ERCC1 isoform expression and DNA repair in non-small-cell lung cancer.

BACKGROUND: The excision repair cross-complementation group 1 (ERCC1) protein is a potential prognostic biomarker of the efficacy of cisplatin-based chemotherapy in non-small-cell lung cancer (NSCLC). Although several ongoing trials are evaluating the level of expression of ERCC1, no consensus has been reached regarding a method for evaluation. METHODS: We used the 8F1 antibody to measure the level of expression of ERCC1 protein by means of immunohistochemical analysis in a validation set of samples obtained from 494 patients in two independent phase 3 trials (the National Cancer Institute of Canada Clinical Trials Group JBR.10 and the Cancer and Leukemia Group B 9633 trial from the Lung Adjuvant Cisplatin Evaluation Biology project). We compared the results of repeated staining of the entire original set of samples obtained from 589 patients in the International Adjuvant Lung Cancer Trial Biology study, which had led to the initial correlation between the absence of ERCC1 expression and platinum response, with our previous results in the same tumors. We mapped the epitope recognized by 16 commercially available ERCC1 antibodies and investigated the capacity of the different ERCC1 isoforms to repair platinum-induced DNA damage. RESULTS: We were unable to validate the predictive effect of immunostaining for ERCC1 protein. The discordance in the results of staining for ERCC1 suggested a change in the performance of the 8F1 antibody since 2006. We found that none of the 16 antibodies could distinguish among the four ERCC1 protein isoforms, whereas only one isoform produced a protein that had full capacities for nucleotide excision repair and cisplatin resistance. CONCLUSIONS: Immunohistochemical analysis with the use of currently available ERCC1 antibodies did not specifically detect the unique functional ERCC1 isoform. As a result, its usefulness in guiding therapeutic decision making is limited. (Funded by Eli Lilly and others.).

Osteopontin and thrombospondin-1 play opposite roles in promoting tumor aggressiveness of primary resected non-small cell lung cancer.

BACKGROUND: Osteopontin (OPN) and thrombospondin-1 (TSP-1) are extracellular matrix proteins secreted by stromal and tumor cells. These proteins appear to have a key role in the tumor microenvironment for cancer development and metastasis. There is little information regarding the prognostic value of the combination of these two proteins in human cancers. Our aim was to clarify clinical significance and prognostic value of each circulating protein and their combination in primary resected non-small cell lung cancer (NSCLC) patients. METHODS: We retrospectively reviewed 171 patients with NSCLC following curative intent surgery from January to December of 2012. Preoperative serums, demographics, clinical and pathological data and molecular profiling were analyzed. Pre-treatment OPN and TSP-1 serum levels were measured by ELISA. Tissue protein expression in primary tumor samples was determined by immunohistochemical analysis. RESULTS: OPN and TSP-1 serum levels were inversely correlated with survival rates. For each 50 units increment of serum OPN, an increased risk of metastasis by 69 % (unadjusted HR 1.69, 95 % CI 1.12-2.56, p = 0.01) and an increased risk of death by 95 % (unadjusted HR 1.95, 95 % CI 1.15-3.32, p = 0.01) were observed. Conversely, for each 10 units increment in TSP-1, the risk of death was decreased by 85 % (unadjusted HR 0.15, 95 % CI 0.03-0.89; p = 0.04). No statistically significant correlation was found between TSP-1 serum level and distant metastasis-free survival (p = 0.2). On multivariate analysis, OPN and TSP-1 serum levels were independent prognostic factors of overall survival (HR 1.71, 95 % CI 1.04-2.82, p = 0.04 for an increase of 50 ng/mL in OPN; HR 0.18, 95 % CI 0.04-0.87, p = 0.03 for an increase of 10 ng/mL in TSP-1). In addition, the combination of OPN and TSP-1 serum levels remained an independent prognostic factor for overall survival (HR 1.31, 95 % CI 1.03-1.67, p = 0.03 for an increase of 6 ng/mL in OPN/TSP-1 ratio). CONCLUSIONS: Our results show that pre-treatment OPN and TSP-1 serum levels may reflect the aggressiveness of the tumor and might serve as prognostic markers in patients with primary resected NSCLC.

Association of Molecular Profiles and Mutational Status With Distinct Histological Lung Adenocarcinoma Subtypes. An Analysis of the LACE-Bio Data.

BACKGROUND: Adjuvant chemotherapy (AC) is indicated for stage II and stage III lung adenocarcinomas (ADC). Using the LACE Bio II database, we analyzed the distribution of various mutations across the subtypes of ADCs and studied the prognostic and predictive roles of PD-L1, TMB, and Tumor Infiltrating Lymphocytes (TILs). MATERIALS AND METHODS: Clinical and genomic data from the LACE Bio II data were extracted. Patients were divided into ADC subtypes, in which the grouping was done based on their known clinical behavior (Lepidic [LEP], Acinar/Papillary [ACI or PAP], Micropapillary/Solid [MIP or SOL], Mucinous [MUC] and Others). Kaplan-Meier (KM) and log-rank test were used to compare survival based on PD-L1, TMB, TILs and combinations of TMB with PD-L1 and TILs. Adjusted Hazard Ratios (HR) were analyzed with Overall Survival (OS), Disease-Free Survival (DFS) and Lung Cancer-Specific Survival (LCSS) as endpoints. RESULTS: A total of 375 ADC patients were identified. MIP/SOL was the subtype most commonly positive for various biomarkers. PD-L1 Negative/high TMB was associated with better outcomes in terms of OS (HR = 0.46 [0.23-0.89], P = .021) and DFS (HR = 0.52 [0.30-0.90], P = .02), relative to PD-L1 Negative/low TMB. High TMB predicted worse outcome with AC use in terms of OS (ratio of hazard ratio rHR = 2.75 [1.07-7.04], P = .035). Marked TILs had better outcome with AC for DFS (rHR = 0.22 [0.06-0.87], P = .031 and LCSS (rHR = 0.08 [0.01-0.66], P = .019) respectively. There was also a beneficial effect of AC among patients with Marked TILs/low TMB in terms of DFS (rHR = 0.06 [0.01-0.53], P = .011). CONCLUSION: High TMB has a prognostic role in resectable lung ADC. The high TMB group had a poor outcome with AC, suggesting that this group may be better served with immune checkpoint therapy.

ERCC1 and RRM1 in the international adjuvant lung trial by automated quantitative in situ analysis.

The excision repair cross completing group 1 gene product (ERCC1) and the regulatory subunit of ribonucleotide reductase (RRM1) have been reported as being prognostic of outcome and predictive of therapeutic efficacy in patients with non-small cell lung cancer. Routinely processed surgical specimens from 784 patients from the International Adjuvant Lung Trial were arrayed as tissue microarrays. In situ protein levels were scored with an automated, quantitative analysis system, dichotomized into high and low marker categories, and analyzed for associations with patients' characteristics, survival, and benefit from adjuvant chemotherapy. Scores for both markers were significantly associated with contributing center (P < 0.001) and skewed, with the bulk of scores being low. High scores were more frequent in women for ERCC1 and RRM1 and in older patients and those with adenocarcinoma for RRM1. Low ERCC1 scores indicated significant benefit from adjuvant chemotherapy [hazard ratio (HR) = 0.73 for chemotherapy versus control, P = 0.02]. Although all other survival associations were not statistically significant, low RRM1 scores trended to indicate benefit from adjuvant chemotherapy (HR = 0.84, P = 0.25), and ERCC1 scores were marginally prognostic of survival (HR = 0.77 for high versus low scores, P = 0.10). We conclude that contributing center and specimen quality substantially affect the levels of both markers. Future trials should incorporate the collection and processing of tumor specimens prospectively on standardized protocols to better reveal the impact of biomarkers on clinically relevant outcomes.

The "Guardian of the Genome"-An Old Key to Unlock the ERCC1 Issue.

Excision Repair Cross-Complementation Group 1 (ERCC1) participates in the repair of DNA intrastrand adducts (ISA) and interstrand cross-links, but its role as a predictive biomarker has never been fully validated. It has now been revealed that p53 mutation status should be considered concomitantly with ERCC1 to predict cisplatin efficacy.See related article by Heyza et al., p. 2523.

Telomere length, telomeric proteins and genomic instability during the multistep carcinogenic process.

Telomeres form specialized structures at the ends of eukaryotic chromosomes, preventing them from being wrongly recognized as DNA damage. The human telomere DNA sequence is a tandem repetition of the sequence TTAGGG. In normal cells, the DNA replication machinery is unable to completely duplicate the telomeric DNA; thus, telomeres are shortened after every cell division. Having reached a critical length, telomeres may be recognized as double strand break DNA lesions, and cells eventually enter senescence. Carcinogenesis is a multistep process involving multiple mutations and chromosomal aberrations. One of the most prevalent aberrations in pre-cancerous lesions is telomere shortening and telomerase activation. We discuss the role and homeostasis of telomeres in normal cells and their implication in the early steps of carcinogenesis. We also discuss various techniques used, and their limitations, in the study of telomeres and genome instability and their role in carcinogenesis and related genomic modifications.

Telomeres and telomerase as targets for anticancer drug development.

In most human cancers, the telomere erosion problem has been bypassed through the activation of a telomere maintenance system (usually activation of telomerase). Therefore, telomere and telomerase are attractive targets for anti-cancer therapeutic interventions. Here, we review a large panel of strategies that have been explored to date, from small inhibitors of the catalytic sub-unit of telomerase to anti-telomerase immunotherapy and gene therapy. The many positive results that are reported from anti-telomere/telomerase assays suggest a prudent optimism for a possible clinical application in a close future. However, we discuss some of the main limits for these approaches of antitumour drug development and why significant work remains before a clinically useful drug can be proposed to patients.

No evidence for viral sequences in five lepidic adenocarcinomas (former "BAC") by a high-throughput sequencing approach.

BACKGROUND: The hypothesis of an infectious etiology of the formerly named bronchiolo-alveolar carcinoma (BAC) has raised controversy. We investigated tumor lung tissues from five patients with former BAC histology using high-throughput sequencing technologies to discover potential viruses present in this type of lung cancer. Around 180 million single reads of 100 bases were generated for each BAC sample. RESULTS: None of the reads showed a significant similarity for Jaagsiekte sheep retrovirus (JSRV) and no other viruses were found except for endogenous retroviruses. CONCLUSIONS: In conclusion, we have demonstrated the absence of JSRV and other known human viruses in five samples of well-characterized lepidic adenocarcinoma.

Loss of PTEN expression is not uncommon, but lacks prognostic value in stage I NSCLC.

The PTEN gene is a tumor-suppressor gene that is inactivated in several types of human tumors. The loss of PTEN expression has been supported as a prognostic marker. Using immunohistochemical analysis, we retrospectively analyzed PTEN expression in specimens from 53 patients with completely resected stage I non-small cell lung cancer (NSCLC) for whom clinical follow-up data were available. Seven of the 53 specimens showed a total lack of staining for PTEN. No statistically significant relationship could be found between PTEN expression and clinicopathological parameters. Although genetic alterations of the PTEN gene are rare in NSCLC, loss of PTEN protein is not an uncommon event in early-stage NSCLC (13.2%). Here, we also report that the level of PTEN protein expression is not an independent prognostic marker in early-stage NSCLC.

PARP inhibitors: an interesting pathway also for non-small cell lung cancer?

Treatment of lung cancer is improving, also based on the identification of molecular characteristics of the tumor, of which some already constitute promising targets. One of the molecular characteristics thought to play an important role in lung cancer is DNA repair dysfunctionality. Deregulated expression of DNA repair proteins, such as PARP, has been studied in lung cancer as a possible biomarker and clinically useful target, but the literature remains relatively poor. Pharmacological inactivation of PARP has allowed the identification of a synthetic lethality with a second DNA repair protein such as BRCA1, but has also shown the potential to sensitize tumors to commonly used cytotoxic agents. The current manuscript reviews data regarding PARP in the context of DNA repair and its different pathways, as well as the clinical data generated until now with PARP inhibitors. A deeper understanding of the DNA damage response in lung malignancies, and particularly a clarification of the crosstalk between DNA repair functionality and genetic stability, is the key to optimize the development of PARP inhibitors in the setting of NSCLC.

Prognostic value of LIPC in non-small cell lung carcinoma.

Non-small cell lung carcinoma (NSCLC) is the most common form of lung cancer and is associated with a high mortality rate worldwide. The majority of individuals bearing NSCLC are treated with surgery plus adjuvant cisplatin, an initially effective therapeutic regimen that, however, is unable to prevent relapse within 5 years after tumor resection in an elevated proportion of patients. The factors that predict the clinical course of NSCLC and its sensitivity to therapy remain largely obscure. One notable exception is provided by pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. PDXK has recently been shown to be required for optimal cisplatin responses in vitro and in vivo and to constitute a bona fide prognostic marker in the NSCLC setting. Together with PDXK, 84 additional factors were identified that influence the response of NSCLC cells to cisplatin, in vitro including the hepatic lipase LIPC. Here, we report that the intratumoral levels of LIPC, as assessed by immunohistochemistry in two independent cohorts of NSCLC patients, positively correlate with disease outcome. In one out of two cohorts studied, the overall survival of NSCLC patients bearing LIPChigh lesions was unaffected, if not slightly worsened, by cisplatin-based adjuvant therapy. Conversely, the overall survival of patients with LIPClow lesions was prolonged by post-operative cisplatin. Pending validation in appropriate clinical series, these results suggest that LIPClow NSCLC patients would be those who mainly benefit from adjuvant cisplatin therapy. Thus, the expression levels of LIPC appear to have an independent prognostic value (and perhaps a predictive potential) in the setting of NSCLC. If these findings were confirmed by additional studies, LIPC expression levels might allow not only for NSCLC patient stratification, but also for the implementation of personalized therapeutic approaches.

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform.

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies.

Cisplatin resistance associated with PARP hyperactivation.

Non-small cell lung carcinoma patients are frequently treated with cisplatin (CDDP), most often yielding temporary clinical responses. Here, we show that PARP1 is highly expressed and constitutively hyperactivated in a majority of human CDDP-resistant cancer cells of distinct histologic origin. Cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PAR(high)) responded to pharmacologic PARP inhibitors as well as to PARP1-targeting siRNAs by initiating a DNA damage response that translated into cell death following the activation of the intrinsic pathway of apoptosis. Moreover, PARP1-overexpressing tumor cells and xenografts displayed elevated levels of PAR, which predicted the response to PARP inhibitors in vitro and in vivo more accurately than PARP1 expression itself. Thus, a majority of CDDP-resistant cancer cells appear to develop a dependency to PARP1, becoming susceptible to PARP inhibitor-induced apoptosis.

Prognostic impact of vitamin B6 metabolism in lung cancer.

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.

[DNA repair pathways and non-small cell lung cancer: clinical perspectives]. / Voies de réparation de l'ADN et cancers bronchiques non à petites cellules : perspectives cliniques.

The role of DNA repair pathways is to maintain cellular integrity. However, genetic instability is a driving force in the development of tumor cells and many tumors are characterized by the loss of functionality in one or several DNA repair pathways. However, if genetic instability trespasses a certain point, it will induce cell death. Therefore, the dysfunctionality of several DNA repair pathways could represent an Achille's heel for the tumor, if such pathways could be pharmacologically targeted. For instance, the inhibition of PARP1, a protein in the base excision repair pathway (BER) is sufficient to induce cell death in cancer cells bearing BRCA1 or BRCA2 mutations, which are essential proteins in the homologous recombination repair pathway (HR). This phenomenon called "synthetic letality" constitutes recent knowledge and we discuss here the possibility that this strategy might be applied to innovative treatment options in lung cancer. Further, several DNA repair proteins could be used in lung cancer as prognostic and/or predictive biomarkers of response to chemotherapy or radiation. Indeed, specific biomarkers of each DNA repair pathway do exist and could guide oncologists in therapeutic decisions (e.g. ERCC1 and cisplatin). Finally, pharmacologic modulation of DNA repair proteins might also be interesting as it might increase therapeutic efficacy of anticancer strategies (DNA-interacting chemotherapy and radiotherapy). Here, we will present the principal DNA repair pathways and associated biomarkers (ERCC1, MSH2, PARP1 and BRCA1/2), and discuss their status in non-small call lung cancer (NSCLC).

Molecular Characteristics of ERCC1-Negative versus ERCC1-Positive Tumors in Resected NSCLC.

PURPOSE: Excision repair cross-complementation group 1 (ERCC1) is a protein involved in repair of DNA platinum adducts and stalled DNA replication forks. We and others have previously shown the influence of ERCC1 expression upon survival rates and benefit of cisplatin-based chemotherapy in patients with resected non-small-cell lung cancer (NSCLC). However, little is known about the molecular characteristics of ERCC1-positive and ERCC1-negative tumors. EXPERIMENTAL DESIGN: We took advantage of a cohort of 91 patients with resected NSCLC, for which we had matched frozen and paraffin-embedded samples to explore the comparative molecular portraits of ERCC1-positive and ERCC1-negative tumors of NSCLC. We carried out a global molecular analysis including assessment of ERCC1 expression levels by using both immunohistochemistry (IHC) and quantitative reverse transcriptase PCR (qRT-PCR), genomic instability, global gene and miRNA expression, and sequencing of selected key genes involved in lung carcinogenesis. RESULTS: ERCC1 protein and mRNA expression were significantly correlated. However, we observed several cases with clear discrepancies. We noted that ERCC1-negative tumors had a higher rate of genomic abnormalities versus ERCC1-positive tumors. ERCC1-positive tumors seemed to share a common DNA damage response (DDR) phenotype with the overexpression of seven genes linked to DDR. The miRNA expression analysis identified miR-375 as significantly underexpressed in ERCC1-positive tumors. CONCLUSIONS: Our data show inconsistencies in ERCC1 expression between IHC and qRT-PCR readouts. Furthermore, ERCC1 status is not linked to specific mutational patterns or frequencies. Finally, ERCC1-negative tumors have a high rate of genomic aberrations that could consequently influence prognosis in patients with resected NSCLC.

Expression of chemokine receptor CCR6 as a molecular determinant of adrenal metastatic relapse in patients with primary lung cancer.

BACKGROUND: Recent studies suggest that chemokines are involved in organ-specific metastatic relapse. We evaluated the potential implications of chemokine receptors in the development of adrenal metastasis after complete resections of primary non-small-cell lung cancer. PATIENTS AND METHODS: We studied a unique cohort of 21 primary lung cancers with matched adrenal metastases for the expression of CX3CR1, CXCR4, CCR6, and CCR7, using immunohistochemistry. RESULTS: Although CXCR4, CX3CR1, and CCR7 were independently expressed in primary and corresponding metastases, CCR6 was clearly overexpressed in adrenal metastases, compared with corresponding primary tumors. Moreover, CCL20, the ligand of CCR6, was preferentially expressed in adrenal tissues that developed metastases. CONCLUSION: We report for the first time (to the best of our knowledge) a potential role for the CCR6 receptor in the organ orientation of the development of metastases in lung cancer. We demonstrated a statistically significant overexpression of CCR6 in adrenal metastases compared with primary lung tumors, indicating that the increased production of CCL20 in adrenal glands might contribute to the selective recruitment of CCR6-expressing cancer cells in lung cancer. This study, in concordance with the data obtained in animal models, suggests that the chemokine receptor family constitutes a biologic support of the "seed and soil" theory.

Are RAS mutations predictive markers of resistance to standard chemotherapy?

KRAS mutations may be predictive of resistance to anti-EGFR monoclonal-based therapy in patients with colorectal cancer (CRC). Screening for KRAS mutations in patients with CRC and non-small-cell lung cancer (NSCLC) may provide additional information on optimizing treatment options with targeted therapies. Only limited studies, however, have assessed the predictive value of KRAS mutations in response to conventional chemotherapy. We reviewed all relevant papers investigating the association of KRAS mutations and conventional chemotherapy-related outcome in NSCLC, CRC, and other solid tumors, both in the adjuvant and advanced settings. Our Review strongly suggests that KRAS mutations have no value in response prediction to conventional chemotherapy in NSCLC, CRC and other solid tumors. Therefore, KRAS mutations should not be used as molecular predictors of response to conventional chemotherapy.

ERCC1 as a risk stratifier in platinum-based chemotherapy for nonsmall-cell lung cancer.

PURPOSE OF REVIEW: Cisplatin-based chemotherapy remains the treatment of choice in advanced nonsmall-cell lung cancer. The development of predictive biomarkers able to identify lung-cancer patients who are most likely to benefit from cisplatin-based chemotherapy would be a powerful tool. Many reports have explored the role of ERCC1 expression in the repair mechanism of cisplatin-induced DNA adducts in cancer cells. RECENT FINDINGS: Using immunohistochemistry in resected tumors, the International Adjuvant Lung Cancer Trial showed that high ERCC1 protein expression was associated with improved survival in patients who did not receive chemotherapy. In contrast, the benefit of adjuvant cisplatin-based chemotherapy was more profound in patients with low ERCC1 expression. Other investigators studying mRNA expression in tumor biopsies from patients treated with cisplatin and gemcitabine showed that patients with low ERCC1 mRNA expression have a longer median survival compared to those with high expression. SUMMARY: High ERCC1 expression is predictive of resistance to platinum-based therapy. Thus, there is solid evidence to support ERCC1 as a useful marker of clinical resistance to platinum-based chemotherapy in the adjuvant setting of nonsmall-cell lung cancer. Meanwhile, optimization of methodology and standardization of technical procedures seem necessary before larger prospective studies can address the same question.