Clinicopathological characterisation of MTAP alterations in gastrointestinal cancers.

BACKGROUND: Methylthioadenosine phosphorylase (MTAP) is an essential metabolic enzyme in the purine and methionine salvage pathway. In cancer, MTAP gene copy number loss (MTAP loss) confers a selective dependency on the related protein arginine methyltransferase 5. The impact of MTAP alterations in gastrointestinal (GI) cancers remains unknown although hypothetically druggable. Here, we aim to investigate the prevalence, clinicopathological features and prognosis of MTAP loss GI cancers. METHODS: Cases with MTAP alterations were retrieved from The Cancer Genome Atlas (TCGA) and a real-world cohort of GI cancers profiled by next-generation sequencing. If MTAP alterations other than loss were found, immunohistochemistry was performed. Finally, we set a case-control study to assess MTAP loss prognostic impact. RESULTS: Findings across the TCGA dataset (N=1363 patients) and our cohort (N=508) were consistent. Gene loss was the most common MTAP alteration (9.4%), mostly co-occurring with CDKN2A/B loss (97.7%). Biliopancreatic and gastro-oesophageal cancers had the highest prevalence of MTAP loss (20.5% and 12.7%, respectively), being mostly microsatellite stable (99.2%). In colorectal cancer, MTAP loss was rare (1.1%), while most MTAP alterations were mutations (5/7, 71.4%); among the latter, only MTAP-CDKN2B truncation led to protein loss, thus potentially actionable. MTAP loss did not confer worse prognosis. CONCLUSIONS: MTAP alterations are found in 5%-10% of GI cancers, most frequently biliopancreatic and gastro-oesophageal. MTAP loss is the most common alteration, identified almost exclusively in MSS, CDKN2A/B loss, upper-GI cancers. Other MTAP alterations were found in colorectal cancer, but unlikely to cause protein loss and drug susceptibility.

Repotrectinib Overcomes F2004V Resistance Mutation in ROS1-Rearranged NSCLC: A Case Report.

ROS1 tyrosine kinase inhibitors (TKIs) were found to provide a substantial clinical benefit for patients with advanced ROS1-positive (ROS1+) NSCLC. Nevertheless, TKI resistance inevitably develops with different mechanisms, preventing prolonged responses. For this reason, next-generation compounds are under clinical development. ROS1 F2004 substitutions have been previously detected on circulating tumor DNA of patients progressing to entrectinib. Hereby, we report the case of a patient with ROS1+ NSCLC in which F2004V-acquired mutation was detected on a site of disease progression, after entrectinib and crizotinib failure. A subsequent treatment with next-generation TKI repotrectinib led to disease response, providing the first clinical evidence of activity of repotrectinib against F2004V resistance mutation.

Long-term Clinical Outcome of Trastuzumab and Lapatinib for HER2-positive Metastatic Colorectal Cancer.

BACKGROUND: ERBB2 amplification occurs in 5% of RAS wild-type metastatic colorectal cancer (mCRC) and it has been shown to be a target for treatment with 2 HER2-directed combinations of trastuzumab and lapatinib or trastuzumab and pertuzumab. We present long-term clinical results of trastuzumab and lapatinib (HERACLES-A trial) at 6.7 years (82 months) follow-up and focus on central nervous system (CNS) recurrences. PATIENTS AND METHODS: Patients had histologically confirmed KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive mCRC. HER2 positivity was assessed by immunohistochemistry and in situ hybridization HERACLES diagnostic criteria. Patients were treated with intravenous trastuzumab 4 mg/kg loading dose, then 2 mg/kg once per week, and oral lapatinib 1000 mg per day until disease progression or toxicity. Patients who presented with symptoms or signs of CNS disease received brain computed tomography scan or magnetic resonance imaging. RESULTS: A total of 35 patients received trastuzumab and lapatinib and 32 were evaluable for response. One patient (3%) achieved complete response (CR), 8 (25%) partial response, and 13 (41%) stable disease. Therefore, response rate was 28%. Median progression-free survival was 4.7 months (95% confidence interval [CI] 3.7-6.1). Median overall survival was 10.0 months (95% CI 7.9-15.8). One patient achieved sustained CR still maintained at 7 years of follow-up. Progression in the central nervous system (CNS) occurred in 6 (19%) of 32 patients. CONCLUSIONS: Long-term (6.7 years) follow-up analysis of HERACLES-A supports using of trastuzumab and lapatinib as treatment reference for KRAS wild-type, chemorefractory HER2-positive mCRC. In this subset of patients, prolongation of survival is accompanied by CNS recurrences that will require diagnostic and therapeutic attention in future studies. Clinicaltrials. Gov identifier: NCT 03225937.

Entrectinib for the treatment of metastatic NSCLC: safety and efficacy.

Introduction: Gene fusions are strong driver alterations in various cancers, increasingly diagnosed with multiple testing techniques. ROS1 fusions can be found in 1-2% of non-small cell lung cancer (NSCLC) and several tyrosine kinase inhibitors (TKIs) have been tested in this oncogene-driven disease. NTRK fusions are characteristic of a few rare types of cancer, also infrequently seen in some common cancers including NSCLC. Entrectinib is a newer ROS1 and NTRK inhibitor developed across different tumor types harboring rearrangements in these genes. Entrectinib was granted FDA accelerated approval in August 2019 for the treatment of ROS1+ NSCLC and NTRK-driven solid tumors.Areas covered: This review covers the mechanism of action, safety, and efficacy of entrectinib in patients with metastatic NSCLC.Expert opinion: Entrectinib is an orally bioavailable TKI of TrkA, TrkB, TrkC, and ROS1, with the ability to cross the blood-brain barrier. Entrectinib was effective and well-tolerated in patients harboring ROS1- or NTRK-rearranged NSCLC treated within phase I and II studies. Entrectinib appears to be the most appropriate treatment choice for TKIs-naïve patients, especially in those presenting brain metastasis. Conversely, in case of systemic progression with the evidence of acquired resistance mutations in ROS1 or Trk proteins, a sequential therapy with entrectinib could not be successful.

HER2 Positivity Predicts Unresponsiveness to EGFR-Targeted Treatment in Metastatic Colorectal Cancer.

BACKGROUND: HER2 amplification is detected in 3% of patients with colorectal cancer (CRC), making tumors in the metastatic setting vulnerable to double pharmacological HER2 blockade. Preclinical findings show that it also might impair response to anti-epidermal growth factor receptor (EGFR) treatment. SUBJECTS AND METHODS: Patients with KRAS exon 2 wild-type metastatic CRC underwent molecular screening of HER2 positivity by HERACLES criteria (immunohistochemistry 3+ or 2+ in ≥50% of cells, confirmed by fluorescence in situ hybridization). A sample of consecutive HER2-negative patients was selected as control. A regression modeling strategy was applied to identify predictors explaining the bulk of HER2 positivity and the association with response to previous anti-EGFR treatment. RESULTS: From August 2012 to April 2018, a total of 100 HER2-positive metastatic CRC tumors were detected out of 1,485 KRAS exon 2 wild-type screened patients (6.7%). HER2-positive patients show more frequently lung metastases (odds ratio [OR], 2.04; 95% confidence interval [CI], 1.15-3.61; p = .014) and higher tumor burden (OR, 1.48; 95% CI, 1.10-2.01; p = .011), and tumors were more likely to be left sided (OR, 0.50; 95% CI, 0.22-1.11; p = .088). HER2-positive patients who received treatment with anti-EGFR agents (n = 79) showed poorer outcome (objective response rate, 31.2% vs. 46.9%, p = .031; progression-free survival, 5.7 months vs. 7 months, p = .087). CONCLUSION: Testing for HER2 should be offered to all patients with metastatic CRC because the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Patients with HER2-amplified metastatic CRC are less likely to respond to anti-EGFR therapy. IMPLICATIONS FOR PRACTICE: Patients with HER2-amplified/overexpressed metastatic colorectal cancer (mCRC) harbor a driver actionable molecular alteration that has been shown in preclinical models to hamper efficacy of the anti-epidermal growth factor receptor (EGFR) targeted therapies. The present study confirmed that this molecular feature was associated with worse objective tumor response and shorter progression-free survival in response to previous anti-EGFR therapies. Moreover, it was found that the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Therefore, HER2 status assessment should be included in the molecular diagnostic workup of all mCRC for speedy referral to clinical trials encompassing HER2-targeted double blockade independently of previous anti-EGFR treatment.

TRKA expression and NTRK1 gene copy number across solid tumours.

AIMS: Neurotrophic Tropomyosin Kinase Receptor 1 (NTRK1) gene encodes for the protein Tropomyosin-related kinase A (TRKA). Deregulated activity of TRKA has been shown to have oncogenic potential. We present here the results of an immunohistochemical (IHC) observational cohort study of TRKA expression together with gene copy number (GCN) assessment in various solid tumours. METHODS: Formalin-fixed, paraffin-embedded consecutive samples of different tumour types were tested for TRKA expression. Samples showing TRKA IHC staining in at least 10% of cells were analysed by fluorescence in situ hybridisation to assess NTRK1 gene rearrangements and/or individual GCN gain. All patients underwent this molecular assessment within the phase I ALKA-001 clinical trial. RESULTS: 1043 samples were tested and annotation for histology was available in 1023. Most of the samples were colorectal adenocarcinoma (CRC) (n=550, 52.7%) and lung adenocarcinoma (n=312, 29.9%). 24 samples (2.3%) were biliary tract carcinoma (BTC). Overall, 17 (1.6%) samples were characterised by TRKA IHC expression (four weak, eight moderate, five strong): 9/17 lung adenocarcinoma, 3/17 CRC, 3/17 BTC, 1/17 thyroid cancer and 1/17 cancer of unknown primary. Of these, 1/17 with strong TRKA IHC staining displayed NTRK1 gene rearrangement and 15/17 NTRK1 GCN gain by FISH. No correlation was found between intensity of TRKA IHC staining and number of copies of NTRK1. CONCLUSIONS: TRKA expression can be found in 1.6% of solid tumours and can be paralleled by NTRK1 gene rearrangements or mostly GCN gain. The prognostic and translational therapeutic impact of the latter remains to be established.

Pooled Analysis of Clinical Outcome of Patients with Chemorefractory Metastatic Colorectal Cancer Treated within Phase I/II Clinical Studies Based on Individual Biomarkers of Susceptibility: A Single-Institution Experience.

BACKGROUND: Patients with metastatic colorectal cancer (mCRC) refractory to standard therapies have a poor prognosis. In this setting, recruitment into clinical trials is warranted, and studies driven by selection according to individual tumor molecular characteristics are expected to provide added value. OBJECTIVE: We retrospectively analyzed data from patients with mCRC refractory to or following failure of standard therapies who were enrolled into phase I/II clinical studies at the Niguarda Cancer Center based on the presence of a specific molecular profile expected to represent the target of susceptibility to the experimental drug(s). PATIENTS AND METHODS: From June 2011 to May 2016, 2044 patients with mCRC underwent molecular screening. Eighty patients (3.9%) were enrolled in ad hoc studies; the median age was 60 years (range 36-86) and the median number of previous treatment lines was five (range 2-8). Molecular characteristics exploited within these studies were MGMT promoter hypermethylation (48.7%), HER2 amplification (28.8%), BRAF V600E mutation (20%), and novel gene fusions involving ALK or NTRK (2.5%). RESULTS: One patient (1%) had RECIST (Response Evaluation Criteria In Solid Tumors) complete response (CR), 13 patients (16.5%) experienced a partial response (PR), and 28 (35%) stable disease (SD). Median progression-free survival (PFS) was 2.8 months (range 2.63-3.83), with 24% of patients displaying PFS >5 months. Median growth modulation index (GMI) was 0.85 (range 0-15.61) and 32.5% of patients had GMI >1.33. KRAS exon 2 mutations were found in 38.5% of patients, and among the 78 patients with known KRAS status, those with wild-type tumors had longer PFS than those with mutated tumors (3.80 [95% CI 2.80-5.03] vs. 2.13 months [95% CI 1.77-2.87], respectively, p = 0.001). Median overall survival (OS) was 7.83 months (range 7.17-9.33) for all patients, and patients with KRAS wild-type tumors had longer OS than those with mutated tumors (7.83 [95% CI 7.33-10.80] vs. 7.18 months [95% CI 5.63-9.33], respectively, p = 0.06). CONCLUSIONS: This single-institution retrospective study indicates that in a heavily pretreated population approximately 4% of mCRC tumors display a potential actionable molecular context suitable for therapeutic intervention. Application of molecular selection is challenging but improves clinical outcome even in later lines of treatment.

Safety and Antitumor Activity of the Multitargeted Pan-TRK, ROS1, and ALK Inhibitor Entrectinib: Combined Results from Two Phase I Trials (ALKA-372-001 and STARTRK-1).

Entrectinib, a potent oral inhibitor of the tyrosine kinases TRKA/B/C, ROS1, and ALK, was evaluated in two phase I studies in patients with advanced or metastatic solid tumors, including patients with active central nervous system (CNS) disease. Here, we summarize the overall safety and report the antitumor activity of entrectinib in a cohort of patients with tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions, naïve to prior TKI treatment targeting the specific gene, and who were treated at doses that achieved therapeutic exposures consistent with the recommended phase II dose. Entrectinib was well tolerated, with predominantly Grades 1/2 adverse events that were reversible with dose modification. Responses were observed in non-small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1-NTRK1-rearranged lung cancer.Significance: Gene fusions of NTRK1/2/3, ROS1, and ALK (encoding TRKA/B/C, ROS1, and ALK, respectively) lead to constitutive activation of oncogenic pathways. Entrectinib was shown to be well tolerated and active against those gene fusions in solid tumors, including in patients with primary or secondary CNS disease. Cancer Discov; 7(4); 400-9. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 339.

Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial.

BACKGROUND: We previously found that dual HER2 blockade with trastuzumab and lapatinib led to inhibition of tumour growth in patient-derived xenografts of HER2-amplified metastatic colorectal cancer. In this study, we aimed to assess the antitumour activity of trastuzumab and lapatinib in patients with HER2-positive colorectal cancer. METHODS: HERACLES was a proof-of-concept, multicentre, open-label, phase 2 trial done at four Italian academic cancer centres. We enrolled adult patients with KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive metastatic colorectal cancer refractory to standard of care (including cetuximab or panitumumab), an Eastern Cooperative Oncology Group performance status of 0 or 1, and at least one measurable lesion. We defined HER2 positivity in tumour samples by use of immunohistochemistry and fluorescence in-situ hybridisation in accordance with our previously validated colorectal cancer-specific diagnostic criteria. Eligible patients received intravenous trastuzumab at 4 mg/kg loading dose followed by 2 mg/kg once per week, and oral lapatinib at 1000 mg per day until evidence of disease progression. The primary endpoint was the proportion of patients achieving an objective response (defined as complete response or partial response), which was assessed by independent central review in the intention-to-treat population. This trial is registered with EudraCT, number 2012-002128-33. FINDINGS: Between Aug 27, 2012, and May 15, 2015, we screened 914 patients with KRAS exon 2 (codons 12 and 13) wild-type metastatic colorectal cancer and identified 48 (5%) patients with HER2-positive tumours, although two died before enrolment. Of these patients, 27 were eligible for the trial. All were evaluable for response. At the time of data cutoff on Oct 15, 2015, with a median follow-up of 94 weeks (IQR 51-127), eight (30%, 95% CI 14-50) of 27 patients had achieved an objective response, with one patient (4%, 95% CI -3 to 11) achieving a complete response, and seven (26%, 95% CI 9-43) achieving partial responses; 12 (44%, 95% CI 25-63) patients had stable disease. Six (22%) of 27 patients had grade 3 adverse events, which consisted of fatigue in four patients, skin rash in one patient, and increased bilirubin concentration in one patient. No grade 4 or 5 adverse events were reported. We detected no drug-related serious adverse events. INTERPRETATION: The combination of trastuzumab and lapatinib is active and well tolerated in treatment-refractory patients with HER2-positive metastatic colorectal cancer. FUNDING: Associazione Italiana Ricerca Cancro (AIRC), Fondazione Oncologia Niguarda Onlus, and Roche.

Sensitivity to Entrectinib Associated With a Novel LMNA-NTRK1 Gene Fusion in Metastatic Colorectal Cancer.

In metastatic colorectal cancer (CRC), actionable genetic lesions represent potential clinical opportunities. NTRK1, 2, and 3 gene rearrangements encode oncogenic fusions of the tropomyosin-receptor kinase (TRK) family of receptor tyrosine kinases in different tumor types. The TPM3-NTRK1 rearrangement is a recurring event in CRC that renders tumors sensitive to TRKA kinase inhibitors in preclinical models. We identified abnormal expression of the TRKA protein in tumor and liver metastases of a CRC patient refractory to standard therapy. Molecular characterization unveiled a novel LMNA-NTRK1 rearrangement within chromosome 1 with oncogenic potential, and the patient was treated with the pan-TRK inhibitor entrectinib, achieving partial response with decrease in hepatic target lesions from 6.8 and 8.2cm in longest diameter to 4.7 and 4.3cm, respectively. To our knowledge, this is the first clinical evidence of efficacy for therapeutic inhibition of TRKA in a solid tumor, illuminating a genomic-driven strategy to identify CRCs reliant on this oncogene to be clinically targeted with entrectinib.

Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer.

BACKGROUND: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. METHODS: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. RESULTS: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1-35 of CAD with exons 20-29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. CONCLUSIONS: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.

Multi-determinants analysis of molecular alterations for predicting clinical benefit to EGFR-targeted monoclonal antibodies in colorectal cancer.

BACKGROUND: KRAS mutations occur in 35-45% of metastatic colorectal cancers (mCRC) and preclude responsiveness to EGFR-targeted therapy with cetuximab or panitumumab. However, less than 20% patients displaying wild-type KRAS tumors achieve objective response. Alterations in other effectors downstream of the EGFR, such as BRAF, and deregulation of the PIK3CA/PTEN pathway have independently been found to give rise to resistance. We present a comprehensive analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression in mCRC patients treated with cetuximab or panitumumab, with the aim of clarifying the relative contribution of these molecular alterations to resistance. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively analyzed objective tumor response, progression-free (PFS) and overall survival (OS) together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumors from cetuximab or panitumumab treated mCRC patients. Among the 106 non-responsive patients, 74 (70%) had tumors with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with > or =2 alterations (p<0.0001). Accordingly, PFS and OS were increasingly worse for patients with tumors harboring none, 1, or > or =2 molecular alteration(s) (p<0.001). CONCLUSIONS/SIGNIFICANCE: When expression of PTEN and mutations of KRAS, BRAF and PIK3CA are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to anti-EGFR therapies can be identified. We propose to define as 'quadruple negative', the CRCs lacking alterations in KRAS, BRAF, PTEN and PIK3CA. Comprehensive molecular dissection of the EGFR signaling pathways should be considered to select mCRC patients for cetuximab- or panitumumab-based therapies.

Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study.

BACKGROUND: The antiepidermal growth factor receptor (antiEGFR) monoclonal antibodies cetuximab and panitumumab have good clinical activity in about 10% of patients with metastatic colorectal cancer that is resistant to chemotherapy. The molecular mechanisms underlying clinical response or resistance to these agents are unknown. METHODS: Tumours from 31 patients with metastatic colorectal cancer who had either an objective response (n=10) or stable disease or progressive disease (n=21) after treatment with cetuximab or panitumumab were screened for genetic changes in EGFR or its immediate intracellular effectors. Specifically, we assessed the EGFR copy number and the mutation profile of the EGFR catalytic domain and of selected exons in KRAS, BRAF, and PIK3CA. RESULTS: Eight of nine of patients with objective responses who were assessable by fluorescence in-situ hybridisation (FISH) had an increased EGFR copy number. By contrast, one of 21 non-responders assessable by FISH had an increased EGFR copy number (p<0.0001 for responders vs non-responders, Fisher's exact test). The mutation status of the EGFR catalytic domain and its immediate downstream effectors PIK3CA, KRAS, and BRAF did not correlate with disease response. In colorectal-cancer cell lines, the concentration of cetuximab that completely inhibited proliferation of cells with amplified EGFR copy number did not affect proliferation of cells with unamplified EGFR. INTERPRETATION: We propose that the response to antiEGFR treatment has a genetic basis and suggest that patients might be selected for treatment on the basis of EGFR copy number.