Katia Bencardino

Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

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.

Blockade of EGFR and MEK Intercepts Heterogeneous Mechanisms of Acquired Resistance to Anti-EGFR Therapies in Colorectal Cancer

Colorectal cancers that become resistant to EGFR inhibitors through a variety of mechanisms can be effectively treated by inhibiting MEK in conjunction with EGFR. Circulating Tumor DNA for Early Detection and Managing Resistance Cancer evolves over time, without any warning signs. Similarly, the development of resistance to therapy generally becomes apparent only when there are obvious signs of tumor growth, at which point the patient may have lost valuable time. Although a repeat biopsy may be able to identify drug-resistant mutations before the tumor has a chance to regrow, it is usually not feasible to do many repeat biopsies. Now, two studies are demonstrating the utility of monitoring the patients’ blood for tumor DNA to detect cancer at the earliest stages of growth or resistance. In one study, Bettegowda and coauthors showed that sampling a patient’s blood may be sufficient to yield information about the tumor’s genetic makeup, even for many early-stage cancers, without a need for an invasive procedure to collect tumor tissue, such as surgery or endoscopy. The authors demonstrated the presence of circulating DNA from many types of tumors that had not yet metastasized or released detectable cells into the circulation. They could detect more than 50% of patients across 14 tumor types at the earliest stages, when these cancers may still be curable, suggesting that a blood draw could be a viable screening approach to detecting most cancers. They also showed that in patients with colorectal cancer, the information derived from circulating tumor DNA could be used to determine the optimal course of treatment and identify resistance to epidermal growth factor receptor (EGFR) blockade. Meanwhile, Misale and colleagues illustrated a way to use this information to overcome treatment resistance. These authors also found that mutations associated with EGFR inhibitor resistance could be detected in the blood of patients with colorectal cancer. In addition, they demonstrated that adding MEK inhibitors, another class of anticancer drugs, can successfully overcome resistance when given in conjunction with the EGFR inhibitors. Thus, the studies from Bettegowda and Misale and their colleagues show the effectiveness of analyzing circulating DNA from a variety of tumors and highlight the potential applications of this technology for early detection, monitoring resistance, and devising treatment plans to overcome resistance. Colorectal cancers (CRCs) that are sensitive to the anti–epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab almost always develop resistance within several months of initiating therapy. We report the emergence of polyclonal KRAS, NRAS, and BRAF mutations in CRC cells with acquired resistance to EGFR blockade. Regardless of the genetic alterations, resistant cells consistently displayed mitogen-activated protein kinase kinase (MEK) and extracellular signal–regulated kinase (ERK) activation, which persisted after EGFR blockade. Inhibition of MEK1/2 alone failed to impair the growth of resistant cells in vitro and in vivo. An RNA interference screen demonstrated that suppression of EGFR, together with silencing of MEK1/2, was required to hamper the proliferation of resistant cells. Indeed, concomitant pharmacological blockade of MEK and EGFR induced prolonged ERK inhibition and severely impaired the growth of resistant tumor cells. Heterogeneous and concomitant mutations in KRAS and NRAS were also detected in plasma samples from patients who developed resistance to anti-EGFR antibodies. A mouse xenotransplant from a CRC patient who responded and subsequently relapsed upon EGFR therapy showed exquisite sensitivity to combinatorial treatment with MEK and EGFR inhibitors. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which reactivate ERK signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in CRC patients who become refractory to anti-EGFR therapies.

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.

Promoter CpG island hypermethylation of the DNA repair enzyme MGMT predicts clinical response to dacarbazine in a phase II study for metastatic colorectal cancer.

Purpose: O6-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein removing mutagenic and cytotoxic adducts from O6-guanine in DNA. Approximately 40% of colorectal cancers (CRC) display MGMT deficiency due to the promoter hypermethylation leading to silencing of the gene. Alkylating agents, such as dacarbazine, exert their antitumor activity by DNA methylation at the O6-guanine site, inducing base pair mismatch; therefore, activity of dacarbazine could be enhanced in CRCs lacking MGMT. We conducted a phase II study with dacarbazine in CRCs who had failed standard therapies (oxaliplatin, irinotecan, fluoropyrimidines, and cetuximab or panitumumab if KRAS wild-type). Experimental Design: All patients had tumor tissue assessed for MGMT as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m2 intravenously every day for four consecutive days, every 21 days, until progressive disease or intolerable toxicity. We used a Simon two-stage design to determine whether the overall response rate would be 10% or more. Secondary endpoints included association of response, progression-free survival, and disease control rate with MGMT status. Results: Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of three cycles of dacarbazine (range 1–12). Grades 3 and 4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), and anemia (18%). Overall, two patients (3%) achieved partial response and eight patients (12%) had stable disease. Disease control rate (partial response + stable disease) was significantly associated with MGMT promoter hypermethylation in the corresponding tumors. Conclusion: Objective clinical responses to dacarbazine in patients with metastatic CRC are confined to those tumors harboring epigenetic inactivation of the DNA repair enzyme MGMT. Clin Cancer Res; 19(8); 2265–72. ©2013 AACR.

Assessment of a HER2 scoring system for colorectal cancer: Results from a validation study

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 enrolment 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.

Effects of gefitinib on serum epidermal growth factor receptor and HER2 in patients with advanced non-small cell lung cancer.

Purpose: The aim of this study was to assess serum extracellular binding domains of epidermal growth factor receptor (EGFR) and HER2 as surrogate markers of Gefitinib (Iressa, ZD1839, AstraZeneca, London, United Kingdom) activity in patients with non-small cell lung cancer. Experimental Design: Serum EGFR and HER2 levels were monitored in blood samples taken within 1 week of starting Gefitinib at day 28 and at every computed tomography scan evaluation. EGFR and HER-2 were assayed in duplicate using commercial sandwich enzyme-linked immunosorbent assay kits (Oncogene Science Bayer Corporation, Cambridge, UK). A logistic regression analysis was performed to evaluate: (1) the relationship between best overall tumor response and basal EGFR and HER2 levels, and (2) the association between best overall tumor response and the differences of EGFR and HER2 levels obtained at the best overall tumor response and at baseline. Results: Forty-six pretreated patients were evaluated, including F/M:11/35, Eastern Cooperative Oncology Group performance status 0–1/2:39/7, IIIB/IV:11/35, and adenocarcinoma/nonadenocarcinoma 29/17. Five partial responses (11%) and 14 stable disease responses (30%) were observed. Median pretreatment EGFR and HER2 were 83.3 ng/ml and 13.7 ng/ml. For baseline EGFR and HER2, the odds ratio of progression was 0.95 [95% confidence interval (CI), 0.91–0.98; P = 0.01] and 0.87 (95% CI, 0.74–1.03; P = 0.11), respectively. The difference between the best overall tumor response and basal EGFR value was predictive for response with a 6% increase in the odds of progression for an increase of 1 ng/ml (odds ratio, 1.06; 95% CI, 1.01–1.11; P = 0.009) and for progression-free survival with a hazard ratio of 1.03 (95% CI, 1.01–1.04; P = 0.003). Conclusion: Modifications of EGFR serum values during treatment seem to reflect Gefitinib activity.

Integrated molecular dissection of the epidermal growth factor receptor (EFGR) oncogenic pathway to predict response to EGFR-targeted monoclonal antibodies in metastatic colorectal cancer

The introduction of KRAS testing as a diagnostic tool to select patients for epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies for metastatic colorectal cancer is widely regarded as a key advance in the field of personalized cancer medicine. Oncologists are now facing emerging issues in the treatment of metastatic colorectal cancer, including: (i) the identification of additional genetic determinants of primary resistance to EGFR-targeted therapy for further improving selection of patients; (ii) the explanation of rare cases of patients carrying KRAS-mutated tumors who have been reported to respond to either cetuximab or panitumumab and (iii) the discovery of mechanisms of secondary resistance to anti-EGFR antibody therapies. Here we discuss the potential role of comprehensive dissection of the key oncogenic nodes in the EGFR signaling cascade to predict resistance and sensitivity to EGFR monoclonal antibodies in metastatic colorectal cancer. Current data suggest that, together with KRAS mutations, the evaluation of BRAF and PIK3CA/PTEN alterations could also be useful for selecting patients with reduced chance to benefit from EGFR-targeted therapy. Furthermore, measuring EGFR gene copy number also appears relevant to positively identify responders. Up until now, each of these markers has been mainly assessed as a single event, often in retrospective analyses and patients’ series. As these molecular alterations display overlapping patterns of occurrence, this adds considerable complexity to the drawing of an algorithm suitable for clinical decision-making. We suggest that in the near future comprehensive molecular analysis of the entire oncogenic pathway triggered by the EGFR should be performed, thus enhancing the prediction ability of individual markers.

Therapeutic implications of resistance to molecular therapies in metastatic colorectal cancer

Metastatic colorectal cancer (mCRC) patients carrying KRAS mutated tumors do not benefit from epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies. Indeed, the mutational status of KRAS is currently a validated predictive biomarker employed to select mCRC patients for EGFR targeted drugs. When patients fail standard 5-fluorouracil-, oxaliplatin-, irinotecan- and bevacizumab-based therapies, EGFR-targeted salvage therapy can be prescribed only for those individuals with KRAS wild-type cancer. Thus, clinicians are now facing the urgent issue of better understanding the biology of KRAS mutant disease, in order to devise novel effective therapies in such defined genetic setting. In addition to KRAS, recent data point out that BRAF and PIK3CA exon 20 mutations hamper response to EGFR-targeted treatment in mCRC, potentially excluding from treatment also patients with these molecular alterations in their tumor. This review will focus on current knowledge regarding the molecular landscape of mCRC including and beyond KRAS, and will summarize novel rationally-developed combinatorial regimens that are being evaluated in early clinical trials.

Tumor MGMT promoter hypermethylation changes over time limit temozolomide efficacy in a phase II trial for metastatic colorectal cancer

BACKGROUND Objective response to dacarbazine, the intravenous form of temozolomide (TMZ), in metastatic colorectal cancer (mCRC) is confined to tumors harboring O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter hypermethylation. We conducted a phase II study of TMZ enriched by MGMT hypermethylation in archival tumor (AT), exploring dynamic of this biomarker in baseline tumor (BT) biopsy and plasma (liquid biopsy). PATIENTS AND METHODS We screened 150 mCRC patients for MGMT hypermethylation with methylation-specific PCR on AT from FFPE specimens. Eligible patients (n = 29) underwent BT biopsy and then received TMZ 200 mg/m(2) days 1-5 q28 until progression. A Fleming single-stage design was used to determine whether progression-free survival (PFS) rate at 12 weeks would be ≥35% [H0 ≤ 15%, type I error = 0.059 (one-sided), power = 0.849]. Exploratory analyses included comparison between MGMT hypermethylation in AT and BT, and MGMT methylation testing by MethylBEAMing in solid (AT, BT) and LB with regard to tumor response. RESULTS The PFS rate at 12 weeks was 10.3% [90% confidence interval (CI) 2.9-24.6]. Objective response rate was 3.4% (90% CI 0.2-15.3), disease control rate 48.3% (90% CI 32.0-64.8), median OS 6.2 months (95% CI 3.8-7.6), and median PFS 2.6 months (95% CI 1.4-2.7). We observed the absence of MGMT hypermethylation in BT in 62.7% of tumors. CONCLUSION Treatment of mCRC with TMZ driven by MGMT promoter hypermethylation in AT samples did not provide meaningful PFS rate at 12 weeks. This biomarker changed from AT to BT, indicating that testing BT biopsy or plasma is needed for refined target selection.