Metastatic Colorectal Cancer Resistance Research Articles

Deciphering treatment resistance in metastatic colorectal cancer: roles of drug transports, EGFR mutations, and HGF/c-MET signaling.

In 2023, colorectal cancer (CRC) is the third most diagnosed malignancy and the third leading cause of cancer death worldwide. At the time of the initial visit, 20% of patients diagnosed with CRC have metastatic CRC (mCRC), and another 25% who present with localized disease will later develop metastases. Despite the improvement in response rates with various modulation strategies such as chemotherapy combined with targeted therapy, radiotherapy, and immunotherapy, the prognosis of mCRC is poor, with a 5-year survival rate of 14%, and the primary reason for treatment failure is believed to be the development of resistance to therapies. Herein, we provide an overview of the main mechanisms of resistance in mCRC and specifically highlight the role of drug transports, EGFR, and HGF/c-MET signaling pathway in mediating mCRC resistance, as well as discuss recent therapeutic approaches to reverse resistance caused by drug transports and resistance to anti-EGFR blockade caused by mutations in EGFR and alteration in HGF/c-MET signaling pathway.

Single-cell sequencing reveals the immune microenvironment landscape related to anti-PD-1 resistance in metastatic colorectal cancer with high microsatellite instability

BackgroundThe objective response rate of microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) patients with first-line anti-programmed cell death protein-1 (PD-1) monotherapy is only 40–45%. Single-cell RNA sequencing (scRNA-seq) enables unbiased analysis of the full variety of cells comprising the tumor microenvironment. Thus, we used scRNA-seq to assess differences among microenvironment components between therapy-resistant and therapy-sensitive groups in MSI-H/mismatch repair-deficient (dMMR) mCRC. Resistance-related cell types and genes identified by this analysis were subsequently verified in clinical samples and mouse models to further reveal the molecular mechanism of anti-PD-1 resistance in MSI-H or dMMR mCRC.MethodsThe response of primary and metastatic lesions to first-line anti-PD-1 monotherapy was evaluated by radiology. Cells from primary lesions of patients with MSI-H/dMMR mCRC were analyzed using scRNA-seq. To identify the marker genes in each cluster, distinct cell clusters were identified and subjected to subcluster analysis. Then, a protein‒protein interaction network was constructed to identify key genes. Immunohistochemistry and immunofluorescence were applied to verify key genes and cell marker molecules in clinical samples. Immunohistochemistry, quantitative real-time PCR, and western blotting were performed to examine the expression of IL-1β and MMP9. Moreover, quantitative analysis and sorting of myeloid-derived suppressor cells (MDSCs) and CD8+ T cells were performed using flow cytometry.ResultsTumor responses in 23 patients with MSI-H/dMMR mCRC were evaluated by radiology. The objective response rate was 43.48%, and the disease control rate was 69.57%. ScRNA-seq analysis showed that, compared with the treatment-resistant group, the treatment-sensitive group accumulated more CD8+ T cells. Experiments with both clinical samples and mice indicated that infiltration of IL-1β-driven MDSCs and inactivation of CD8+ T cells contribute to anti-PD-1 resistance in MSI-H/dMMR CRC.ConclusionsCD8+ T cells and IL-1β were identified as the cell type and gene, respectively, with the highest correlation with anti-PD-1 resistance. Infiltration of IL-1β-driven MDSCs was a significant factor in anti-PD-1 resistance in CRC. IL-1β antagonists are expected to be developed as a new treatment for anti-PD-1 inhibitor resistance.

In silico analysis to identify miR-1271-5p/PLCB4 (phospholipase C Beta 4) axis mediated oxaliplatin resistance in metastatic colorectal cancer

Oxaliplatin (OXA) is the first-line chemotherapy drug for metastatic colorectal cancer (mCRC), and the emergence of drug resistance is a major clinical challenge. Although there have been numerous studies on OXA resistance, but its underlying molecular mechanisms are still unclear. This study aims to identify key regulatory genes and pathways associated with OXA resistance. The Gene Expression Omnibus (GEO) GSE42387 dataset containing gene expression profiles of parental and OXA-resistant LoVo cells was applied to explore potential targets. GEO2R, STRING, CytoNCA (a plug-in of Cytoscape), and DAVID were used to analyze differentially expressed genes (DEGs), protein–protein interactions (PPIs), hub genes in PPIs, and gene ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. R2 online platform was used to run a survival analysis of validated hub genes enriched in KEGG pathways. The ENCORI database predicted microRNAs for candidate genes. A survival analysis of those genes was performed, and validated using the OncoLnc database. In addition, the 'clusterProfiler' package in R was used to perform gene set enrichment analysis (GSEA). We identified 395 DEGs, among which 155 were upregulated and 240 were downregulated. In total, 95 DEGs were screened as hub genes after constructing the PPI networks. Twelve GO terms and three KEGG pathways (steroid hormone biosynthesis, malaria, and pathways in cancer) were identified as being significant in the enrichment analysis of hub genes. Twenty-one hub genes enriched in KEGG pathways were defined as key genes. Among them AKT3, phospholipase C Beta 4 (PLCB4), and TGFB1 were identified as OXA-resistance genes through the survival analysis. High expressions of AKT3 and TGFB1 were each associated with a poor prognosis, and lower expression of PLCB4 was correlated with worse survival. Further, high levels of hsa-miR-1271-5p, which potentially targets PLCB4, were associated with poor overall survival in patients with CRC. Finally, we found that PLCB4 low expression was associated with MAPK signaling pathway and VEGF signaling pathway in CRC. Our results demonstrated that hsa-miR-1271-5p/PLCB4 in the pathway in cancer could be a new potential therapeutic target for mCRC with OXA resistance.

Overcoming EGFR Resistance in Metastatic Colorectal Cancer Using Vitamin C: A Review.

Targeted monoclonal antibody therapy against Epidermal Growth Factor Receptor (EGFR) is a leading treatment modality against metastatic colorectal cancer (mCRC). However, with the emergence of KRAS and BRAF mutations, resistance was inevitable. Cells harboring these mutations overexpress Glucose Transporter 1 (GLUT1) and sodium-dependent vitamin C transporter 2 (SVCT2), which enables intracellular vitamin C transport, leading to reactive oxygen species generation and finally cell death. Therefore, high dose vitamin C is proposed to overcome this resistance. A comprehensive search strategy was adopted using Pubmed and MEDLINE databases (up to 11 August 2022). There are not enough randomized clinical trials to support its use in the clinical management of mCRC, except for a subgroup analysis from a phase III study. High dose vitamin C shows a promising role in overcoming EGFR resistance in mCRC with wild KRAS mutation with resistance to anti-epidermal growth factor inhibitors and in patients with KRAS and BRAF mutations.

Recent and Future Strategies to Overcome Resistance to Targeted Therapies and Immunotherapies in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cause of cancer-related deaths worldwide, and 20% of patients with CRC present at diagnosis with metastases. The treatment of metastatic CRC is based on a fluoropyrimidine-based chemotherapy plus additional agents such as oxaliplatin and irinotecan. To date, on the basis of the molecular background, targeted therapies (e.g., monoclonal antibodies against epidermal growth factor receptor or inhibiting angiogenesis) are administered to improve the treatment of metastatic CRC. In addition, more recently, immunological agents emerged as effective in patients with a defective mismatch repair system. The administration of targeted therapies and immunotherapy lead to a significant increase in the survival of patients; however these drugs do not always prove effective. In most cases the lack of effectiveness is due to the development of primary resistance, either a resistance-inducing factor is already present before treatment or resistance is acquired when it occurs after treatment initiation. In this review we describe the most relevant targeted therapies and immunotherapies and expand on the reasons for resistance to the different approved or under development targeted drugs. Then we showed the possible mechanisms and drugs that may lead to overcoming the primary or acquired resistance in metastatic CRC.

Comprehensive characterization of PTPRT expression in colorectal cancer (CRC).

3538 Background: PTPRT is a protein coding gene involved in signal transduction and cellular adhesion. It acts as a tumor suppressor gene and mutated PTPRT has been implicated in the early metastasis of CRC. PTPRT mutations have been reported as independent potential biomarkers for bevacizumab resistance in metastatic CRC and linked to improved response and survival of patients treated with checkpoint inhibitors in several tumors. Here we characterized the molecular features and clinical outcomes associated with PTPRT gene expression in CRC. Methods: 15025 CRC tested with NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS) by Caris Life Sciences (Phoenix, AZ) were analyzed. Top quartile transcripts per million for PTPRT expression were considered high (H) and bottom quartile low (L). Cell infiltration in the tumor microenvironment (TME) was estimated by QuantiSEQ. X2, Fisher-Exact, Mann-Whitney tests were used and significance determined as P-value adjusted for multiple comparisons ( Q <.05). Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: PTPRT expression was higher in left- than right-sided CRC and in metastases than primary/local tumors; it was highest in CMS4 and lowest in CMS1 (all Q <.05). PTPRT mutants had lower expression than wild type (0.037 vs 0.064, Q <.05). Overall, PTPRT-H had a lower rate of TMB-H (5% vs 17%), deficient mismatch repair (dMMR) (3% vs 13%) and PD-L1 (2% vs 6%) (H vs L, all Q <.05). In the proficient MMR (pMMR) cohort, PTPRT-H remained inversely correlated with TMB and PD-L1, was negatively associated with rates of mutated KRAS, PIK3CA, SMAD4, FBXW7, and positively associated with mutated TP53 and CDX2 amplification (all Q <.05). Expression of immune related genes was higher in PTPRT-H CRC, including CD274, CD80, CD86, CTLA4, HAVCR2, IDO1, IFNG, LAG3, PDCD1, and PDCD1LG2, regardless of MMR status (all Q <.05). PTPRT-H was associated with higher immune cell infiltration in the TME including B cells, M2 macrophages, neutrophils, NK, Tregs, CD4+ and CD8+ T cells, and myeloid dendritic cells (fold change/FC: 1.21-7.1), but lower M1 macrophages (FC: 0.76), regardless of MMR status (all Q <.05) with the only exception of CD8+ T cells in dMMR. PTPRT expression above median was associated with better OS (overall: HR 0.69, 95% CI [0.64-0.74]; pMMR: HR 0.67 [0.63-0.73]), longer time on treatment of bevacizumab (overall: HR 0.80 [0.74-0.87], pMMR: HR 0.80 [0.74-0.87]), and shorter time on immunotherapy treatment in the dMMR cohort (HR 2.13 [1.33-3.45]). Conclusions: Our data show a strong association between PTPRT expression and distinct molecular features (including CMS and immune biomarkers), TME cell infiltration and targeted treatment outcomes in CRC. These findings support PTPRT as a candidate prognostic and predictive biomarker for bevacizumab and immunotherapy treatment, and as a potential target in CRC.

Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer.

Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.

COLOMATE challenge to overcome resistance in metastatic colorectal cancer.

COLOMATE challenge to overcome resistance in metastatic colorectal cancer.

Circulating Tumour DNA Sequencing Identifies a Genetic Resistance-Gap in Colorectal Cancers with Acquired Resistance to EGFR-Antibodies and Chemotherapy.

Simple SummaryRecent studies have shown the potential of next generation sequencing (NGS) for the identification of genetic variants in tumour DNA that has been released into the bloodstream (ctDNA). However, such variants are often rare in the sample and error correction is required to filter out false calls. In this study we used error corrected ctDNA-sequencing to identify genetic drivers of resistance in metastatic colorectal cancer (mCRC) patients that had become resistant to combined chemotherapy and EGFR-antibody treatment. Our data showed that ctDNA-seq could detect common and novel resistance mechanisms in cases of both primary and acquired resistance. ctDNA-seq could therefore facilitate patient stratification to novel therapies and avoid ineffective treatment with EGFR-antibodies. Furthermore, the data revealed a lack of detectable genetic resistance in a large fraction of the cancer cell population, indicating a need to investigate other, potentially non-genetic, resistance mechanisms.Epidermal growth factor receptor antibodies (EGFR-Abs) confer a survival benefit in patients with RAS wild-type metastatic colorectal cancer (mCRC), but resistance invariably occurs. Previous data showed that only a minority of cancer cells harboured known genetic resistance drivers when clinical resistance to single-agent EGFR-Abs had evolved, supporting the activity of non-genetic resistance mechanisms. Here, we used error-corrected ctDNA-sequencing (ctDNA-Seq) of 40 cancer genes to identify drivers of resistance and whether a genetic resistance-gap (a lack of detectable genetic resistance mechanisms in a large fraction of the cancer cell population) also occurs in RAS wild-type mCRCs treated with a combination of EGFR-Abs and chemotherapy. We detected one MAP2K1/MEK1 mutation and one ERBB2 amplification in 2/3 patients with primary resistance and KRAS, NRAS, MAP2K1/MEK1 mutations and ERBB2 aberrations in 6/7 patients with acquired resistance. In vitro testing identified MAP2K1/MEK1 P124S as a novel driver of EGFR-Ab resistance. Mutation subclonality analyses confirmed a genetic resistance-gap in mCRCs treated with EGFR-Abs and chemotherapy, with only 13.42% of cancer cells harboring identifiable resistance drivers. Our results support the utility of ctDNA-Seq to guide treatment allocation for patients with resistance and the importance of investigating further non-canonical EGFR-Ab resistance mechanisms, such as microenvironmentally-mediated resistance. The detection of MAP2K1 mutations could inform trials of MEK-inhibitors in these tumours.

Molecular mechanisms of cetuximab resistance in metastatic colorectal cancer

Molecular mechanisms of cetuximab resistance in metastatic colorectal cancer

C-MYC Expression Is a Possible Keystone in the Colorectal Cancer Resistance to EGFR Inhibitors.

Alterations in the transcriptional factor c-MYC could be involved in the anti-EGFR resistance in metastatic colorectal cancer (mCRC). The c-MYC expression was evaluated in 121 RAS and BRAF wild-type mCRC before treatment with anti-EGFR+Folfiri therapy and in 33 subsequent metastases collected during target therapy (TT) or in TT resistance phase. We analyzed the expression and the functional role of some c-MYC linked miRNAs (miR-31-3p, miR-143 and miR-145) in our patient group and in two CRC cell lines, also performing a c-MYC target PCR array. Patients with higher c-MYC expression (HME) showed a significant lower PFS and OS when compared to those with low c-MYC expression (LME). HME pattern was significantly more frequent in the metastases after TT and significantly associated to anti-EGFR molecular resistance alterations. We also found a significant correlation between the expression of the above-mentioned c-MYC linked miRNAs, c-MYC level and anti-EGFR resistance. Moreover, expression gene profiling pointed out the pivotal role of c-MYC in CRC-related cell-cycle, apoptosis, signal transduction and cell-growth pathways. c-MYC expression might distinguish patients with a lower PFS and OS in anti-EGFR treated mCRC. The individuation of some miRNAs involved in the c-MYC pathway regulation and the downstream c-MYC effector genes could provide a new possible target to overcome the anti-EGFR resistance in mCRC.

Glucocorticoid Receptor Modulates EGFR Feedback upon Acquisition of Resistance to Monoclonal Antibodies.

Evidences of a crosstalk between Epidermal Growth Factor Receptor (EGFR) and Glucocorticoid Receptor (GR) has been reported, ranging from the modulation of receptor levels or GR mediated transcriptional repression of EGFR target genes, with modifications of epigenetic markers. The present study focuses on the involvement of EGFR positive and negative feedback genes in the establishment of cetuximab (CTX) resistance in metastatic Colorectal Cancer (CRC) patients. We evaluated the expression profile of the EGFR ligands TGFA and HBEGF, along with the pro-inflammatory cytokines IL-1B and IL-8, which were previously reported to be negatively associated with monoclonal antibody response, both in mice and patient specimens. Among EGFR negative feedback loops, we focused on ERRFI1, DUSP1, LRIG3, and LRIG1. We observed that EGFR positive feedback genes are increased in CTX-resistant cells, whereas negative feedback genes are reduced. Next, we tested the expression of these genes in CTX-resistant cells upon GR modulation. We unveiled that GR activation leads to an increase in ERRFI1, DUSP1, and LRIG1, which were shown to restrict EGFR activity, along with a decrease in the EGFR activators (TGFA and IL-8). Finally, in a cohort of xenopatients, stratified for response to cetuximab, we observed an inverse association between the expression level of LRIG1 and CRC progression upon CTX treatment. Our model implies that combining GR modulation to EGFR inhibition may yield an effective treatment strategy in halting cancer progression.

Abstract 3617: Prediction of irinotecan and flurouracil efficacy in metastatic colorectal cancer by early measurement of circulating tumor DNA

Abstract Background: Chemotherapy resistance in metastatic colorectal cancer (mCRC) is a major challenge, and development of biomarkers for determining therapy efficacy is vital to ensure optimal palliative treatment. Recently, quantification of changes in circulating tumor DNA (ctDNA) levels has attracted substantial attention as a predictive marker. Here, we provide evidence that ctDNA has the potential to be used as an early marker of therapeutic efficacy of irinotecan and fluorouracil (FOLFIRI). Experimental Procedures: Patients diagnosed with mCRC, and with indication for first-line combination chemotherapy, were prospectively enrolled in this phase II study. Blood was drawn at baseline, day 7, 14, 21, 40 and then monthly until progression. Response evaluation was done at eight-week intervals by CT scanning using RECIST criteria. Chemotherapy was administered at day one and two every two weeks. The levels of cell-free DNA (cfDNA) and ctDNA were determined using sensitive digital droplet PCR assays. ctDNA assays were designed either to mutations identified by targeted sequencing or on basis of known tumor mutations detected in primary or metastatic lesions using a panel of mutation-specific qPCR based assays (KRAS, NRAS, BRAF). Results: Twenty-four patients were enrolled. A KRAS, NRAS or BRAF mutation was observed in tumor tissue of 16 patients. In 12 of these the mutations were confirmed in baseline samples and used for assessing ctDNA. For the remaining 12 patients, a mutation for ctDNA quantification was identified by targeted tumor sequencing. Patients with high baseline ctDNA levels (75th centile) had significantly shorter progression-free survival (PFS) than patients with low baseline ctDNA levels. After first treatment cycle all patients experienced a decline in ctDNA levels demonstrating initial response to treatment; however, the reduction in ctDNA was not associated with prolonged PFS or overall survival (OS). Conversely, change in ctDNA levels from first to second cycle of treatment (three weeks after treatment start) predicted FOLFIRI efficacy. Patients with increasing ctDNA levels at this time point had a significantly shorter PFS and a shorter OS than patients with stable or decreasing ctDNA levels. Conclusion: In agreement with the literature, we show that baseline tumor DNA levels predicted PFS. The initial observed decline in ctDNA levels upon administration of FOLFIRI indicates that all tumors had cancer cells sensitive to treatment; however, after elimination of these, resistant clones expanded, leading to treatment resistance. Importantly, our study demonstrates that FOLFIRI efficacy can be evaluated as early as three weeks after treatment initiation by measurement of ctDNA levels. This enables early detection of treatment failure and allows for rapid adaptation of the treatment strategy. Citation Format: Iben Lyskjær, Camilla J. Kronborg, Mads H. Rasmussen, Boe S. Sørensen, Mona Rosenkilde, Michael Knudsen, Søren R. Krag, Karen-Lise G. Spindler, Claus L. Andersen. Prediction of irinotecan and flurouracil efficacy in metastatic colorectal cancer by early measurement of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3617.

Nuclear IGF-1R predicts chemotherapy and targeted therapy resistance in metastatic colorectal cancer

Background:Although chemotherapy is the cornerstone treatment for patients with metastatic colorectal cancer (mCRC), acquired chemoresistance is common and constitutes the main reason for treatment failure. Monoclonal antibodies against insulin-like growth factor-1 receptor (IGF-1R) have been tested in pre-treated mCRC patients, but results have been largely deceiving.Methods:We analysed time to progression, overall survival, and the mutational status of RAS, BRAF and nuclear p-IGF-1R expression by immunohistochemistry, in 470 metastatic CRC patients. The effect of IGF-1R activation and distribution was also assessed using cellular models of CRC and RNAi for functional validation.Results:Nuclear IGF-1R increased in metastatic tumours compared to paired untreated primary tumours, and significantly correlated with poor overall survival in mCRC patients. In vitro, chemo-resistant cell lines presented significantly higher levels of IGF-1R expression within the nuclear compartment, and PIAS3, a protein implicated also in the sumoylation process of intranuclear proteins, contributed to IGF-1R nuclear sequestration, highlighting the essential role of PIAS3 in this process. Intriguingly, we observed that ganitumab, an IGF-1R blocking-antibody used in several clinical trials, and dasatinib, an SRC inhibitor, increased the nuclear localisation of IGF-1R.Conclusions:Our study demonstrates that IGF-1R nuclear location might lead to chemotherapy and targeted agent resistance.

Highlights of This Issue

Factors influencing the efficacy of abiraterone in suppressing tumor growth in castration-resistant prostate cancer (CRPC) are not well understood. Based on its steroidal structure, Mostaghel and colleagues hypothesized that abiraterone may be transported by the SLCO family of androgen transporters. In vitro, prostate cancer cells expressing SLCO2B1 showed higher abiraterone uptake when compared with controls. In a neoadjuvant trial of abiraterone prior to prostatectomy, genetic variation in SLCO2B1 was associated with tissue abiraterone levels and rates of minimal residual disease. These data suggest variation in SLCO genes may serve as predictors of response to abiraterone treatment in men with CRPC.KRAS mutation was reported as a contributor to acquired cetuximab resistance in metastatic colorectal cancer. To explore whether other mutations in genes downstream of EGFR confer acquired resistance to cetuximab-based therapy, Xu and colleagues designed a strategy to identify low-frequency candidate mutations that are associated with acquired cetuximab resistance by sequencing circulating tumor DNA taken from patient plasma samples obtained before and during treatment. Five novel mutations in exon 19 and hotspot PIK3CA mutations were identified, which contribute to acquired cetuximab resistance. The role of PIK3CA mutations in acquired cetuximab resistance appears to be comparable with that of KRAS mutations.In patients treated with pembrolizumab, tumor burden increase of less than 20% from baseline during therapy was noted in 55% of the patients and was associated with longer overall survival, proposing a practical prognostic marker that may objectively guide treatment decisions. Pseudoprogression was noted in 4 among 107 patients (4%), including one patient without measurable tumor burden at baseline, which is indicative of a need for increased attention to nonmeasurable tumor burden in the context of immunotherapy.Reliable and reproducible methods for detecting PD-L1 expression in tumors is crucial for identifying patients who respond to anti-PD-1 therapy. The PD-L1 IHC 22C3 pharmDx is a companion diagnostic developed by Dako to identify non-small cell lung cancer patients who are likely to derive benefit from treatment with pembrolizumab. Using this assay, Cooper and colleagues investigated the inter- and intra-observer reproducibility of pathologists' assessment of PD-L1 staining and the impact of training on reproducibility. They demonstrated good agreement at both 1% and 50% cut-points. A one-hour training session had minimal impact on the reproducibility.

Abstract 2795: Integrating multiomics discovery approaches to identify biomarkers of therapeutic resistance in metastatic colorectal cancer through analyses of multiple sequential tumor and liquid biopsies; Qcroc01: NCT00984048

Abstract Colorectal cancer (CRC) is the 2nd leading cause of cancer related-death in Canada. Clinical responses of metastatic (m)CRC to first-line treatment range from 35 to 60%, but even responders inevitably develop therapeutic resistance. Studies aiming at understanding mechanisms of resistance have largely investigated primary tumors. However, selective pressures during therapy can lead to the expansion of resistant clones and tumor heterogeneity. This highlights the need to characterize the molecular changes of metastasis and plasma over time of treatment and response to decipher tumor evolution and therapeutic resistance mechanisms. In this multicenter study, 52 tissue samples from liver metastasis were collected at baseline (pre-biopsies) and at the time of resistance (post-biopsies) in responder and non-responder mCRC patients (n=44) undergoing the same standard first-line treatments. Multiple post-biopsies also have been harvested in 4 patients, to allow the assessment of tumor heterogeneity and as well as the evolution of the genomic complexity after treatment exposure. Analyses were carried out across multiple omic platforms to identify resistant signatures and characterize molecular changes during treatment. Biopsies were profiled using exome and transcriptome sequencing as well as high-density SNP array analysis to capture chromosomal anomalies, loss of heterozygosity (LOH) and copy number variations (CNV). Additionally, serial blood samples were collected for proteomic, ctDNA and cytokine analysis. Our preliminary analysis of transcriptomes performed on serial biopsies from a set of 11 patients identified genes consistently overexpressed at resistance. Cytogenetics analysis showed similar genomic profiles of matched pre- and post-biopsies and allowed the establishment of LOH and CNV catalogues of liver metastasis, while exome sequencing revealed cumulative somatic mutations over time of treatment, which suggests subclonal and acquired “driver” mutations of resistance. Plasma-derived ctDNA analysis was performed to investigate the mutational status during treatment and whether they correlate with their relative levels in biopsies. Immune gene expression analysis of a test set of 27 metastases revealed strong clustering of 7 metastases due to overexpression of transcripts related to active immune response, allowing to define novel subgroups of patients based on immune response status. Our study, using a multi-omic strategy and integration of independent molecular platforms to profile liver metastasis samples of responder and non-responder mCRC patients, constitutes an innovative approach to identify clinical biomarkers and molecular signature of resistance, which may enhance individualization of cancer medicine and customized therapy. Citation Format: Karen Gambaro, Maud Marques, Ryan Morin, Claudia Kleinman, Michael Witcher, Simon Turcotte, Benoit Samson, Bernard Lespérance, Yoo-Joung Ko, Richard Dalfen, Eve St-Hilaire, Lucas Sideris, Felix Couture, Sabine Tejpar, Ronald Burkes, Mohammed Harb, Francine Aubin, Thierry Alcindor, Errol Camlioglu, Adriana Aguilar, Mathilde Couetoux du Tertre1, Suzan McNamara, Adrian Gologan, Petr Kavan, Gerald Batist. Integrating multiomics discovery approaches to identify biomarkers of therapeutic resistance in metastatic colorectal cancer through analyses of multiple sequential tumor and liquid biopsies; Qcroc01: NCT00984048 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2795. doi:10.1158/1538-7445.AM2017-2795

Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor's biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.

Increased Detection Sensitivity for KRAS Mutations Enhances the Prediction of Anti-EGFR Monoclonal Antibody Resistance in Metastatic Colorectal Cancer

KRAS mutations represent the main cause of resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs) in metastatic colorectal cancer (mCRC). We evaluated whether highly sensitive methods for KRAS investigation improve the accuracy of predictions of anti-EGFR MoAbs efficacy. We retrospectively evaluated objective tumor responses in mCRC patients treated with cetuximab or panitumumab. KRAS codons 12 and 13 were examined by direct sequencing, MALDI-TOF MS, mutant-enriched PCR, and engineered mutant-enriched PCR, which have a sensitivity of 20%, 10%, 0.1%, and 0.1%, respectively. In addition, we analyzed KRAS codon 61, BRAF, and PIK3CA by direct sequencing and PTEN expression by immunohistochemistry. In total, 111 patients were considered. Direct sequencing revealed mutations in codons 12 and 13 of KRAS in 43/111 patients (39%) and BRAF mutations in 9/111 (8%), with almost all of these occurring in nonresponder patients. Using highly sensitive methods, we identified up to 13 additional KRAS mutations compared with direct sequencing, all occurring in nonresponders. By analyzing PIK3CA and PTEN, we found that of these 13 patients, 7 did not show any additional alteration in the PI3K pathway. The application of highly sensitive methods for the detection of KRAS mutations significantly improves the identification of mCRC patients resistant to anti-EGFR MoAbs.

Co-expression of matrix metalloproteinase-7 (MMP-7) and phosphorylated insulin growth factor receptor I (pIGF-1R) correlates with poor prognosis in patients with wild-type KRAS treated with cetuximab or panitumumab: A GEMCAD study

Background: By transactivacion, phosphorylated insulin growth factor receptor I (IGF-1R) can activate epidermal growth factor receptor (EGFR). MMP-7, produced by colorectal cancer cells, also can activate IGF-1R by degrading IGFBP-3 and releasing IGF-I.Methods: A cohort of patients (pts) with advanced colorectal cancer (CRC), under second- or third-line treatment with cetuximab or panitumumab, was tested using immunohistochemistry for expression of the activated form of IGF-1R (p-IGF-1R) and MMP-7. KRAS and BRAF mutation status was determined by sequencing and allelic discrimination analysis, respectively. Analyses were performed in primary CRC tumor samples or metastases, and the association of immunohistochemistry findings, mutational results, and treatment outcomes was investigated in both univariate and multivariate analyses.Results: Expression of activated IGF-1R and MMP-7 was observed in 51 and 49% of pts, respectively. Co-expression of MMP-7 and pIGF-1R (double positivity, DP) was observed in 28 pts (25%). There was no association between KRAS or BRAF mutational status and DP (p=0.52). Pts with DP responded more poorly to first-line chemotherapy (p=0.005) and to anti-EGFR treatment (p=0.01) than non-DP pts. In wild type (WT) KRAS pts, those with DP have poorer PFS (2.7 months vs. 3.5m, p=0.036; HR 1.98, 95% CI 1.05-3.75) and OS (6.4 months vs. 8.6 m, p=0.010; HR 2.33, 95%CI 1.23-4.43) in the adjusted multivariate analysis.Conclusions: Our study suggests that concomitant expression of MMP-7 and activation of p-IGF-1R (DP) correlates with poor prognosis in WT KRAS pts treated with anti-EGFR.See commentary: MMP7 and activation of IGF-1R: A new insight into anti-EGFR therapeutic resistance in metastatic colorectal cancer

Molecular determinants of anti-EGFR sensitivity and resistance in metastatic colorectal cancer.

Since 2004, the clinical impact of monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) on patients with metastatic colorectal cancer (MCRC) has been clearly established. The combination of these biological agents with conventional chemotherapy has led to a significant improvement in response rate, progression-free survival and overall survival in first-line as well as in second- or third-line treatment of MCRC. However, the high variability of response and outcome in MCRC patients treated with these anti-EGFR mAbs has highlighted the need of identifying clinical and/or molecular predictive markers to ensure appropriate use of targeted therapies. The presence of somatic KRAS mutations has been clearly identified as a predictive marker of resistance to anti-EGFR in MCRC, and the use of anti-EGFR mAbs is now restricted to patients with no detectable KRAS mutation. Several studies have indicated that amplification of EGFR, overexpression of the EGFR ligands and inactivation of the anti-oncogene TP53 are associated with sensitivity to anti-EGFR mAbs, whereas mutations of BRAF and PIK3CA and loss of PTEN expression are associated with resistance. Besides these somatic variations, germline polymorphisms such as those affecting genes involved in the EGFR pathway or within the immunoglobulin receptors may also modulate response to anti-EGFR mAbs. Until now, all these markers are not completely validated and only KRAS genotyping is mandatory in routine practice for use of the anti-EGFR mAbs in MCRC.