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Background The ALEX study demonstrated significantly improved progression-free survival (PFS) with alectinib versus crizotinib in treatment-naive ALK-positive non-small-cell lung cancer (NSCLC) at the primary data cut-off (9 February 2017). We report mature PFS (cut-off: 30 November 2018) and overall survival (OS) data up to 5 years (cut-off: 29 November 2019). Patients and methods Patients with stage III/IV ALK-positive NSCLC were randomized to receive twice-daily alectinib 600 mg (n = 152) or crizotinib 250 mg (n = 151) until disease progression, toxicity, withdrawal or death. Primary end point: investigator-assessed PFS. Secondary end points included objective response rate, OS and safety. Results Mature PFS data showed significantly prolonged investigator-assessed PFS with alectinib [hazard ratio (HR) 0.43, 95% confidence interval (CI) 0.32–0.58; median PFS 34.8 versus 10.9 months crizotinib]. Median duration of OS follow-up: 48.2 months alectinib, 23.3 months crizotinib. OS data remain immature (37% of events). Median OS was not reached with alectinib versus 57.4 months with crizotinib (stratified HR 0.67, 95% CI 0.46–0.98). The 5-year OS rate was 62.5% (95% CI 54.3–70.8) with alectinib and 45.5% (95% CI 33.6–57.4) with crizotinib, with 34.9% and 8.6% of patients still on study treatment, respectively. The OS benefit of alectinib was seen in patients with central nervous system metastases at baseline [HR 0.58 (95% CI 0.34–1.00)] and those without [HR 0.76 (95% CI 0.45–1.26)]. Median treatment duration was longer with alectinib (28.1 versus 10.8 months), and no new safety signals were observed. Conclusions Mature PFS data from ALEX confirmed significant improvement in PFS for alectinib over crizotinib in ALK-positive NSCLC. OS data remain immature, with a higher 5-year OS rate with alectinib versus crizotinib. This is the first global randomized study to show clinically meaningful improvement in OS for a next-generation tyrosine kinase inhibitor versus crizotinib in treatment-naive ALK-positive NSCLC. Clinical trials number NCT02075840.

Human epidermal growth factor receptor 2 (HER2) dysregulation is associated with tumorigenesis in gastric/gastroesophageal junction cancer; however, the number of patients with HER2-positive disease is unclear, possibly due to differing scoring criteria/assays. Data are also lacking for early disease. We aimed to assess the HER2-positivity rate using approved testing criteria in a large, real-life multinational population. HER2-positivity was defined as an immunohistochemistry staining score of 3+, or immunohistochemistry 2+ and HER2 amplification detected by in situ hybridization. A total of 4949 patients were enrolled and results showed that 14.2% of 4920 samples with immunohistochemistry results were HER2-positive. HER2-positivity was significantly higher in males (16.1% vs. 9.6% in females), in gastroesophageal versus stomach tumors (22.1% vs. 12.9%), in biopsy versus surgical samples (18.3% vs. 13.0%), in intestinal tumor subtypes versus diffuse (21.5% vs. 4.8%) and mixed types (21.5% vs. 8.5%) (P

Alectinib, a highly selective inhibitor of anaplastic lymphoma kinase (ALK), has shown systemic and central nervous system (CNS) efficacy in the treatment of ALK-positive non-small-cell lung cancer (NSCLC). We investigated alectinib as compared with crizotinib in patients with previously untreated, advanced ALK-positive NSCLC, including those with asymptomatic CNS disease. In a randomized, open-label, phase 3 trial, we randomly assigned 303 patients with previously untreated, advanced ALK-positive NSCLC to receive either alectinib (600 mg twice daily) or crizotinib (250 mg twice daily). The primary end point was investigator-assessed progression-free survival. Secondary end points were independent review committee-assessed progression-free survival, time to CNS progression, objective response rate, and overall survival. During a median follow-up of 17.6 months (crizotinib) and 18.6 months (alectinib), an event of disease progression or death occurred in 62 of 152 patients (41%) in the alectinib group and 102 of 151 patients (68%) in the crizotinib group. The rate of investigator-assessed progression-free survival was significantly higher with alectinib than with crizotinib (12-month event-free survival rate, 68.4% [95% confidence interval (CI), 61.0 to 75.9] with alectinib vs. 48.7% [95% CI, 40.4 to 56.9] with crizotinib; hazard ratio for disease progression or death, 0.47 [95% CI, 0.34 to 0.65]; P

Purpose Women receiving trastuzumab with chemotherapy are at risk for trastuzumab-related cardiac dysfunction (TRCD). We explored the prognostic value of cardiac markers (troponins I and T, N-terminal prohormone of brain natriuretic peptide [NT-proBNP]) to predict baseline susceptibility to develop TRCD. We examined whether development of cardiac end points or significant left ventricular ejection fraction (LVEF) drop was associated with markers’ increases. Patients and Methods Cardiac marker assessments were coupled with LVEF measurements at different time points for 533 patients from the Herceptin Adjuvant (HERA) study who agreed to participate in this study. Patients with missing marker assessments were excluded, resulting in 452 evaluable patients. A primary cardiac end point was defined as symptomatic congestive heart failure of New York Heart Association class III or IV, confirmed by a cardiologist, and a significant LVEF drop, or death of definite or probable cardiac causes. A secondary cardiac end point was defined as a confirmed significant asymptomatic or mildly symptomatic LVEF drop. Results Elevated baseline troponin I (> 40 ng/L) and T (> 14 ng/L), occurring in 56 of 412 (13.6%) and 101 of 407 (24.8%) patients, respectively, were associated with an increased significant LVEF drop risk (univariate analysis: hazard ratio, 4.52; P < .001 and hazard ratio, 3.57; P < .001, respectively). Few patients had their first elevated troponin value recorded during the study (six patients for troponin I and 25 patients for troponin T). Two patients developed a primary and 31 patients a secondary cardiac end point (recovery rate of 74%, 23 of 31). For NT-proBNP, higher increases from baseline were seen in patients with significant LVEF drop. Conclusion Elevated troponin I or T before trastuzumab is associated with increased risk for TRCD. A similar conclusion for NT-proBNP could not be drawn because of the lack of a well-established elevation threshold; however, higher increases from baseline were seen in patients with TRCD compared with patients without.

At the prior data cutoff (February 9, 2017) the ALEX trial showed superior investigator-assessed progression-free survival (PFS) for alectinib versus crizotinib in untreated, anaplastic lymphoma kinase (ALK)-positive, advanced NSCLC (hazard ratio = 0.47, 95% confidence interval: 0.34-0.65, p0.001). The median PFS in the alectinib arm was not reached versus 11.1 months with crizotinib. Retrospective analyses suggest that the echinoderm microtubule-associated protein-like 4 gene-ALK variant (EML4-ALK) may influence ALK-inhibitor treatment benefit. We present updated analyses, including exploratory subgroup analysis by EML4-ALK variant, after an additional 10 months' follow-up (cutoff December 1, 2017).Patients were randomized to receive twice-daily alectinib, 600 mg, or crizotinib, 250 mg, until disease progression, toxicity, death, or withdrawal. PFS was determined by the investigators. Baseline plasma and tissue biomarker samples were analyzed by using hybrid-capture, next-generation sequencing to determine EML4-ALK variant.Baseline characteristics were balanced. Investigator-assessed PFS was prolonged with alectinib (stratified hazard ratio = 0.43, 95% confidence interval: 0.32-0.58). The median PFS times were 34.8 months with alectinib and 10.9 months with crizotinib. EML4-ALK fusions were detectable in 129 patient plasma samples and 124 tissue samples; variants 1, 2, and 3/ab did not affect PFS, objective response rate, or duration of response. Investigator-assessed PFS was longer for alectinib than for crizotinib across EML4-ALK variants 1, 2, and 3a/b in plasma and tissue. Despite longer treatment duration (27.0 months in the case of alectinib versus 10.8 months in the case of crizotinib), the safety of alectinib compared favorably with that of crizotinib.Alectinib continues to demonstrate superior investigator-assessed PFS versus crizotinib in untreated ALK-positive NSCLC, irrespective of EML4-ALK variant.

Newly diagnosed invasive breast cancers should be evaluated for Human Epidermal Growth Factor Receptor 2 (HER2) status by immunohistochemistry (IHC) and/or in situ hybridisation (ISH) to determine eligibility for trastuzumab or other HER2-targeted therapies. Previous reports of high discordance rates between IHC and ISH have raised concerns over the accuracy of HER2 testing, especially when IHC is conducted locally. This study aimed to determine the rate of false-negative IHC results at three pathology centres (one central, two local) in Greece by central retesting of 240 prospectively collected invasive breast cancers scored as IHC 0/1+ at initial testing. All samples were from female patients (median age 58.0 years). Initial IHC tests utilised either the CB11 (159/239; 66.5%) or 4B5 (80/239; 33.5%) antibodies and were scored as 0 in 105/240 cases (43.8%) and 1+ in 135/240 cases (56.3%). All samples were centrally retested by automated silver in situ hybridisation (SISH). Of 237 samples with SISH staining suitable for assessment, 223 (94.1%; 95% confidence interval 90.3–96.5%) were classed as SISH-negative (HER2:chromosome enumeration probe 17 (CEP17) HER2:CEP17 >2.2), providing a false-negative rate of 3.4%. A further four samples (1.7%) exhibited equivocal amplification status (HER2:CEP17 1.8–2.2) and two (0.8%) showed polysomy of chromosome 17. The proportion of SISH-negative results did not significantly differ between the IHC 0 and 1+ subgroups (95.2% vs. 93.2%; p=0.505). In conclusion, the low observed rate of false-negative IHC results in this study supports the use of IHC for initial HER2 status assessment in local or central laboratories in Greece.

Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

Introduction: Increase in targeted therapies has resulted in the need for a single assay capable of detecting diverse biomarkers indicated for these agents. Comprehensive genomic profiling (CGP) provides such a solution, but due to the complexity and number of assays available today, standardization of validation has become critically important. We present FoundationOne CDx, the first NGS-based comprehensive companion diagnostics (CDx) platform developed and performed in compliance with FDA 21 CFR part 820. The assay interrogates 324 genes, and has CDx indications in five tumor types associated with 17 targeted therapies (Table 1). The versatile assay design will facilitate streamlined development of future CDx indications. Methods: DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB). Results: Clinical validity was established such that the concordance between CGP and approved CDx were statistically non-inferior to that of two runs of approved CDx. For analytical validity, limit of detection (LoD) was at allele frequency 4% for known substitutions and indels. LoD was 16% tumor content for copy number amplifications, 30% for homozygous deletions, 11% for rearrangements, 12% for MSI, and 20% for TMB. Concordance with an orthogonal NGS platform was 94.6% for substitutions and indels. Within-assay reproducibility had PPA 99.4%. Conclusion: Rapid expansion of targeted therapies and CDx has necessitated a new approach and urgency to defining performance standards. We developed a comprehensive CDx assay and demonstrated clinical and analytical validity to support and accelerate using CGP for routine clinical care. Table 1. Companion Diagnostic IndicationsIndicationBiomarkerTherapyNon-small cell lung cancer (NSCLC)EGFR exon 19 deletions and EGFR exon 21 L858R alterationsafatinib, gefitinib, or erlotinibEGFR exon 20 T790M alterationsosimertinibALK rearrangementsalectinib, crizotinib, or ceritinibBRAF V600Edabrafenib in combination with trametinibMelanomaBRAF V600Edabrafenib, vemurafenibBRAF V600E and V600Ktrametinib, cobimetinib, in combination with vemurafenibBreast cancerERBB2 (HER2) amplificationtrastuzumab, ado-trastuzumab-emtansine, or pertuzumabColorectal cancerKRAS wild-type (absence of mutations in codons 12 and 13)cetuximabKRAS and NRAS wild-type (absence of mutations in exons 2, 3, and 4)panitumumabOvarian cancerBRCA1/2 alterationsrucaparib Citation Format: James X. Sun, Yali Li, Coren Milbury, Joel Skoletsky, Christine Burns, Wai-ki Yip, Jun Luo, Ninad Dewal, Adrienne Johnson, Kyle Gowen, Jing Tong, Yuting He, Jie He, Pei Ma, Jared White, Steve Roels, John Truesdell, Eric Peters, Houston Gilbert, Charlie Wu, Erica Schleifman, Johannes Noe, Carl Barrett, Kenneth Thress, Suzanne Jenkins, Julia Elvin, Geoff Otto, Doron Lipson, Jeffrey Ross, Vincent Miller, Philip Stephens, Michael Doherty, Christine Vietz. A clinically validated comprehensive companion diagnostic platform for care of patients with advanced cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4757.

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.

Background & Aims: Hepatic bile salt secretion is an essential function of vertebrate liver. Rat and mouse bile salt export pump (Bsep) are adenosine triphosphate (ATP)-dependent bile salt transporters. Mutations in human BSEP were identified as the cause of progressive familial intrahepatic cholestasis type 2. BSEP protein is highly identical with its rat and mouse orthologs and has not yet been functionally characterized; the effect of BSEP mutations on its function has also not been studied. Therefore, the aim of this study was to functionally characterize human BSEP. Methods: Complementary DNA for BSEP was isolated from human liver and expressed with the baculovirus system in Sf9 cells. ATP-dependent bile salt transport assays were performed with Sf9 cell vesicles expressing BSEP and a rapid filtration assay. Results: Cloning of human BSEP required the inactivation of a bacterial cryptic promoter motif within its coding region. BSEP expressed in Sf9 cells transports different bile salts in an ATP-dependent manner with Michaelis constant values as follows: taurocholate, 7.9 ± 2.1 μmol/L; glycocholate, 11.1 ± 3.3 μmol/L; taurochenodeoxycholate, 4.8 ± 1.7 μmol/L; tauroursodeoxycholate, 11.9 ± 1.8 μmol/L. The rank order of the intrinsic clearance of bile salts was taurochenodeoxycholate > taurocholate > tauroursodeoxycholate > glycocholate. Conclusions: This study characterizes human BSEP as an ATP-dependent bile salt export pump with transport properties similar to its rat and mouse orthologs. Expression of BSEP in Sf9 cells will enable functional characterization of the consequences of mutations in the human BSEP gene. GASTROENTEROLOGY 2002;123:1659-1666

LBA9008 Background: Alectinib, a TKI targeting ALK, has shown robust efficacy in crizotinib-naïve/resistant ALK+ NSCLC. J-ALEX showed superiority of alectinib 300mg BID vs crizotinib in Japanese pts with crizotinib-naïve ALK+ NSCLC (progression-free survival [PFS] HR 0.34, p

Background/Aims Inherited dysfunction of the bile salt export pump BSEP ( ABCB11 ) causes a progressive and a benign form of familial intrahepatic cholestasis, denominated as PFIC2 and BRIC2, respectively. We functionally characterized novel ABCB11 mutations encountered in two patients with a PFIC2 and a BRIC2 phenotype, respectively. Methods BSEP expression was determined in liver biopsies by immunohistochemistry. ABCB11 mutations were functionally characterized by taurocholate transport in SF9 cells transfected with human ABCB11 . Results The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((+3)A>C) combined with an early stop codon at position 930 (R930X), while the BRIC2 patient was compound heterozygous for two nonsynonymous mutations in exon 9 (E297G) and exon 12 (R432T), respectively. Hepatic BSEP expression was absent in PFIC2 and preserved in BRIC2. In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. Conclusions The intron 4 (+3)A>C, R930X and R432T represent previously undescribed mutations of the ABCB11 gene that confer a PFIC2 and a BRIC2 phenotype, respectively. By combining functional in-vitro characterization with immunohistochemical detection of variant BSEP we provide direct evidence for the role of ABCB11 mutations in the pathogenesis of different forms of intrahepatic cholestasis.