Transcriptome-wide gene expression outlier analysis pinpoints therapeutic vulnerabilities in colorectal cancer.

Multiple strategies are continuously being explored to expand the drug target repertoire in solid tumors. We devised a novel computational workflow for transcriptome-wide gene expression outlier analysis that allows the systematic identification of both overexpression and underexpression events in cancer cells. Here, it was applied to expression values obtained through RNA sequencing in 226 colorectal cancer (CRC) cell lines that were also characterized by whole-exome sequencing and microarray-based DNA methylation profiling. We found cell models displaying an abnormally high or low expression level for 3533 and 965 genes, respectively. Gene expression abnormalities that have been previously associated with clinically relevant features of CRC cell lines were confirmed. Moreover, by integrating multi-omics data, we identified both genetic and epigenetic alternations underlying outlier expression values. Importantly, our atlas of CRC gene expression outliers can guide the discovery of novel drug targets and biomarkers. As a proof of concept, we found that CRC cell lines lacking expression of the MTAP gene are sensitive to treatment with a PRMT5-MTA inhibitor (MRTX1719). Finally, other tumor types may also benefit from this approach.

Tolerance to colibactin correlates with homologous recombination proficiency and resistance to irinotecan in colorectal cancer cells.

The bacterial genotoxin colibactin promotes colorectal cancer (CRC) tumorigenesis, but systematic assessment of its impact on DNA repair is lacking, and its effect on response to DNA-damaging chemotherapeutics is unknown. We find that CRC cell lines display differential response to colibactin on the basis of homologous recombination (HR) proficiency. Sensitivity to colibactin is induced by inhibition of ATM, which regulates DNA double-strand break repair, and blunted by HR reconstitution. Conversely, CRC cells chronically infected with colibactin develop a tolerant phenotype characterized by restored HR activity. Notably, sensitivity to colibactin correlates with response to irinotecan active metabolite SN38, in both cell lines and patient-derived organoids. Moreover, CRC cells that acquire colibactin tolerance develop cross-resistance to SN38, and a trend toward poorer response to irinotecan is observed in a retrospective cohort of CRCs harboring colibactin genomic island. Our results shed insight into colibactin activity and provide translational evidence on its chemoresistance-promoting role in CRC.

Correction: Ciardiello et al. Biomarker-Guided Anti-EGFR Rechallenge Therapy in Metastatic Colorectal Cancer. Cancers 2021, 13, 1941.

In the original publication [...].

Clinical Utility of Liquid Biopsy to Detect BRAF and NRAS Mutations in Stage III/IV Melanoma Patients by Using Real-Time PCR.

BACKGROUND: Liquid biopsy is a potentially useful tool for melanoma patients, also for detecting BRAS/NRAS mutations, even if the tissue analysis remains the current standard. METHODS: In this work, we tested ctDNA on plasma samples from 56 BRAF-V600/NRAS mutant stage III/IV melanoma patients using a real-time quantitative PCR (qPCR)-based platform. The study population was divided into two cohorts: the first including 26 patients who had undergone radical resection (resected cohort) and the second including 30 patients who had unresected measurable disease (advanced cohort). Moreover, for 10 patients in the advanced cohort, ctDNA assessment was repeated at specified timepoints after baseline testing. Data were analyzed and correlated to the clinicopathologic characteristics and outcomes. RESULTS: In the baseline cohort, a higher tissue-plasma concordance was seen in patients with high burden of disease (sum of diameters ≥30 mm, ≥2 metastatic sites, elevated LDH levels); furthermore, monitoring of these patients through ctDNA analysis was informative for therapeutic responses. On the other hand, the low sensitivity of this technique did not allow for clinically valuable prediction of relapses in radically resected stage III/IV patients. CONCLUSIONS: Overall, our data suggest that qPCR-based ctDNA analysis could be informative in a subset of locally advanced and metastatic melanoma patients with specific clinical-radiological characteristics, supporting further investigations in this setting.

Temozolomide Treatment Alters Mismatch Repair and Boosts Mutational Burden in Tumor and Blood of Colorectal Cancer Patients.

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, whereas MMR-deficient (MMRd) tumors often respond to immune-checkpoint blockade. We previously reported that the treatment of colorectal cancer preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB), and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-methylguanine-DNA-methyltransferase (MGMT)-deficient, MMR-proficient, RAS-mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes, and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A subset of patients whose tumors displayed the MSH6 mutation, the TMZ mutational signature, and increased TMB achieved disease stabilization upon pembrolizumab treatment. SIGNIFICANCE: MMR-proficient mCRCs are unresponsive to immunotherapy. We provide the proof of concept that inactivation of MMR genes can be achieved pharmacologically with TMZ and molecularly monitored in the tissue and blood of patients with mCRC. This strategy deserves additional evaluation in mCRC patients whose tumors are no longer responsive to standard-of-care treatments. See related commentary by Willis and Overman, p. 1612. This article is highlighted in the In This Issue feature, p. 1599.

Liquid biopsies to monitor and direct cancer treatment in colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent and deadly cancers worldwide. Despite recent improvements in treatment and prevention, most of the current therapeutic options are weighted by side effects impacting patients' quality of life. Better patient selection towards systemic treatments represents an unmet clinical need. The recent multidisciplinary and molecular advancements in the treatment of CRC patients demand the identification of efficient biomarkers allowing to personalise patient care. Currently, core tumour biopsy specimens represent the gold-standard biological tissue to identify such biomarkers. However, technical feasibility, tumour heterogeneity and cancer evolution are major limitations of this single-snapshot approach. Genotyping circulating tumour DNA (ctDNA) has been addressed as potentially overcoming such limitations. Indeed, ctDNA has been retrospectively demonstrated capable of identifying minimal residual disease post-surgery and post-adjuvant treatment, as well as spotting druggable molecular alterations for tailoring treatments in metastatic disease. In this review, we summarise the available evidence on ctDNA applicability in CRC. Then, we review ongoing clinical trials assessing how liquid biopsy can be used interventionally to guide therapeutic choice in localised, locally advanced and metastatic CRC. Finally, we discuss how its widespread could transform CRC patients' management, dissecting its limitations while suggesting improvement strategies.

Cetuximab Rechallenge Plus Avelumab in Pretreated Patients With RAS Wild-type Metastatic Colorectal Cancer: The Phase 2 Single-Arm Clinical CAVE Trial.

IMPORTANCE: Rechallenge therapy with anti-epidermal growth factor receptor (EGFR) drugs has been suggested in patients with chemo-refractory RAS wild-type (WT) metastatic colorectal cancer (mCRC) after initial response to anti-EGFR-based first-line treatment. The association of treatment with cetuximab plus avelumab with overall survival (OS) may be worthy of investigation in this setting. OBJECTIVE: To assess the efficacy and safety of cetuximab rechallenge therapy plus avelumab. DESIGN, SETTING, AND PARTICIPANTS: This single-arm, multicenter phase 2 trial enrolled patients from August 2018 to February 2020. Eligible patients with RAS WT mCRC had a complete or partial response to first-line chemotherapy plus anti-EGFR drugs, developed acquired resistance, and failed second-line therapy. Baseline circulating tumor DNA (ctDNA) for KRAS, NRAS, BRAF, and EGFR-S492R mutation analysis was done. INTERVENTIONS: Patients received avelumab (10 mg/kg every 2 weeks) and cetuximab (400 mg/m2 and, subsequently, 250 mg/m2 weekly) until disease progression or unacceptable toxic effects. MAIN OUTCOMES AND MEASURES: The primary end point was OS. Secondary end points were progression-free survival (PFS), overall response rate (ORR), and safety. RESULTS: Seventy-seven patients were enrolled (42 men, 35 women; median age, 63 years); 71 had microsatellite stable tumors (MSS), 3 microsatellite instability-high tumors (MSI-H), 3 unknown. The study met the primary end point, with median OS (mOS) of 11.6 months (95% CI, 8.4-14.8 months). Median PFS (mPFS) was 3.6 months (95% CI, 3.2-4.1 months). Common grade-3 adverse events were cutaneous eruption, 11 (14%), and diarrhea, 3 (4%). For 67 of 77 (87%) patients, baseline analysis of plasma circulating tumor DNA (ctDNA) for KRAS, NRAS, BRAF, and EGFR-S492R variations was feasible. Forty-eight patients had WT disease, whereas 19 had mutations. Patients with RAS/BRAF WT ctDNA had mOS of 17.3 months (95% CI, 12.5-22.0 months) compared with 10.4 months (95% CI, 7.2-13.6 months) in patients with mutated ctDNA (hazard ratio [HR], 0.49; 95% CI, 0.27-0.90; P = .02). The mPFS was 4.1 months (95% CI, 2.9-5.2 months) in RAS/BRAF WT patients compared with 3.0 months (95% CI, 2.6-3.5 months) in patients with mutated ctDNA (HR, 0.42; 95% CI, 0.23-0.75; P = .004). CONCLUSIONS AND RELEVANCE: The findings of this single-arm phase 2 trial suggest that cetuximab plus avelumab is an active, well tolerated rechallenge therapy in RAS WT mCRC. Plasma ctDNA analysis before treatment may allow selection of patients who could benefit. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04561336.

Biomarker-Guided Anti-Egfr Rechallenge Therapy in Metastatic Colorectal Cancer.

The prognosis of patients with metastatic colorectal cancer (mCRC) who progressed to the first and the second lines of treatment is poor. Thus, new therapeutic strategies are needed. During the last years, emerging evidence suggests that retreatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MAbs) in the third line of mCRC patients, that have previously obtained clinical benefit by first-line therapy with anti-EGFR MAbs plus chemotherapy, could lead to prolonged survival. The rationale beyond this "rechallenge" strategy is that, after disease progression to first line EGFR-based therapy, a treatment break from anti-EGFR drugs results in RAS mutant cancer cell decay, restoring the sensitivity of cancer cells to cetuximab and panitumumab. In fact, rechallenge treatment with anti-EGFR drugs has shown promising clinical activity, particularly in patients with plasma RAS and BRAF wild type circulating tumor DNA, as defined by liquid biopsy analysis at baseline treatment. The aim of this review is to analyze the current knowledge on rechallenge and to investigate the role of novel biomarkers that can guide the appropriate selection of patients that could benefit from this therapeutic strategy. Finally, we discuss on-going trials and future perspectives.

Precision oncology in metastatic colorectal cancer - from biology to medicine.

Remarkable progress has been made in the development of biomarker-driven targeted therapies for patients with multiple cancer types, including melanoma, breast and lung tumours, although precision oncology for patients with colorectal cancer (CRC) continues to lag behind. Nonetheless, the availability of patient-derived CRC models coupled with in vitro and in vivo pharmacological and functional analyses over the past decade has finally led to advances in the field. Gene-specific alterations are not the only determinants that can successfully direct the use of targeted therapy. Indeed, successful inhibition of BRAF or KRAS in metastatic CRCs driven by activating mutations in these genes requires combinations of drugs that inhibit the mutant protein while at the same time restraining adaptive resistance via CRC-specific EGFR-mediated feedback loops. The emerging paradigm is, therefore, that the intrinsic biology of CRC cells must be considered alongside the molecular profiles of individual tumours in order to successfully personalize treatment. In this Review, we outline how preclinical studies based on patient-derived models have informed the design of practice-changing clinical trials. The integration of these experiences into a common framework will reshape the future design of biology-informed clinical trials in this field.

Dual inhibition of TGFß and AXL as a novel therapy for human colorectal adenocarcinoma with mesenchymal phenotype.

A subset of colorectal cancer (CRC) with a mesenchymal phenotype (CMS4) displays an aggressive disease, with an increased risk of recurrence after surgery, reduced survival, and resistance to standard treatments. It has been shown that the AXL and TGFß signaling pathways are involved in epithelial-to-mesenchymal transition, migration, metastatic spread, and unresponsiveness to targeted therapies. However, the prognostic role of the combination of these biomarkers and the anti-tumor effect of AXL and TGFß inhibition in CRC still has to be assessed. To evaluate the role of AXL and TGFß as negative biomarker in CRC, we conducted an in-depth in silico analysis of CRC samples derived from the Gene Expression Omnibus. We found that AXL and TGFß receptors are upregulated in CMS4 tumors and are correlated with an increased risk of recurrence after surgery in stage II/III CRC and a reduced overall survival. Moreover, we showed that AXL receptor is differently expressed in human CRC cell lines. Dual treatment with the TGFß galunisertib and the AXL inhibitor, bemcentinib, significantly reduced colony formation and migration capabilities of tumor cells and displayed a strong anti-tumor activity in 3D spheroid cultures derived from patients with advanced CRC. Our work shows that AXL and TGFß receptors identify a subgroup of CRC with a mesenchymal phenotype and correlate with poor prognosis. Dual inhibition of AXL and TGFß could represent a novel therapeutic strategy for patients with this aggressive disease.

Vulnerability to low-dose combination of irinotecan and niraparib in ATM-mutated colorectal cancer.

BACKGROUND: Despite the advancements in new therapies for colorectal cancer (CRC), chemotherapy still constitutes the mainstay of the medical treatment. For this reason, new strategies to increase the efficacy of chemotherapy are desirable. Poly-ADP-Ribose Polymerase inhibitors (PARPi) have shown to increase the activity of DNA damaging chemotherapeutics used in the treatment of CRC, however previous clinical trials failed to validate these results and pointed out dose-limiting toxicities that hamper the use of such combinations in unselected CRC patients. Nevertheless, in these studies little attention was paid to the mutational status of homologous recombination repair (HRR) genes. METHODS: We tested the combination of the PARPi niraparib with either 5-fluorouracil, oxaliplatin or irinotecan (SN38) in a panel of 12 molecularly annotated CRC cell lines, encompassing the 4 consensus molecular subtypes (CMSs). Synergism was calculated using the Chou-Talalay method for drug interaction. A correlation between synergism and genetic alterations in genes involved in homologous recombination (HR) repair was performed. We used clonogenic assays, mice xenograft models and patient-derived 3D spheroids to validate the results. The induction of DNA damage was studied by immunofluorescence. RESULTS: We showed that human CRC cell lines, as well as patient-derived 3D spheroids, harboring pathogenic ATM mutations are significantly vulnerable to PARPi/chemotherapy combination at low doses, regardless of consensus molecular subtypes (CMS) and microsatellite status. The strongest synergism was shown for the combination of niraparib with irinotecan, and the presence of ATM mutations was associated to a delay in the resolution of double strand breaks (DSBs) through HRR and DNA damage persistence. CONCLUSIONS: This work demonstrates that a numerically relevant subset of CRCs carrying heterozygous ATM mutations may benefit from the combination treatment with low doses of niraparib and irinotecan, suggesting a new potential approach in the treatment of ATM-mutated CRC, that deserves to be prospectively validated in clinical trials.

Resistance to anti-epidermal growth factor receptor in metastatic colorectal cancer: What does still need to be addressed?

Colorectal cancer (CRC) represents a global health problem, being one of the most diagnosed and aggressive tumors. Cetuximab and panitumumab monoclonal antibodies (mAbs) in combination with chemotherapy are an effective strategy for patients with RAS Wild Type (WT) metastatic colorectal cancer (mCRC). However, tumors are often unresponsive or develop resistance. In the last years, molecular alterations in principal oncogenes (RAS, BRAF, PI3KCA, PTEN) in the downstream pathway of the epidermal growth factor receptor (EGFR) and in other receptors (HER2, MET) that converge on MAPK-ERK signalling have been identified as novel mechanisms of resistance to anti-EGFR strategies. However, further efforts are needed to better stratify CRCs and ensure more individualized treatments. Herein, we describe the consolidated molecular drivers of resistance and the therapeutic strategies available so far, with an overview on potential biomarkers of response that could be integrated in clinical practice.

Strategic Combinations to Prevent and Overcome Resistance to Targeted Therapies in Oncology.

Recent advances in the understanding of underlying molecular signaling mechanisms of cancer susceptibility and progression have led to an increase in the use of targeted therapies for cancer treatment. Despite improvements in survival with new treatment options in oncology, resistance to therapy is a major obstacle to the long-term effectiveness of targeted agents in metastatic cancer treatment, culminating in insensitivity to treatment and tumor outgrowth. Adaptive resistance can play an important role in primary and upfront resistance to therapy as well as in secondary or acquired resistance. By focusing on colorectal and breast tumors, we discuss how therapeutic combinations based on specific drivers of tumor biology can be used to overcome resistance. We present how monitoring tumor dynamics over time may allow early adaptation of treatment. Breast cancer is the most common malignancy in women worldwide, and the majority of these cancers are sensitive to endocrine therapy (ET) blocking the production of or response to estrogen. However, primary and acquired resistance limits efficacy. Recent combinations of agents targeted to pathways that drive tumor growth resistance with ET have resulted in remarkable improvements in disease response and control, improving survival in some settings. In this review, we summarize adaptive resistance mechanisms, approaches to combination strategies, and dynamic tumor monitoring to improve efficacy and overcome resistance. We provide examples of combination therapy to enhance the efficacy of targeted therapies in breast and colorectal tumors.

Clinical Practice Use of Liquid Biopsy to Identify RAS/BRAF Mutations in Patients with Metastatic Colorectal Cancer (mCRC): A Single Institution Experience.

Tumor heterogeneity represents a possible cause of error in detecting predictive genetic alterations on tumor tissue and can be overcome by testing alterations in circulating tumor DNA (ctDNA) using liquid biopsy. We assessed 72 consecutive patients with a diagnosis of metastatic colorectal cancer (mCRC) using Idylla™ Biocartis, a fully automated platform that evaluates the most frequent mutations of KRAS, NRAS and BRAF genes. We correlated the results of liquid biopsy and standard tissue-based next generation sequencing (NGS) analyses to patient clinical features. The overall agreement was 81.94%. Concordance was 85.71% and 96.15% in treatment-naïve patients and in the patient subgroup with liver metastases, respectively. In liver metastases positive, treatment-naïve patients, sensitivity, specificity and positive predictive value (PPV) were 92.31%, 100% and 100%, respectively. Circulating mutational fraction (CMF) was significantly higher in patients with liver metastases and high carcinoembryonic antigen (CEA) levels. In a subgroup of patients pre-treated with anti-Epidermal Growth Factor Receptor (EGFR) agents, emerging KRAS mutations were evidenced in 33% of cases. Testing RAS/BRAF mutations on plasma using the Idylla™ Biocartis platform is feasible and reliable in mCRC patients in clinical practice.

A case report of a severe fluoropyrimidine-related toxicity due to an uncommon DPYD variant.

INTRODUCTION: Fluoropyrimidines such as 5-fluorouracil (5-FU) and its orally active prodrug, capecitabine, are widely used in the treatment of gastrointestinal cancer, including colorectal cancer. Dihydropyrimidine dehydrogenase (DPD) plays an important role in the 5-FU metabolism. Dihydropyrimidine dehydrogenase gene (DPYD) is a highly polymorphic gene with several hundreds of reported genetic variants and DPD activity levels vary considerably among individuals, with different 5-FU-related efficacy and toxicity. About 5% of the population is deficient in DPD enzyme activity. The most well studied DPYD variant is the IVS14+1G>A, also known as DPYD *2A. In this report, we present a case of a patient with a double heterozygote DPYD variant (DPYD activity score: 0,5 according to Clinical Pharmacogenetics Implementation Consortium) who experienced a severe fluoropyrimidine-related toxicity resolved without any consequence. PATIENT CONCERNS: A 46-years-old Caucasian man with diagnosis of left colon adenocarcinoma underwent left hemicolectomy on July 2017: pT3 G3 N1c M0. According to the disease stage, he started an adjuvant therapy with XELOX using capecitabine at 50% of total dose, because of his DPYD IVS14+1G>A variant, detected before the treatment. DIAGNOSIS: After few days, despite of this dose reduction, he experienced life-threatening adverse events such as mucositis G3, diarrhea G3, neutropenia G4, thrombocytopenia G4, and hyperbilirubinemia G3 according to Common Terminology Criteria for Adverse Events v 5.0. INTERVENTIONS: As first, we set up an intensive rehydration therapy, antibiotic and antifungal prophylaxis, Granulocyte-Colony Stimulating Factors, and supportive blood transfusions. Additional genetic tests revealed a double heterozygote variant of DPYD gene (DPYD IVS14+1G>A and 2846A>T) which is a very rare situation and only 3 cases are described in literature, all of them concluded with patient's death. OUTCOMES: After 3 weeks of intensive therapy, the patient was fully recovered. Furthermore, all the whole-body CT scans performed since discharge from the hospital until now, have confirmed no evidence of disease. CONCLUSIONS: Recent studies demonstrated that screening strategy for the most common DPYD variants allowed for avoiding toxicities and saving money. This report underlines the importance of genotyping DPYD before treatment and emphasizes the role of genotype-guided dose individualization.

Immunotherapy of colorectal cancer: Challenges for therapeutic efficacy.

A better knowledge of the complex interactions between cancer cells and the immune system has led to novel immunotherapy approaches. Treatment with selective anti-PD1, anti-PD-L1 and/or anti-CTLA-4 monoclonal antibodies (mAbs) has been a revolution in the therapeutic scenario of several cancer types, with the highest clinical efficacy in melanoma and in lung cancer. Colorectal cancer is one of the tumours in which immunotherapy has been shown less effective. Whereas in deficient mismatch repair (MMR) or in highly microsatellite instable (MSI-H) metastatic colorectal cancer there is clear clinical evidence for a therapeutic role of immune checkpoint inhibitors, the vast majority of patients with proficient MMR or with microsatellite stable (MSS) tumours do not benefit from immunotherapy. Defining the molecular mechanisms for immunogenicity in metastatic colorectal cancer is needed in order to develop predictive biomarkers and effective therapeutic combination strategies. A major challenge will be to identify, among the heterogeneous spectrum of this disease, those patients with specific tumour and tumour infiltrating stroma molecular and functional characteristics, that could be effectively treated with immunotherapy. In this review, we discuss the role of immune response in the context of metastatic colorectal cancer. We summarize the available clinical data with the use of anti PD-1/PD-L1 mAbs as single agents or in combination with anti CTLA-4 mAbs in MSI-H patients. Finally, we address the challenges and the potential strategies for rendering the more frequent microsatellite stable (MSS) tumours "immune-competent" and, therefore, amenable for effective immunotherapy interventions.

EPHA2 Is a Predictive Biomarker of Resistance and a Potential Therapeutic Target for Improving Antiepidermal Growth Factor Receptor Therapy in Colorectal Cancer.

The EPHA2 tyrosine kinase receptor is implicated in tumor progression and targeted therapies resistance. We evaluated EPHA2 as a potential resistance marker to the antiepidermal growth factor receptor (EGFR) monoclonal antibody cetuximab in colorectal cancer. We studied activation of EPHA2 in a panel of human colorectal cancer cell lines sensitive or resistant to anti-EGFR drugs. The in vitro and in vivo effects of ALW-II-41-27 (an EPHA2 inhibitor) and/or cetuximab treatment were tested. Formalin-fixed paraffin-embedded tumor specimens from 82 RAS wild-type (WT) metastatic colorectal cancer patients treated with FOLFIRI + cetuximab as first-line therapy in the CAPRI-GOIM trial were assessed for EPHA2 expression by immunohistochemistry and correlated with treatment efficacy. EPHA2 was differentially activated in colorectal cancer cell lines. Combined treatment with ALW-II-41-27 plus cetuximab reverted primary and acquired resistance to cetuximab, causing cell growth inhibition, inducing apoptosis and cell-cycle G1-G2 arrest. In tumor xenograft models, upon progression to cetuximab, ALW-II-41-27 addition significantly inhibited tumor growth. EPHA2 protein expression was detected in 55 of 82 tumor samples, frequently expressed in less-differentiated and left-sided tumors. High levels of EPHA2 significantly correlated with worse progression-free survival [8.6 months; confidence interval (CI) 95%, 6.4-10.8; vs. 12.3 months; CI 95%, 10.4-14.2; P = 0.03] and with increased progression rate (29% vs. 9%, P = 0.02). A specific EPHA2 inhibitor reverts in vitro and in vivo primary and acquired resistance to anti-EGFR therapy. EPHA2 levels are significantly associated with worse outcome in patients treated with FOLFIRI + cetuximab. These results highlight EPHA2 as a potential therapeutic target in metastatic colorectal cancer.

Receptor tyrosine kinase-dependent PI3K activation is an escape mechanism to vertical suppression of the EGFR/RAS/MAPK pathway in KRAS-mutated human colorectal cancer cell lines.

BACKGROUND: Previous studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEK-inhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. However, rapid onset of resistance is a limit to combination therapies in KRAS mutated CRC. METHODS: We generated four different KRAS mutated CRC cell lines resistant to a combination of cetuximab (an anti-EGFR antibody) and refametinib (a selective MEK-inhibitor) after continuous exposure to increasing concentration of the drugs. We characterized these resistant cell lines by evaluating the expression and activation status of a panel of receptor tyrosine kinases (RTKs) and intracellular transducers by immunoblot and qRT-PCR. Oncomine comprehensive assay and microarray analysis were carried out to investigate new acquired mutations or transcriptomic adaptation, respectively, in the resistant cell lines. Immunofluorescence assay was used to show the localization of RTKs in resistant and parental clones. RESULTS: We found that PI3K-AKT pathway activation acts as an escape mechanism in cell lines with acquired resistance to combined inhibition of EGFR and MEK. AKT pathway activation is coupled to the activation of multiple RTKs such as HER2, HER3 and IGF1R, though its pharmacological inhibition is not sufficient to revert the resistant phenotype. PI3K pathway activation is mediated by autocrine loops and by heterodimerization of multiple receptors. CONCLUSIONS: PI3K activation plays a central role in the acquired resistance to the combination of anti-EGFR and MEK-inhibitor in KRAS mutated colorectal cancer cell lines. PI3K activation is cooperatively achieved through the activation of multiple RTKs such as HER2, HER3 and IGF1R.

Sequential HER2 blockade as effective therapy in chemorefractory, HER2 gene-amplified, RAS wild-type, metastatic colorectal cancer: learning from a clinical case.

BACKGROUND: Constitutive activation of HER2-dependent intracellular signalling by HER2 gene amplification or by HER2 mutations has been demonstrated as a mechanism of primary and secondary cancer resistance to cetuximab or panitumumab in preclinical and clinical models of metastatic colorectal cancer (mCRC). Both HER2 Amplification for Colorectal Cancer Enhanced Stratification (HERACLES) cohort A and My Pathway clinical trials provided clinical evidence that anti-HER2 therapies could be active in these patients. PATIENT AND METHODS: HER2 gene amplification and HER2 protein overexpression analysis were performed in tumour tissue by fluorescence in situ hybridisation and immunohistochemistry. HER2 positivity was defined according to HERACLES CRC-specific HER2 scoring criteria. DNA analysis for multiple assessment of gene mutations or amplifications was carried out with the next-generation sequencing (NGS) Ion AmpliSeq Colon and Lung Cancer Panel and by using a more extensive targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay). RESULTS: We report the clinical case of a patient with HER2 gene amplified and RAS/BRAF wild-type mCRC who experienced a long lasting and relevant clinical efficacy from sequential anti-HER2 therapies (trastuzumab plus lapatinib, pertuzumab plus trastuzumab, trastuzumab emtansine, trastuzumab plus capecitabine) achieving a cumulative clinical benefit of 29 months, after failure of the first three lines of standard treatments, which included all the potentially active drugs in mCRC, and which accounted for only 14 months of disease control. HER gene amplification was confirmed by NGS on two different metastatic lesions during the evolution of the disease. CONCLUSION: The clinical case highlights the role of HER2 gene amplification as a key genetic driver of cancer development and progression in mCRC and suggests that sequential HER2 blockade could be a potential therapeutic strategy.