A Phase II Exploratory Study to Identify Biomarkers Predictive of Clinical Response to Regorafenib in Patients with Metastatic Colorectal Cancer Who Have Failed First-Line Therapy.

Single-agent regorafenib is approved in Canada for metastatic colorectal cancer (mCRC) patients who have failed previous lines of therapy. Identifying prognostic biomarkers is key to optimizing therapeutic strategies for these patients. In this clinical study (NCT01949194), we evaluated the safety and efficacy of single-agent regorafenib as a second-line therapy for mCRC patients who received it after failing first-line therapy with an oxaliplatin or irinotecan regimen with or without bevacizumab. Using various omics approaches, we also investigated putative biomarkers of response and resistance to regorafenib in metastatic lesions and blood samples in the same cohort. Overall, the safety profile of regorafenib seemed similar to the CORRECT trial, where regorafenib was administered as ≥ 2 lines of therapy. While the mutational landscape showed typical mutation rates for the top five driver genes (APC, KRAS, BRAF, PIK3CA, and TP53), KRAS mutations were enriched in intrinsically resistant lesions. Additional exploration of genomic-phenotype associations revealed several biomarker candidates linked to unfavorable prognoses in patients with mCRC using various approaches, including pathway analysis, cfDNA profiling, and copy number analysis. However, further research endeavors are necessary to validate the potential utility of these promising genes in understanding patients' responses to regorafenib treatment.

Copy number and transcriptome alterations associated with metastatic lesion response to treatment in colorectal cancer.

BACKGROUND: Therapeutic resistance is the main cause of death in metastatic colorectal cancer. To investigate genomic plasticity, most specifically of metastatic lesions, associated with response to first-line systemic therapy, we collected longitudinal liver metastatic samples and characterized the copy number aberration (CNA) landscape and its effect on the transcriptome. METHODS: Liver metastatic biopsies were collected prior to treatment (pre, n = 97) and when clinical imaging demonstrated therapeutic resistance (post, n = 43). CNAs were inferred from whole exome sequencing and were correlated with both the status of the lesion and overall patient progression-free survival (PFS). We used RNA sequencing data from the same sample set to validate aberrations as well as independent datasets to prioritize candidate genes. RESULTS: We identified a significantly increased frequency gain of a unique CN, in liver metastatic lesions after first-line treatment, on chr18p11.32 harboring 10 genes, including TYMS, which has not been reported in primary tumors (GISTIC method and test of equal proportions, FDR-adjusted p = 0.0023). CNA lesion profiles exhibiting different treatment responses were compared and we detected focal genomic divergences in post-treatment resistant lesions but not in responder lesions (two-tailed Fisher's Exact test, unadjusted p ≤ 0.005). The importance of examining metastatic lesions is highlighted by the fact that 15 out of 18 independently validated CNA regions found to be associated with PFS in this study were only identified in the metastatic lesions and not in the primary tumors. CONCLUSION: This investigation of genomic-phenotype associations in a large colorectal cancer liver metastases cohort identified novel molecular features associated with treatment response, supporting the clinical importance of collecting metastatic samples in a defined clinical setting.

Discovery of a putative blood-based protein signature associated with response to ALK tyrosine kinase inhibition.

BACKGROUND: ALK tyrosine kinase inhibition has become a mainstay in the clinical management of ALK fusion positive NSCLC patients. Although ALK mutations can reliably predict the likelihood of response to ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, they cannot reliably predict response duration or intrinsic/extrinsic therapeutic resistance. To further refine the application of personalized medicine in this indication, this study aimed to identify prognostic proteomic biomarkers in ALK fusion positive NSCLC patients to crizotinib. METHODS: Twenty-four patients with advanced NSCLC harboring ALK fusion were administered crizotinib in a phase IV trial which included blood sampling prior to treatment. Targeted proteomics of 327 proteins using MRM-MS was used to measure plasma levels at baseline (including pre-treatment and early treatment blood samples) and assess potential clinical association. RESULTS: Patients were categorized by duration of response: long-term responders [PFS ≥ 24 months (n = 7)], normal responders [3 < PFS < 24 months (n = 10)] and poor responders [PFS ≤ 3 months (n = 5)]. Several proteins were identified as differentially expressed between long-term responders and poor responders, including DPP4, KIT and LUM. Next, using machine learning algorithms, we evaluated the classification potential of 40 proteins. Finally, by integrating the different analytic methods, we selected 22 proteins as potential candidates for a blood-based prognostic signature of response to crizotinib in NSCLC patients harboring ALK fusion. CONCLUSION: In conjunction with ALK mutation, the expression of this proteomic signature may represent a liquid biopsy-based marker of long-term response to crizotinib in NSCLC. Expanding the utility of prognostic biomarkers of response duration could influence choice of therapy, therapeutic sequencing, and potentially the need for alternative or combination therapy.Trial registration ClinicalTrials.gov, NCT02041468. Registered 22 January 2014, https://clinicaltrials.gov/ct2/show/NCT02041468?term=NCT02041468&rank=1.

Proteogenomics of Colorectal Cancer Liver Metastases: Complementing Precision Oncology with Phenotypic Data.

: Hotspot testing for activating KRAS mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for KRASwildtype tumors anti-EGFR response rates are <30%, while mutated-KRAS does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of KRASwildtype and KRASG12V CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRASG12V. For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47-1.01 fmol/µg total protein) and healthy tissues (0.13-0.64 fmol/µg). In KRASG12V-mCRC, G12V-mutation levels were 42-100%, while one patient had only 10% KRASG12V but 90% KRASwildtype. This might represent a missed therapeutic opportunity: based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumor-phenotyping indicates the patient might have benefitted from anti-EGFR therapy.

Analysis of the Genomic Landscape in ALK+ NSCLC Patients Identifies Novel Aberrations Associated with Clinical Outcomes.

Rearrangements in the anaplastic lymphoma kinase (ALK) gene are found in approximately 5% of non-small cell lung carcinoma (NSCLC). Here, we present a comprehensive genomic landscape of 11 patients with ALK+ NSCLC and investigate its relationship with response to crizotinib. Using whole-exome sequencing and RNAseq data, we identified four rare ALK fusion partners (HIP1, GCC2, ERC1, and SLC16A7) and one novel partner (CEP55). At the mutation level, TP53 was the most frequently mutated gene and was only observed in patients with the shortest progression-free survival (PFS). Of note, only 4% of the genes carrying mutations are present in more than 1 patient. Analysis of somatic copy number aberrations (SCNA) demonstrated that a gain in EML4 was associated with longer PFS, and a loss of ALK or gain in EGFR was associated with shorter PFS. This study is the first to report a comprehensive view of the ALK+ NSCLC copy number landscape and to identify SCNA regions associated with clinical outcome. Our data show the presence of TP53 mutation as a strong prognostic indication of poor clinical response in ALK+ NSCLC. Furthermore, new and rare ALK fusion partners were observed in this cohort, expanding our knowledge in ALK+ NSCLC.