Mutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of ...universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC).
Genome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (
,
,
,
). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy.
Methylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for
, 68.5% for
, 69.7% for
, 69.1% for
and 65.1% for
. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival.
This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.
We sought to develop criteria for ERBB2-positivity (HER2) in colorectal cancer to ensure accurate identification of ERBB2-amplified metastatic colorectal cancer patients suitable for enrollment in a ...phase II trial of ERBB2-targeted therapy (HERACLES trial). A two-step approach was used. In step 1, a consensus panel of pathologists adapted existing protocols for use in colorectal cancer to test ERBB2 expression and amplification. Collegial revision of an archival test cohort of colorectal cancer samples led to specific recommendations for adapting current breast and gastric cancer criteria for scoring ERBB2 in colorectal cancer. In step 2, from September 2012 to January 2015, colorectal-specific ERBB2 testing protocols and ERBB2 scoring criteria were used to centrally screen for ERBB2-positive KRAS wild-type colorectal cancer patients to be enrolled in the HERACLES trial (clinical validation cohort). In both archival test (N=256) and clinical validation (N=830) cohorts, a clinically sizeable 5% fraction of KRAS wild-type colorectal cancer patients was found to be ERBB2-positive according to the colorectal cancer-specific ERBB2 scoring criteria. ERBB2-positive tumors showed ERBB2 immunostaining consisting of intense membranous ERBB2 protein expression, corresponding to homogenous ERBB2 amplification, in >50% of cells. None of the immunohistochemistry 0 or 1+ cases was amplified. Concordance between SISH and FISH was 100%. In conclusion, we propose specific criteria for defining ERBB2-positivity in colorectal cancer (HERACLES Diagnostic Criteria). In a phase II trial of trastuzumab and lapatinib in a cetuximab-resistant population, HERACLES Diagnostic Criteria shaped the selection of patients and defined ERBB2 as a predictive marker for response to ERBB2-targeted therapy in metastatic colorectal cancer.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether ...tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patient's progressing liver metastasis following prolonged response to cetuximab revealed a MEK1(K57T) mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS(Q61H) mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient.
Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.
Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during ...treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.
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•ERBB2, RAS, PIK3CA mutations are associated with resistance to HER2 blockade in mCRC•A liquid biopsy test would have identified >85% primary resistance cases•Lesion size and contribution to plasma ctDNA were correlated•Patterns of lesion-specific mutations and TCR were longitudinally compared in blood
Siravegna et al. identify genetic events associated with ERBB2 amplified metastatic colorectal cancers resistant to trastuzumab plus lapatinib treatment and reveal lesion-private evolutionary patterns. Analyses of metastases from a patient unveil metastasis-specific evolution and pharmacologic vulnerabilities.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples ...representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.
The prognostic value of Toll-like receptor 3 (TLR3) is debated in cancer, differing between tumor types, methods, and cell types. We recently showed for the first time that TLR3 expression on early ...stage non-small-cell lung cancer (NSCLC) results associated with a good prognosis. Here, we provide experimental evidences explaining the molecular reason behind TLR3's favorable prognostic role. We demonstrated that TLR3 activation in vitro induces apoptosis in lung cancer cell lines and, accordingly, that TLR3 expression is associated with caspase-3 activation in adenocarcinoma NSCLC specimens, both evaluated by immunohistochemistry. Moreover, we showed that TLR3 expression on cancer cells contributes to activate the CD103+ lung dendritic cell subset, that is specifically associated with processing of antigens derived from apoptotic cells and their presentation to CD8+ T lymphocytes. These findings point to the relevant role of TLR3 expression on lung cancer cells and support the use of TLR3 agonists in NSCLC patients to re-activate local innate immune response.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Micro-Abstract Treatment of metastatic colorectal cancer includes resection of liver metastases, however, no biomarkers drive selection of patients. We performed a meta-analysis including 1833 ...patients treated with complete liver resection, showing that Kirsten rat sarcoma viral oncogene homolog and b-viral oncogene homolog B1 mutations are negatively associated with survival. This supports integration of mutational status into a combined score to better identify candidates for resection of colorectal cancer liver metastases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Molecular targeted drugs are clinically effective anti-cancer therapies. However, tumours treated with single agents usually develop resistance. Here we use colorectal cancer (CRC) as a model to ...study how the acquisition of resistance to EGFR-targeted therapies can be restrained. Pathway-oriented genetic screens reveal that CRC cells escape from EGFR blockade by downstream activation of RAS-MEK signalling. Following treatment of CRC cells with anti-EGFR, anti-MEK or the combination of the two drugs, we find that EGFR blockade alone triggers acquired resistance in weeks, while combinatorial treatment does not induce resistance. In patient-derived xenografts, EGFR-MEK combination prevents the development of resistance. We employ mathematical modelling to provide a quantitative understanding of the dynamics of response and resistance to these single and combination therapies. Mechanistically, we find that the EGFR-MEK Combo blockade triggers Bcl-2 and Mcl-1 downregulation and initiates apoptosis. These results provide the rationale for clinical trials aimed at preventing rather than intercepting resistance.
Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin ...directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of
18
F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the "in vivo" relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
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