or tumour immune infiltrates is unknown. PATIENTS AND METHODS: Patients with locally advanced HNSCC treated at Gustave Roussy (Villejuif, France) between 2006 and 2013 by induction TPF followed by surgery were retrospectively considered. Patients with paired samples (pre-TPF and post-TPF) were kept for further analysis. PD-L1 expression was quantified by immunohistochemistry according to a validated protocol. The objective of the study was to compare PD-L1 expression on tumour cells (TC) and immune cells (IC) (positivity threshold of ≥5%) before and after TPF. CD8+ and Foxp3+ lymphocytes densities before and after TPF were also quantified. RESULTS: Out of 313 patients receiving induction TPF, 86 underwent surgery; paired samples were available for 21 of them. Baseline PD-L1 expression was ≥5% in two and five samples for TC and IC, respectively. A significant increase of PD-L1 expression was observed after TPF, with 15 samples (71%) presenting a positive staining in IC after induction chemotherapy (P=0.003; Wilcoxon rank-sum test) and eight samples (38%) in TC (P=0.005; Wilcoxon rank-sum test). Tumour-infiltrating CD8+ mean densities also significantly increased post-TPF (P=0.01). There was no significant difference in Foxp3+ expression, CD8/Foxp3 ratio or correlation with outcome. CONCLUSION: TPF induction chemotherapy in advanced HNSCC increases PD-L1 positivity on tumour-infiltrating ICs, as well as CD8+ lymphocytes density. These results warrant independent validation on larger datasets and might help therapeutic strategy in advanced HNSCC.

MMS19 as a potential predictive marker of adjuvant chemotherapy benefit in resected non-small cell lung cancer.

BACKGROUND: Resectable non-small cell lung cancer (NSCLC) treatment options most often consist of surgical resection along with adjuvant chemotherapy (ACT). The benefit of ACT however is modest and is accompanied by important side effects. OBJECTIVE: One central quest in the field is therefore the identification of a predictive marker of the response to ACT. METHODS: We applied an unbiased approach based on high content analysis of expression data generated from a discovery patient cohort. RESULTS: We identified MMS19, a component of the cytoplasmic Iron-Sulfur Assembly (CIA) machinery important for the Nucleotide Excision Repair (NER) pathway as a pivotal gene for cisplatin toxicity. We then confirmed the association between MMS19 expression and the response to Cisplatin treatment in a panel of NSCLC cell lines. Finally we validated these pre-clinical data in a subgroup of JBR.10 trial patients through a hypothesis-driven analysis, and showed that MMS19 levels associated with ACT benefit. CONCLUSIONS: We therefore propose the expression level of MMS19 as a candidate predictive marker of ACT benefit in resected NSCLC patients.

Translational regulation of the mRNA encoding the ubiquitin peptidase USP1 involved in the DNA damage response as a determinant of Cisplatin resistance.

Cisplatin (cis-diaminedichloroplatin (II), CDDP) is part of the standard therapy for a number of solid tumors including Non-Small-Cell Lung Cancer (NSCLC). The initial response observed is in most cases only transient and tumors quickly become refractory to the drug. Tumor cell resistance to CDDP relies on multiple mechanisms, some of which still remain unknown. In search for such mechanisms, we examined the impact of CDDP on mRNA translation in a sensitive and in a matched resistant NSCLC cell line. We identified a set of genes whose mRNAs are differentially translated in CDDP resistant vs. sensitive cells. The translation of the mRNA encoding the Ubiquitin-Specific Peptidase 1 (USP1), a Ubiquitin peptidase with important function in multiple DNA repair pathways, is inhibited by CDDP exposure in the sensitive cells, but not in the resistant cells. This lack of down-regulation of USP1 expression at the translational level plays a primary role in CDDP resistance since inhibition of USP1 expression or activity by siRNA or the small molecule inhibitor ML323, respectively is sufficient to re-sensitize resistant cells to CDDP. We involved the USP1 mRNA translation as a major mechanism of CDDP resistance in NSCLC cells and suggest that USP1 could be evaluated as a candidate predictive marker and as a therapeutic target to overcome CDDP resistance. More generally, our results indicate that analysis of gene expression at the level of mRNA translation is a useful approach to identify new determinants of CDDP resistance.

19q13-ERCC1 gene copy number increase in non--small-cell lung cancer.

BACKGROUND: Excision repair cross complementing 1 gene expression level has potential as a prognostic and predictive marker of the efficacy of chemotherapy in NSCLC. The effect of ERCC1 gene copy number (CN) variation (CNV) on ERCC1 expression and the clinical outcome of patients with NSCLC are not known. MATERIALS AND METHODS: Copy number variation of the 19q13.3 region carrying the ERCC1 gene, classified as gene amplification (GA) or high polysomy (HP), was evaluated on 235 formalin-fixed and paraffin-embedded tumors from resected NSCLC patient samples and 16 NSCLC cell lines using FISH. We analyzed the potential correlations between FISH status and ERCC1 expression, patient's outcome, and cisplatin sensitivity in the cohort or cell lines. RESULTS: An increase of 19q13.3 gene CN was detected in 60 cases (25.5%) including 27 cases with GA and 33 cases with HP. A nonsignificant trend for higher ERCC1 expression in HP patients compared with GA and patients with low CNV was found (P = .06). In patients not treated with chemotherapy, FISH negative status cases had longer disease-free survival (DFS) compared with patients with 19q13-ERCC1 GA (P = .02). A 3-fold increase in IC50 of cisplatin in cell lines with high 19q13-ERCC1 CN compared with cells without CNV was shown. CONCLUSION: ERCC1 CN increase assessed using FISH did not determine ERCC1 expression status but yields potential prognostic information on DFS in untreated patients with NSCLC. The clinical relevance of an association of 19q13-ERCC1 FISH status and chemosensitivity or prognosis in patients needs further investigation and validation.

Synergistic interaction between cisplatin and PARP inhibitors in non-small cell lung cancer.

The antineoplastic agent cis-diammineplatinum(II) dichloride (cisplatin, CDDP) is part of the poorly effective standard treatment of non-small cell lung carcinoma (NSCLC). Here, we report a novel strategy to improve the efficacy of CDDP. In conditions in which CDDP alone or either of two PARP inhibitors, PJ34 hydrochloride hydrate or CEP 8983, used as standalone treatments were inefficient in killing NSCLC cells, the combination of CDDP plus PJ34 or that of CDDP plus CEP 8983 were found to kill a substantial fraction of the cells. This cytotoxic synergy could be recapitulated by combining CDDP and the siRNA-mediated depletion of the principal PARP isoform, PARP1, indicating that it is mediated by on-target effects of PJ34 or CEP 8983. CDDP and PARP inhibitors synergized in inducing DNA damage foci, mitochondrial membrane permeabilization leading to cytochrome c release, and dissipation of the inner transmembrane potential, caspase activation, plasma membrane rupture and loss of clonogenic potential in NSCLC cells. Collectively, our results indicate that CDDP can be advantageously combined with PARP inhibitors to kill several NSCLC cell lines, independently from their p53 status. Combined treatment with CDDP and PARP inhibitors elicits the intrinsic pathway of apoptosis.

PARP1 impact on DNA repair of platinum adducts: preclinical and clinical read-outs.

Evaluation of DNA repair proteins might provide meaningful information in relation to prognosis and chemotherapy efficacy in Non-Small Cell Lung Cancer (NSCLC) patients. The role of Poly(ADP-Ribose) Polymerase (PARP) in DNA repair of platinum adducts has not been firmly established. We used a DNA repair functional test based on antibody recognition of cisplatin intrastrand platinum adducts on DNA. We evaluated the effect of PARP inhibition on DNA repair functionality in a panel of cisplatin cell lines treated by the clinical-grade pharmacological inhibitor CEP8983 (a 4-methoxy-carbazole derivate) and the commercially available inhibitor PJ34 (phenanthridinone). We determined PARP1 protein expression in whole tumor sections from the International Adjuvant Lung cancer Trial (IALT)-bio study and tested a 3-marker PARP1/MSH2/ERCC1 algorithm combining PARP1 tumor status with previously published data. Chemosensitivity of cisplatin in NSCLC cell lines was correlated with the accumulation of cisplatin DNA adducts (P=0.0004). Further, the pharmacological inhibition of PARP induced a 1.7 to 2.3-fold increase in platinum adduct accumulation (24h) in A549 cell line suggesting a slow-down of platinum DNA-adduct repair capacity. In parallel, PARP1 inhibition increased the sensitivity to cisplatin treatment. In patient samples, PARP1 expression levels did not influence patient survival or the effect of platinum-based post-operative chemotherapy in the global IALT-bio population (interaction P=0.79). Among cases with high expression of all three markers (triple positive), untreated patients had prolonged survival with a median DFS of 7.8 years, (HR=0.34, 95%CI [0.19-0.61], adjusted P=0.0003) compared to triple negative patients (1.4 years). Remarkably, triple positive patients suffered from a detrimental effect (4.9-year reduction of median DFS) by post-operative cisplatin-based chemotherapy (HR=1.79, 95%CI [1.01-3.17], adjusted P=0.04, chemotherapy vs. control). Combinatorial sub-group analysis of the 3 markers further suggested that PARP1 tumor positivity might constitute a molecular context with high theranostic interest of ERCC1 and MSH2 in NSCLC. In conclusion, our data confirm that platinum DNA adduct accumulation is linked to chemosensitivity, which increase by pharmacological PARP inhibitors points to a role of PARP-dependent DNA repair in the process. We further suggest DNA repair biomarkers should be analyzed in a larger context of multiple DNA repair pathway regulation.

ERCC1 and RRM1: ready for prime time?

The quest for markers of sensitivity to cytotoxic agents has been ongoing for decades. In non-small-cell lung cancer, platinum compounds represent the cornerstone of systemic therapy. They target DNA and induce damage that cancer cells struggle to overcome. Somatic excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), and ribonucleotide reductase M1 (RRM1) expression levels have been extensively explored as markers of DNA repair capacity in tumor cells. Although low ERCC1 and/or RRM1 expression is generally associated with sensitivity to platinum, the results published in retrospective and prospective studies are not always consistent. Against this background, we will examine in this review the function of these two biomarkers as well as the tools available for their assessment and the associated technical issues. Their prognostic and predictive values will be summarized and considered in terms of customizing systemic therapy according to biomarker (ERCC1 and RRM1) expression levels. We will also discuss why the use of both markers should at this point be restricted to clinical research and underline that functional readouts of DNA repair will help boost future strategies for biomarker discovery in the field.

The potential of exploiting DNA-repair defects for optimizing lung cancer treatment.

The tumor genome is commonly aberrant as a consequence of mutagenic insult and incomplete DNA repair. DNA repair as a therapeutic target has recently received considerable attention owing to the promise of drugs that target tumor-specific DNA-repair enzymes and potentiate conventional cytotoxic therapy through mechanism-based approaches, such as synthetic lethality. Treatment for non-small-cell lung cancer (NSCLC) consists mainly of platinum-based chemotherapy regimens and improvements are urgently needed. Optimizing treatment according to tumor status for DNA-repair biomarkers, such as ERCC1, BRCA1 or RRM1, could predict response to platinum, taxanes and gemcitabine-based therapies, respectively, and might improve substantially the response of individual patients' tumors. Finally, recent data on germline variation in DNA-repair genes may also be informative. Here, we discuss how a molecular and functional DNA-repair classification of NSCLC may aid clinical decision making and improve patient outcome.

MutS homologue 2 and the long-term benefit of adjuvant chemotherapy in lung cancer.

PURPOSE: We sought to determine the long-term (median follow-up, 7.5 years) predictive power of human MutS homologue 2 (MSH2) immunohistochemical expression in patients who enrolled in the International Adjuvant Lung Trial. EXPERIMENTAL DESIGN: We tested the interaction between MSH2 and the allocated treatment (chemotherapy versus observation) in a Cox model adjusted on clinicopathologic variables. The significance level was set at 0.01. RESULTS: MSH2 levels were low in 257 (38%) and high in 416 (62%) tumors. The benefit from chemotherapy was likely different according to MSH2 (interaction test, P = 0.06): there was a trend for chemotherapy to prolong overall survival when MSH2 was low [hazard ratio (HR), 0.76; 95% confidence interval (95% CI), 0.59-0.97; P = 0.03], but not when MSH2 was high (HR, 1.12; 95% CI, 0.81-1.55; P = 0.48). In the control arm, the HR was 0.66 (95% CI, 0.49-0.90; P = 0.01) when MSH2 was high. When combining MSH2 with excision repair cross-complementing group 1 (ERCC1) into four subgroups, the benefit of chemotherapy decreased with the number of markers expressed at high levels (P = 0.01). A similar decrease was noted when combining MSH2 and P27 (P = 0.01). Chemotherapy prolonged overall survival in the combined low MSH2/low ERCC1 subgroup (HR, 0.65; 95% CI, 0.47-0.91; P = 0.01) and in the combined low MSH2/low P27 subgroup (HR, 0.65; 95% CI, 0.46-0.93; P = 0.01). CONCLUSIONS: MSH2 expression is a borderline significant predictor of a long-term benefit from adjuvant cisplatin-based chemotherapy in patients with completely resected lung cancer. MSH2 combined with ERCC1 or P27 may identify patients most likely to benefit durably from chemotherapy.

Inhibition of Chk1 kills tetraploid tumor cells through a p53-dependent pathway.

Tetraploidy constitutes an adaptation to stress and an intermediate step between euploidy and aneuploidy in oncogenesis. Tetraploid cells are particularly resistant against genotoxic stress including radiotherapy and chemotherapy. Here, we designed a strategy to preferentially kill tetraploid tumor cells. Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts. Chk1 inhibition abolished the spindle assembly checkpoint and caused premature and abnormal mitoses that led to p53 activation and cell death at a higher frequency in tetraploid than in diploid cells. Similarly, abolition of the spindle checkpoint by knockdown of Bub1, BubR1 or Mad2 induced p53-dependent apoptosis of tetraploid cells. Chk1 inhibition reversed the cisplatin resistance of tetraploid cells in vitro and in vivo, in xenografted human cancers. Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells. Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.