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PURPOSE Emerging evidence suggests a correlation between the tumor mutational burden (TMB) and the response to programmed cell death-1 protein (PD-1) monotherapy across multiple cancer types. In skin cancers, as high TMB is mostly because of ultraviolet (UV) exposure, we hypothesized a correlation between the primary melanoma cutaneous location according to sun exposure and response to anti–PD-1 monotherapy. METHODS The aim of this study was to analyze, in advanced melanoma, the relationship between TMB, locations according to sun exposure, and response to PD-1 inhibitors. We conducted a prospective multicentric analysis, by sequencing the most recent metastatic sample before PD-1 inhibitors using FoundationOne assay. RESULTS One hundred two patients were included, with TMB available for 94 cases. In univariate and multivariate linear regression, TMB was significantly associated with sun-exposed areas of the primary melanoma location and with age (coefficients of the association with log-TMB: non-UV location, –1.05; chronic sun-exposed area, 1.12; P value for the location, < 10–5; age, 0.021 per year, P value for age, .002). Molecular UV signature present on the metastatic site was associated with higher TMB (P = .003). Melanomas bearing a high TMB had a higher probability of response to PD-1 inhibitors compared with melanomas with a low TMB, with a dose-dependent effect following an exponential curve and a negative odds ratio of 0.40 (95% CI, 0.20 to 0.72, P = .004) between log-TMB and 6-month progression. CONCLUSION Cumulative sun exposure related to skin location and molecular UV signature present on the metastatic site appear to be relevant biomarkers directly linked to TMB. Because TMB is not yet available to all for routine clinical use, the location of the primary melanoma in a sun-exposed area may play an important role in clinical decisions regarding therapeutic choice., The location of the primary melanoma in a sun-exposed area can help choosing first-line advanced melanoma treatment.

Highlights • NUT carcinoma is a rare but aggressive solid tumor that can be diagnosed by presence of the BRD4-NUT fusion. • This series presents 31 cases of solid tumors that harbor BRD4-NUT but often carry other diagnoses such NSCLC—NOS and NSCLC-SCC. • Despite lack of PD-L1 expression and a low tumor mutational burden, two index cases responded to either nivolumab or atezolizumab+chemotherapy with partial response or better with 4–5 month duration of response. • The unexpected response to checkpoint inhibitors could be explained by a very high affinity of the fusion peptide at the junction of BRD4 and NUT to the MHC complex as recently suggested for an exceptional response to an immune checkpoint inhibitor in a fusion bearing low TMB, low PD-L1 expression head and neck carcinoma., Background The translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment. Case presentation Thirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLC—NOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0–4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases – including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis. Conclusions CGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.

PURPOSE Combined hepatocellular cholangiocarcinoma (cHCC-CCA) is a rare, aggressive primary liver carcinoma, with morphologic features of both hepatocellular carcinomas (HCC) and liver cholangiocarcinomas (CCA). METHODS The genomic profiles of 4,975 CCA, 1,470 HCC, and 73 cHCC-CCA cases arising from comprehensive genomic profiling in the course of clinical care were reviewed for genomic alterations (GA), tumor mutational burden, microsatellite instability status, genomic loss of heterozygosity, chromosomal aneuploidy, genomic ancestry, and hepatitis B virus status. RESULTS In cHCC-CCA, GA were most common in TP53 (65.8%), TERT (49.3%), and PTEN (9.6%), and 24.6% cHCC-CCA harbored potentially targetable GA. Other GA were predominantly associated with either HCC or CCA, including, but not limited to, TERT, FGFR2, IDH1, and presence of hepatitis B virus. On the basis of these features, a machine learning (ML) model was trained to classify a cHCC-CCA case as CCA-like or HCC-like. Of cHCC-CCA cases, 16% (12/73) were ML-classified as CCA-like and 58% (42/73) cHCC-CCA were ML-classified as HCC-like. The ML model classified more than 70% of cHCC-CCA as CCA-like or HCC-like on the basis of genomic profiles, without additional clinico-pathologic input. CONCLUSION These findings demonstrate the use of ML for classification as based on a targeted exome panel used during routine clinical care. Classification of cHCC-CCA by genomic features alone creates insights into the biology of the disease and warrants further investigation for relevance to clinical care.

e21592 Background: While 10% of melanomas occur in a context suggesting hereditary predisposition, a clear molecular explanation has only been established for approximately 20% of families. In the course of clinical care, we identified a new CTNNA1 truncating germline mutation in a family affected by multiple early-onset melanomas. Methods: NGS and CGH-array were performed on the index case’s melanoma, followed by Sanger sequencing of the germline DNA of relatives. Immunochemistry (IHC) was employed to evaluate the level of αE-catenin (encoded by CTNNA1) in the family's samples. Stable CTNNA1 knockout human melanoma cell lines were generated to investigate the functional effects of CTNNA1 loss. Functional assays, including colony formation, 3-D tumor spheroid formation, wound healing, and transwell invasion were performed, as well as electron microscopy and RNAsequencing (RNAseq). CTNNA1 mutational status was determined in several databases and further sequencing of CTNNA1 in a DNA bank of families with multiple melanomas was done. Results: While the allele frequency in the index patient’s tumoral DNA was compatible with a germline mutation, the CTNNA1 F611fs*10 mutation was subsequently found cosegregating with individuals affected by melanoma in the family. CGH array on tumor DNA identified a segmental loss on chromosome 5, leading to a loss of heterozygosity of CTNNA1, resulting in a loss of αE-catenin observed by IHC. Clinically, the mean age of first melanoma diagnosis was 29,7 years (range: 18-56), 2 were metastatic, and the others were SSM (superficial spreading melanoma) in situ (n = 3) or Breslow index 0,56 mm (n = 1). Functional assays performed on CTNNA1 KO melanoma cell lines showed a loss of cell-to-cell adhesion phenotype, in accordance with the altered adherens junctions observed by electron microscopy, and the specific pathway enrichments observed by RNAseq. This specific phenotype could be rescued by transfection with a plasmid containing wild-type CTNNA1, as opposed to the CTNNA1 F611fs* plasmid. Germline CTNNA1 mutations are rare as none could be further identified in a DNA bank of 27 multiple melanoma families. In a database of 4743 melanomas somatically sequenced for CTNNA1, 131 of them had a CTNNA1 alteration (2,76%), with a median tumor mutational burden of 44 mut/MB of DNA (range from 0 to 451). Among them, 15 alterations were predicted to be inactivating, including 7 associated with a BRAF or NRAS activating mutation. Conclusions: Altogether, our results strongly support that CTNNA1 loss of function predisposes to melanoma formation characterized by a decreased cell adhesion. Since germline CTNNA1 alterations have already been implicated in lobular breast cancers and hereditary diffuse gastric cancers, CTNNA1 likely constitutes a tumor suppressor gene involved in familial melanoma, thus broadening the spectrum of syndromes associated with this gene.

Little is known about the genomic landscape of FGFR2‐altered gastroesophageal adenocarcinomas. This article attempts to bridge that gap, with a focus on concurrent alterations that may affect sensitivity to FGFR2‐directed therapies., Background. With the exception of trastuzumab, therapies directed at receptor tyrosine kinases (RTKs) in gastroesophageal adenocarcinomas (GEA) have had limited success. Recurrent fibroblast growth factor receptor 2 (FGFR2) alterations exist in GEA; however, little is known about the genomic landscape of FGFR2‐altered GEA. We examined FGFR2 alteration frequency and frequency of co‐occurring alterations in GEA. Subjects, Materials, and Methods. A total of 6,667 tissue specimens from patients with advanced GEA were assayed using hybrid capture‐based genomic profiling. Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA, and microsatellite instability was determined on 95 or 114 loci. Descriptive statistics were used to compare subgroups. Results. We identified a total of 269 (4.0%) FGFR2‐altered cases consisting of FGFR2‐amplified (amp; 193, 72% of FGFR2‐altered), FGFR2‐mutated (36, 13%), FGFR2‐rearranged (re; 23, 8.6%), and cases with multiple FGFR2 alterations (17, 6.3%). Co‐occurring alterations in other GEA RTK targets including ERBB2 (10%), EGFR (8%), and MET (3%) were observed across all classes of FGFR2‐altered GEA. Co‐occurring alterations in MYC (17%), KRAS (10%), and PIK3CA (5.6%) were also observed frequently. Cases with FGFR2amp and FGFR2re were exclusively microsatellite stable. The median TMB for FGFR2‐altered GEA was 3.6 mut/mb, not significantly different from a median of 4.3 mut/mb seen in FGFR2 wild‐type samples. Conclusion. FGFR2‐altered GEA is a heterogenous subgroup with approximately 20% of FGFR2‐altered samples harboring concurrent RTK alterations. Putative co‐occurring modifiers of FGFR2‐directed therapy including oncogenic MYC, KRAS, and PIK3CA alterations were also frequent, suggesting that pretreatment molecular analyses may be needed to facilitate rational combination therapies and optimize patient selection for clinical trials. Implications for Practice. Actionable receptor tyrosine kinase alterations assayed within a genomic context with therapeutic implications remain limited to HER2 amplification in gastroesophageal adenocarcinomas (GEA). Composite biomarkers and heterogeneity assessment are critical in optimizing patients selected for targeted therapies in GEA. Comprehensive genomic profiling in FGFR2‐altered GEA parallels the heterogeneity findings in HER2‐amplified GEA and adds support to the utility of genomic profiling in advanced gastroesophageal adenocarcinomas.

CGP results from >60,000 cases were screened to identify NTRK fusion events from cases of neuroendocrine tumors. 2417 NET patients from diverse anatomic sites were identified. From this dataset, six cases harbored NTRK fusions which included intra- and inter-chromosomal translocations. A NTRK fusion frequency of approximately 0.3% was found across all subtypes of NETs. Three cases involved translocations of NTRK1 with unique fusion partners (GPATCH4, PIP5K1A, CCDC19). Co-occurring alterations occurred in five cases. NTRK alterations were identified in nearly the full spectrum of NETs, including from the small intestine, pancreas, lung, and others. With the late stage clinical development of NTRK TKIs (including entrectinib and larotrectinib), these findings may further inform targeted approaches to therapy in NET.

Background Invasive lobular carcinoma (ILC) as a disease entity distinct from invasive ductal carcinoma (IDC) has merited focused studies of the genomic landscape, but those to date are largely limited to the assessment of early-stage cancers. Given that genomic alterations develop as acquired resistance to endocrine therapy, studies on refractory ILC are needed. Patients and methods Tissue from 336 primary-enriched, breast-biopsied ILC and 485 estrogen receptor (ER)-positive IDC and metastatic biopsy specimens from 180 ILC and 191 ER-positive IDC patients was assayed with hybrid-capture-based comprehensive genomic profiling for short variant, indel, copy number variants, and rearrangements in up to 395 cancer-related genes. Results Whereas ESR1 alterations are enriched in the metastases of both ILC and IDC compared with breast specimens, NF1 alterations are enriched only in ILC metastases (mILC). NF1 alterations are predominantly under loss of heterozygosity (11/14, 79%), are mutually exclusive with ESR1 mutations [odds ratio = 0.24, P 20 mutations/mb; P

PURPOSE BRCA1 or BRCA2 loss of function results in homologous recombination deficiency (HRD), which is targetable by poly (ADP-ribose) polymerase (PARP) inhibitors and other DNA-damaging agents. In cancers associated with germline BRCA1/2 alterations ( BRCA1/2-associated cancers: breast, ovarian, pancreatic, prostate), BRCA1/2 alterations result in HRD and are biomarkers for PARP inhibitor use. In other (non– BRCA1/2-associated) cancer types, the association between BRCA1/2 alteration and HRD is less clear. METHODS A total of 234,154 tumor samples were sequenced by hybrid capture-based comprehensive genomic profiling. Somatic, germline, and zygosity status was determined computationally. BRCA1/2 alterations were classified as predicted germline/somatic and biallelic/monoallelic. Genome-wide loss of heterozygosity (gLOH) was evaluated as a marker of HRD. RESULTS BRCA1/2 alterations were observed at a 4.7% frequency. BRCA1/2 mutations were predicted germline in 57.4% of BRCA1/2-associated and 37.2% of non– BRCA1/2-associated cancers. The fraction of BRCA1/2-altered cases that were biallelic was 68.7%, with a higher biallelic fraction in BRCA1/2-associated (89.9%) versus non– BRCA1/2-associated cancers (43.6%). Differences in tissue distribution of biallelic BRCA1 versus BRCA2 alterations were noted, including a higher rate of biallelic BRCA2 alteration in prostate cancer. Biallelic BRCA1/2 alteration was observed at a 3.2% frequency ( BRCA1/2-associated cancers, 8.9%; non– BRCA1/2-associated cancers, 1.3%) and > 1% frequency in at least 13 cancer types. Across cancer types, biallelic BRCA1/2 alteration was associated with increased gLOH versus monoallelic or wild-type BRCA1/2; predicted germline or somatic mutations were both associated with elevated gLOH. CONCLUSION Biallelic BRCA1/2 alterations were associated with elevated gLOH in diverse cancer types, including those not traditionally associated with BRCA1/2 cancer syndromes. Biomarker development for PARP inhibitors should integrate methods to distinguish biallelic from monoallelic BRCA1/2 status, and biallelic BRCA1/2 alteration should be broadly evaluated across cancer types as a biomarker for underlying HRD and PARP inhibitor sensitivity.

Background: Characterization of the different genomic alterations (GAs) in urothelial carcinoma (UC), by site of origin, may identify contrasting therapeutic opportunities and inform distinct putative pathogenetic mechanisms. Objective: To describe the genomic landscape of UC based on the anatomic site of the primary tumor. Design, setting, and participants: In total, 479 upper tract UC (UTUC) and 1984 bladder UC (BUC) patients underwent comprehensive genomic profiling (CGP) to evaluate all classes of GAs, tumor mutational burden (TMB), and microsatellite instability (MSI) status. Targetable GAs and signatures were assessed according to the European Society for Medical Oncology Scale for Clinical Actionability of molecular Targets (ESCAT). Intervention: Hybrid-capture–based CGP. Outcome measurements and statistical analysis: Descriptive analyses and differences between anatomic subgroups were reported. Results and limitations: In total, 39% of patients with UC harbored one or more tier 1–2 GAs, suggesting potential benefit from approved or investigational therapies. UTUC cases were enriched in FGFR3 short variant (SV) GA (20% vs 13%) and HRAS SV GA (7.3% vs 3%), the latter attributed specifically to enrichment in renal pelvis UC (9.5%) versus ureteral UC (1.8%, p = 0.002). RB1 GAs were more frequent in BUC than in UTUC (21% vs 7.8% p < 0.001). Non-FGFR3 kinase fusions were observed in 1% of patients, including BRAF/RAF1 fusions in 0.5%. BRAF mutations/fusions were observed in 2% of cases and were mutually exclusive with FGFR3 GA (p = 0.002). There were no differences of TMB high/MSI high for primary tumor and metastatic sites, but UTUC was enriched for MSI high (3.4%) relative to BUC (0.8%, p < 0.001). Conclusions: Differences in the genomic landscapes of UTUC and BUC were modest; however, patients with UTUC were enriched for FGFR3 and HRAS SV relative to BUC. Further investigation on UC, stratified by the site of origin, is warranted. In addition, these results suggest an opportunity for the routine incorporation of CGP prior to systemic therapy initiation in metastatic UC. Patient summary: Genomic profiling of advanced urothelial carcinoma can inform several therapeutic opportunities for patients, particularly those with upper tract urothelial carcinoma, an infrequent and generally aggressive tumor entity with nonoverlapping clinical features compared with its bladder counterpart, which is often treated based on the data extrapolated from bladder cancer.

Treatment with immune checkpoint inhibitors (ICPIs) extends survival in a proportion of patients across multiple cancers. Tumor mutational burden (TMB)-the number of somatic mutations per DNA megabase (Mb)-has emerged as a proxy for neoantigen burden that is an independent biomarker associated with ICPI outcomes. Based on findings from recent studies, TMB can be reliably estimated using validated algorithms from next-generation sequencing assays that interrogate a sufficiently large subset of the exome as an alternative to whole-exome sequencing. Biological processes contributing to elevated TMB can result from exposure to cigarette smoke and ultraviolet radiation, from deleterious mutations in mismatch repair leading to microsatellite instability, or from mutations in the DNA repair machinery. A variety of clinical studies have shown that patients with higher TMB experience longer survival and greater response rates following treatment with ICPIs compared with those who have lower TMB levels; this includes a prospective randomized clinical trial that found a TMB threshold of ≥10 mutations per Mb to be predictive of longer progression-free survival in patients with non-small cell lung cancer. Multiple trials are underway to validate the predictive values of TMB across cancer types and in patients treated with other immunotherapies. Here we review the rationale, algorithm development methodology, and existing clinical data supporting the use of TMB as a predictive biomarker for treatment with ICPIs. We discuss emerging roles for TMB and its potential future value for stratifying patients according to their likelihood of ICPI treatment response. IMPLICATIONS FOR PRACTICE: Tumor mutational burden (TMB) is a newly established independent predictor of immune checkpoint inhibitor (ICPI) treatment outcome across multiple tumor types. Certain next-generation sequencing-based techniques allow TMB to be reliably estimated from a subset of the exome without the use of whole-exome sequencing, thus facilitating the adoption of TMB assessment in community oncology settings. Analyses of multiple clinical trials across several cancer types have demonstrated that TMB stratifies patients who are receiving ICPIs by response rate and survival. TMB, alongside other genomic biomarkers, may provide complementary information in selecting patients for ICPI-based therapies.

Background The genomic landscape of primary clear cell renal cell carcinoma (ccRCC) has been well described. However, little is known about cohort genomic alterations (GA) landscape in ccRCC metastases, or how it compares to primary tumours in aggregate. The genomic landscape of metastases may have biological, clinical, and therapeutic implications. Methods We collected targeted next-generation sequencing mutation calls from two independent cohorts and described the metastases GA landscape and descriptively compared it to the GA landscape in primary tumours. Results The cohort 1 (n = 578) consisted of 349 primary tumours and 229 metastases. Overall, the most common mutations in the metastases were VHL (66.8%), PBRM1 (41.87%), and SETD2 (24.7%). TP53 was more frequently mutated in metastases compared to primary tumours (14.85% versus 8.9%; p = 0.031). No other gene had significant difference in the cohort frequency of mutations between the metastases and primary tumours. Mutation burden was not significantly different between the metastases and primary tumours or between metastatic sites. The second cohort (n = 257) consisted of 177 primary tumours and 80 metastases. No differences in frequency of mutations or mutational burden were observed between primaries and metastases. Conclusions These data support the theory that ccRCC primary tumours and metastases encompass a uniform distribution of common genomic alterations tested by next-generation sequencing targeted panels. This study does not address variability between matched primary tumours and metastases or the change in genomic alterations over time and after sequential systemic therapies.

// Ming Yin 1, 6 , Petros Grivas 2, 7 , Hamid Emamekhoo 3 , Prateek Mendiratta 2 , Siraj Ali 4 , JoAnn Hsu 5 , Monali Vasekar 1 , Joseph J. Drabick 1 , Sumanta Pal 5 and Monika Joshi 1 1 Department of Medicine, Division of Hematology-Oncology, Penn State Cancer Institute, Hershey, PA, USA 2 Department of Hematology and Medical Oncology, Cleveland Clinic Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA 3 Department of Medicine, Division of Hematology-Oncology, University of Wisconsin Carbone Cancer Center, WI, USA 4 Foundation Medicine, Cambridge, MA, USA 5 Department of Medical Oncology, City of Hope Comprehensive Cancer Center, Duarte, CA, USA 6 Department of Medicine, Division of Oncology, Ohio State University Comprehensive Cancer Center, Columbus, OH, USA 7 Department of Medicine, Division of Oncology, University of Washington, Seattle, WA, USA Correspondence to: Monika Joshi, email: mjoshi@pennstatehealth.psu.edu Keywords: biomarkers; prognosis; bladder cancer; genomic alterations; next generation sequencing Received: November 30, 2017 Accepted: March 02, 2018 Published: March 30, 2018 ABSTRACT Background: Mutations of DNA repair genes, e.g. ATM/RB1 , are frequently found in urothelial cancer (UC) and have been associated with better response to cisplatin-based chemotherapy. Further external validation of the prognostic value of ATM/RB1 mutations in UC can inform clinical decision making and trial designs. Results: In the discovery dataset, ATM/RB1 mutations were present in 24% of patients and were associated with shorter OS (adjusted HR 2.67, 95% CI, 1.45–4.92, p = 0.002). There was a higher mutation load in patients carrying ATM/RB1 mutations (median mutation load: 6.7 versus 5.5 per Mb, p = 0.072). In the validation dataset, ATM/RB1 mutations were present in 22.2% of patients and were non-significantly associated with shorter OS (adjusted HR 1.87, 95% CI, 0.97–3.59, p = 0.06) and higher mutation load (median mutation load: 8.1 versus 7.2 per Mb, p = 0.126). Materials and Methods: Exome sequencing data of 130 bladder UC patients from The Cancer Genome Atlas (TCGA) dataset were analyzed as a discovery cohort to determine the prognostic value of ATM/RB1 mutations. Results were validated in an independent cohort of 81 advanced UC patients. Cox proportional hazard regression analysis was performed to calculate the hazard ratio (HR) and 95% confidence interval (CI) to compare overall survival (OS). Conclusions: ATM/RB1 mutations may be a biomarker of poor prognosis in unselected UC patients and may correlate with higher mutational load. Further studies are required to determine factors that can further stratify prognosis and evaluate predictive role of ATM/RB1 mutation status to immunotherapy and platinum-based chemotherapy.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture–based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode–based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%–99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%–95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%–71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments–certified/College of American Pathologists–accredited/New York State–approved laboratory.

Background Genomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative. Patients and methods Hybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer. Results At least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%). Conclusions GAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.

// Roman Groisberg 1, 2 , David S. Hong 1 , Vijaykumar Holla 3 , Filip Janku 1 , Sarina Piha-Paul 1 , Vinod Ravi 4 , Robert Benjamin 4 , Shreyas Kumar Patel 4 , Neeta Somaiah 4 , Anthony Conley 4 , Siraj M. Ali 6 , Alexa B. Schrock 6 , Jeffrey S. Ross 6 , Philip J. Stephens 6 , Vincent A. Miller 6 , Shiraj Sen 1, 2 , Cynthia Herzog 5 , Funda Meric-Bernstam 1 and Vivek Subbiah 1 1 Department of Investigational Cancer Therapeutics (A Phase I Program), Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 2 Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 3 Khalifa Institute for Personalized Cancer Therapy (IPCT), The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 4 Department of Sarcoma Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 5 Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 6 Foundation Medicine Inc, Cambridge, Massachusetts 02139, USA Correspondence to: Vivek Subbiah, email: vsubbiah@mdanderson.org Keywords: sarcoma, targeted therapy, phase I trials Received: January 29, 2017 Accepted: March 08, 2017 Published: April 05, 2017 ABSTRACT Background: There are currently no United States Food and Drug Administration approved molecularly matched therapies for sarcomas except gastrointestinal stromal tumors. Complicating this is the extreme diversity, heterogeneity, and rarity of these neoplasms. Few therapeutic options exist for relapsed and refractory sarcomas. In clinical practice many oncologists refer patients for genomic profiling hoping for guidance on treatment options after standard therapy. However, a systematic analysis of actionable mutations has yet to be completed. We analyzed genomic profiling results in patients referred to MD Anderson Cancer Center with advanced sarcomas to elucidate the frequency of potentially actionable genomic alterations in this population. Methods: We reviewed charts of patients with advanced sarcoma who were referred to investigational cancer therapeutics department and had CLIA certified comprehensive genomic profiling (CGP) of 236 or 315 cancer genes in at least 50ng of DNA. Actionable alterations were defined as those identifying anti-cancer drugs on the market, in registered clinical trials, or in the Drug-Gene Interaction Database. Results: Among the 102 patients analyzed median age was 45.5 years (range 8-76), M: F ratio 48:54. The most common subtypes seen in our study were leiomyosarcoma (18.6%), dedifferentiated liposarcoma (11%), osteosarcoma (11%), well-differentiated liposarcoma (7%), carcinosarcoma (6%), and rhabdomyosarcoma (6%). Ninety-five out of 102 patients (93%) had at least one genomic alteration identified with a mean of six mutations per patient. Of the 95 biopsy samples with identifiable genomic alterations, the most commonly affected genes were TP53 (31.4%), CDK4 (23.5%), MDM2 (21.6%), RB1 (18.6%), and CDKN2A/B (13.7%). Notable co-segregating amplifications included MDM2-CDK4 and FRS2-FGF. Sixteen percent of patients received targeted therapy based on CGP of which 50% had at least stable disease. Conclusions: Incorporating CGP into sarcoma management may allow for more precise diagnosis and sub-classification of this diverse and rare disease, as well as personalized matching of patients to targeted therapies such as those available in basket clinical trials.

In non-small-cell lung cancer (NSCLC) refractory to standard therapy and which lacks well-known oncogenic drivers, genomic profiling can still identify genomic alterations that may suggest potential sensitivity to targeted therapy. PTEN mutation in NSCLC may be sensitizing to analogs of rapamycin such as everolimus or temsirolimus, but more investigation is needed. We report the case of a patient with metastatic NSCLC harboring a PTEN mutation as well as high tumor mutational burden and PD-L1 positivity with a durable response to temsirolimus, but refractory to a checkpoint inhibitor. Even in the event of failure of treatment with checkpoint inhibitors in the background of a case with a higher tumor mutational burden and PD-L1 positivity, targeting specific genomic alterations may still result in patient benefit., Video abstract

Colorectal cancer (CRC) is increasingly appreciated as a heterogeneous disease, with factors such as microsatellite instability (MSI), cancer subsite within the colon versus rectum, and age of diagnosis associated with specific disease course and therapeutic response. Activating oncogenic mutations in KRAS and NRAS are common in CRC, driving tumor progression and influencing efficacy of both cytotoxic and targeted therapies. The RAS mutational spectrum differs substantially between tumors arising from distinct tissues. Structure-function analysis of relatively common somatic RAS mutations in G12, Q61, and other codons is characterized by differing potency and modes of action. Here we show the mutational profile of KRAS, NRAS, and the less common HRAS in 13,336 CRC tumors, comparing the frequency of specific mutations based on age of diagnosis, MSI status, and colon versus rectum subsite. We identify mutation hotspots, and unexpected differences in mutation spectrum, based on these clinical parameters., Activating oncogenic mutations in KRAS and NRAS are common in colorectal cancer, which is a heterogenous disease. Here, the authors show that the RAS mutation spectrum is markedly different between colon and rectal cancer, and also different based on age of diagnosis and microsatellite instability.

International audience; BACKGROUND: BRAF and MEK inhibitors are approved for BRAF V600-mutated advanced melanoma, with response rates of up to 70%. Responses to targeted therapies have also been observed for diverse non-V600 BRAF alterations. Thus, sensitive, accurate, and broad detection of BRAF alterations is critical to match patients with available targeted therapies.MATERIALS AND METHODS: Pathology reports were reviewed for 385 consecutive melanoma cases with BRAF mutations or rearrangements identified using a hybrid capture-based next-generation sequencing comprehensive genomic profiling (CGP) assay during the course of clinical care.RESULTS: Records of prior BRAF molecular testing were available for 79 (21%) cases. Of cases with BRAF V600 mutations, 11/57 (19%) with available data were negative by prior BRAF testing. Prior negative BRAF results were also identified in 16/20 (80%) cases with non-V600 mutations, 2 of which harbored multiple BRAF alterations, and in 2/2 (100%) cases with activating BRAF fusions. Clinical outcomes for a subset of patients are presented.CONCLUSION: CGP identifies diverse activating BRAF alterations in a significant fraction of cases with prior negative testing. Given the proven clinical benefit of BRAF/MEK inhibitors in BRAF-mutated melanoma, CGP should be considered for patients with metastatic melanoma, particularly if other testing is negative.IMPLICATIONS FOR PRACTICE: Published guidelines for melanoma treatment recommend BRAF mutational analysis, but little guidance is provided as to selection criteria for testing methodologies, or as to clinical implications for non-V600 alterations. This study found that hybrid capture-based next-generation sequencing can detect BRAF alterations in samples from a significant fraction of patients with advanced melanoma with prior negative BRAF results. This study highlights the need for oncologists and pathologists to be critically aware of coverage and sensitivity limitations of various assays, particularly regarding non-V600E alterations, of which many are potentially targetable.

Background Alterations in the DNA damage response (DDR) pathway confer sensitivity to certain chemotherapies, radiation, and other DNA damage repair targeted therapies. BRCA1/2 are the most well-studied DDR genes, but recurrent alterations are described in other DDR pathway members across cancers. Deleterious DDR alterations may sensitize tumor cells to poly (ADP-ribose) polymerase inhibition, but there are also increasing data suggesting that there may also be synergy with immune checkpoint inhibitors. The relevance of DDR defects in gastrointestinal (GI) cancers is understudied. We sought to characterize DDR-defective GI malignancies and to explore genomic context and tumor mutational burden (TMB) to provide a platform for future rational investigations. Materials and Methods Tumor samples from 17,486 unique patients with advanced colorectal, gastroesophageal, or small bowel carcinomas were assayed using hybrid-capture-based comprehensive genomic profiling including sequencing of 10 predefined DDR genes: ARID1A, ATM, ATR, BRCA1, BRCA2, CDK12, CHEK1, CHEK2, PALB2, and RAD51. TMB (mutations per megabase [mut/Mb]) was calculated from up to 1.14 Mb of sequenced DNA. Clinicopathologic features were extracted and descriptive statistics were used to explore genomic relationships among identified subgroups. Results DDR alterations were found in 17% of cases: gastric adenocarcinoma 475/1,750 (27%), small bowel adenocarcinoma 148/666 (22%), esophageal adenocarcinoma 467/2,501 (19%), and colorectal cancer 1,824/12,569 (15%). ARID1A (9.2%) and ATM (4.7%) were the most commonly altered DDR genes in this series, followed by BRCA2 (2.3%), BRCA1 (1.1%), CHEK2 (1.0%), ATR (0.8%), CDK12 (0.7%), PALB2 (0.6%), CHEK1 (0.1%) and RAD51 (0.1%). More than one DDR gene alteration was found in 24% of cases. High microsatellite instability (MSI-H) and high TMB (TMB-H, ≥20 mut/Mb) were found in 19% and 21% of DDR-altered cases, respectively. Of DDR-altered/TMB-H cases, 87% were also MSI-H. However, even in the microsatellite stable (MSS)/DDR-wild-type (WT) versus MSS/DDR-altered, TMB-high was seen more frequently (0.4% vs. 3.3%, P < .00001.) Median TMB was 5.4 mut/Mb in the MSS/DDR-altered subset versus 3.8 mut/Mb in the MSS/DDR-WT subset (P ≤ .00001), and ATR alterations were enriched in the MSS/TMB-high cases. Conclusion This is the largest study to examine selected DDR defects in tubular GI cancers and confirms that DDR defects are relatively common and that there is an association between the selected DDR defects and a high TMB in more than 20% of cases. Microsatellite stable DDR-defective tumors with elevated TMB warrant further exploration. Implications for Practice Deleterious DNA damage response (DDR) alterations may sensitize tumor cells to poly (ADP-ribose) polymerase inhibition, but also potentially to immune checkpoint inhibitors, owing to accumulation of mutations in DDR-defective tumors. The relevance of DDR defects in gastrointestinal (GI) cancers is understudied. This article characterizes DDR-defective GI malignancies and explores genomic context and tumor mutational burden to provide a platform for future rational investigations.

Purpose: The third-generation EGFR inhibitor, osimertinib, is the first mutant-selective inhibitor that has received regulatory approval for the treatment of patients with EGFR-mutant lung cancer. Despite the development of highly selective third-generation inhibitors, acquired resistance remains a significant clinical challenge. Recently, we and others have identified a novel osimertinib resistance mutation, G724S, which was not predicted in in vitro screens. Here, we investigate how G724S confers resistance to osimertinib. Experimental Design: We combine structure-based predictive modeling of G724S in combination with the 2 most common EGFR-activating mutations, exon 19 deletion (Ex19Del) and L858R, with in vitro drug-response models and patient genomic profiling. Results: Our simulations suggest that the G724S mutation selectively reduces osimertinib-binding affinity in the context of Ex19Del. Consistent with our simulations, cell lines transduced with Ex19Del/G724S demonstrate resistance to osimertinib, whereas cells transduced with L858R/G724S are sensitive to osimertinib. Subsequent clinical genomic profiling data further suggest G724S occurs with Ex19Del but not L858R. Furthermore, we demonstrate that Ex19Del/G724S retains sensitivity to afatinib, but not to erlotinib, suggesting a possible therapy for patients at the time of disease relapse. Conclusions: Altogether, these data suggest that G724S is an allele-specific resistance mutation emerging in the context of Ex19Del but not L858R. Our results fundamentally reframe the problem of targeted therapy resistance from one focused on the “drug–resistance mutation” pair to one focused on the “activating mutation–drug–resistance mutation” trio. This has broad implications across clinical oncology.

// Sun Min Lim 1,2,* , Hyung Soon Park 3,* , Sangwoo Kim 4 , Sora Kim 4 , Siraj M. Ali 5 , Joel R. Greenbowe 5 , In Seok Yang 4 , Nak-Jung Kwon 6 , Jae Lyun Lee 7 , Min-Hee Ryu 7 , Jin-Hee Ahn 7 , Jeeyun Lee 8 , Min Goo Lee 3 , Hyo Song Kim 1 , Hyunki Kim 9 , Hye Ryun Kim 1 , Yong Wha Moon 1,2 , Hyun Cheol Chung 1 , Joo-Hang Kim 2 , Yoon-Koo Kang 7 and Byoung Chul Cho 1 1 Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea 2 Division of Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea 3 Department of Pharmacology and Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea 4 Severance Biomedical Science Institute and Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea 5 Foundation Medicine Inc, Cambridge, MA, USA 6 MacroGen Inc., Seoul, Korea 7 Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 8 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 9 Department of Pathology, Yonsei University College of Medicine, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Yoon-Koo Kang, email: // Byoung Chul Cho, email: // Keywords : everolimus, NF1, TSC1, mTOR, next-generation sequencing Received : November 27, 2015 Accepted : January 25, 2016 Published : February 07, 2016 Abstract Background: Given the modest responses to everolimus, a mTOR inhibitor, in multiple tumor types, there is a pressing need to identify predictive biomarkers for this drug. Using targeted ultra-deep sequencing, we aimed to explore genomic alterations that confer extreme sensitivity to everolimus. Results: We collected formalin-fixed paraffin-embedded tumor/normal pairs from 39 patients (22 with exceptional clinical benefit, 17 with no clinical benefit) who were treated with everolimus across various tumor types (13 gastric cancers, 15 renal cell carcinomas, 2 thyroid cancers, 2 head and neck cancer, and 7 sarcomas). Ion AmpliSeq TM Comprehensive Cancer Panel was used to identify alterations across all exons of 409 target genes. Tumors were sequenced to a median coverage of 552x. Cancer genomes are characterized by 219 somatic single-nucleotide variants (181 missense, 9 nonsense, 7 splice-site) and 22 frameshift insertions/deletions, with a median of 2.1 mutations per Mb (0 to 12.4 mutations per Mb). Overall, genomic alterations with activating effect on mTOR signaling were identified in 10 of 22 (45%) patients with clinical benefit and these include MTOR, TSC1, TSC2, NF1 , PIK3CA and PIK3CG mutations. Recurrently mutated genes in chromatin remodeling genes ( BAP1; n = 2, 12%) and receptor tyrosine kinase signaling ( FGFR4; n = 2, 12%) were noted only in patients without clinical benefit. Conclusions: Regardless of different cancer types, mTOR-pathway-activating mutations confer sensitivity to everolimus. Targeted sequencing of mTOR pathway genes facilitates identification of potential candidates for mTOR inhibitors.

The subset of metastatic colorectal adenocarcinomas that harbor BRAF V600E mutations are aggressive tumors with significantly shortened survival and limited treatment options. Here we present a colorectal cancer patient whose disease progressed through standard chemotherapy and who developed liver metastasis. Comprehensive genomic profiling (FoundationOne(®)) identified a BRAF V600E mutation in the liver lesion, as well as other genomic alterations consistent with colorectal cancers. Combination therapy of dabrafenib and trametinib with standard cytotoxic chemotherapy resulted in a durable major ongoing response for the patient. This report illustrates the utility of comprehensive genomic profiling with personalized targeted therapy for aggressive metastatic colorectal adenocarcinomas.

Purpose Dabrafenib and trametinib are approved for the management of advanced non–small-cell lung cancers (NSCLCs) that harbor BRAF V600E mutations. Small series and pan-cancer analyses have identified non-V600 alterations as therapeutic targets. We sought to examine a large genomic data set to comprehensively characterize non-V600 B RAF alterations in lung cancer. Patients and Methods A total of 23,396 patients with lung cancer provided data to assay with comprehensive genomic profiling. Data were reviewed for predicted pathogenic BRAF base substitutions, short insertions and deletions, copy number changes, and rearrangements. Results Adenocarcinomas represented 65% of the occurrences; NSCLC not otherwise specified (NOS), 15%; squamous cell carcinoma, 12%; and small-cell lung carcinoma, 5%. BRAF was altered in 4.5% (1,048 of 23,396) of all tumors; 37.4% (n = 397) were BRAF V600E, 38% were BRAF non-V600E activating mutations, and 18% were BRAF inactivating. Rearrangements were observed at a frequency of 4.3% and consisted of N-terminal deletions (NTDs; 0.75%), kinase domain duplications (KDDs; 0.75%), and BRAF fusions (2.8%). The fusions involved three recurrent fusion partners: ARMC10, DOCK4, and TRIM24. BRAF V600E was associated with co-occurrence of SETD2 alterations, but other BRAF alterations were not and were instead associated with CDKN2A, TP53, and STK11 alterations ( P < .05). Potential mechanisms of acquired resistance to BRAF V600E inhibition are demonstrated. Conclusion This series characterized the frequent occurrence (4.4%) of BRAF alterations in lung cancers. Recurrent BRAF alterations in NSCLC adenocarcinoma are comparable to the frequency of other NSCLC oncogenic drivers, such as ALK, and exceed that of ROS1 or RET. This work supports a broad profiling approach in lung cancers and suggests that non-V600E BR AF alterations represent a subgroup of lung cancers in which targeted therapy should be considered.

Castleman disease (CD) is a rare lymphoproliferative disorder subclassified as unicentric CD (UCD) or multicentric CD (MCD) based on clinical features and the distribution of enlarged lymph nodes with characteristic histopathology. MCD can be further subtyped based on human herpes virus 8 (HHV8) infection into HHV8-associated MCD, HHV8 − /idiopathic MCD (iMCD), and polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin change (POEMS)–associated MCD. In a subset of cases of UCD, an associated follicular dendritic cell sarcoma (FDCS) may be seen. Although numerous reports of the clinical and histologic features of UCD, MCD, and FDCS exist, an understanding of the genetic and epigenetic landscape of these rare diseases is lacking. Given this paucity of knowledge, we analyzed 15 cases of UCD and 3 cases of iMCD by targeted next-generation sequencing (NGS; 405 genes) and 3 cases of FDCS associated with UCD hyaline vascular variant (UCD-HVV) by whole-exome sequencing. Common amplifications of ETS1 , PTPN6 , and TGFBR2 were seen in 1 iMCD and 1 UCD case; the iMCD case also had a somatic DNMT3A L295Q mutation. This iMCD patient also showed clinicopathologic features consistent with a specific subtype known as Castleman-Kojima disease (thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly [TAFRO] clinical subtype). Additionally, 1 case of UCD-HVV showed amplification of the cluster of histone genes on chromosome 6p. FDCS associated with UCD-HVV showed mutations and copy number changes in known oncogenes, tumor suppressors, and chromatin structural-remodeling proteins.

Background Genomic fusions of the anaplastic lymphoma kinase gene (ALK) are a well-established therapy target in non-small cell lung cancer (NSCLC). From a survey of 114,200 clinical cases, we determined the prevalence of ALK rearrangements (rALK) in non-NSCLC tumors and report their responsiveness to therapies targeting ALK. Materials and Methods Comprehensive genomic profiling of 114,200 relapsed and metastatic malignancies, including both solid tumors and hematolymphoid cancers, was performed using a hybrid-capture, adaptor ligation-based next-generation sequencing assay. Results Of 114,200 clinical samples, 21,522 (18.8%) were NSCLC and 92,678 (81.2%) were other tumor types. Of the 876 (0.8%) cases with ALK fusions (fALK) or rALK, 675 (77.1%) were NSCLC and 201 (22.9%) were other tumor types. ALK fusions were significantly more frequent in NSCLC (3.1%) than non-NSCLC (0.2%; p Conclusion ALK rearrangements can be identified in a wide variety of epithelial and mesenchymal malignancies beyond NSCLC. Anti-ALK therapies can be effective in non-NSCLC tumors driven by fALK, and further study of therapies targeting ALK in clinical trials involving a wider variety of cancer types appears warranted.

// Jeeyun Lee 1, * , Hee Cheol Kim 2, * , Jung Yong Hong 3, * , Kai Wang 4 , Sun Young Kim 1 , Jiryeon Jang 1 , Seung Tae Kim 1 , Joon Oh Park 1 , Ho Yeong Lim 1 , Won Ki Kang 1 , Young Suk Park 1 , Jiyun Lee 1 , Woo Yong Lee 2 , Yoon Ah Park 2 , Jung Wook Huh 2 , Seong Hyeon Yun 2 , In-Gu Do 5 , Seok Hyung Kim 5 , Sohail Balasubramanian 4 , Philip J. Stephens 4 , Jeffrey S. Ross 4, 6 , Gang Gary Li 7 , Zachary Hornby 7 , Siraj M. Ali 4 , Vincent A. Miller 4 , Kyoung-Mee Kim 5, 8 , Sai-Hong Ignatius Ou 9 1 Department of Medicine, Division of Hematology-Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 2 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 3 Department of Internal Medicine, Chung-Ang University College of Medicine, Dongjak-Gu, Seoul, Republic of Korea 4 Foundation Medicine Inc, Cambridge, Massachusetts, USA 5 Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 6 Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York, USA 7 Ignyta Inc, San Diego, California, USA 8 Innovative Cancer Medicine Institute, Samsung Medical Center, Seoul, Korea 9 Chao Family Comprehensive Cancer Center, University of California Irvine School of Medicine, Orange, California, USA * These authors have contributed equally to this work Correspondence to: Sai-Hong Ignatius Ou, e-mail: Ignatius.ou@uci.edu Kyoung-Mee Kim, e-mail: kkmkys@skku.edu Keywords: colorectal carcinoma, anaplastic lymphoma kinase (ALK) rearrangement, immunohistochemistry, next generation sequencing Received: April 02, 2015 Accepted: June 19, 2015 Published: July 01, 2015 ABSTRACT Purpose: Anaplastic lymphoma kinase (ALK) rearrangement has been detected in colorectal carcinoma (CRC) using advanced molecular diagnostics tests including exon scanning, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). We investigated if immunohistochemistry (IHC) can be used to detect ALK rearrangement in gastrointestinal malignancies. Experimental designs: Tissue microarrays (TMAs) from consecutive gastric carcinoma (GC) and CRC patients who underwent surgical resection at Samsung Medical Center, Seoul, Korea were screened by IHC using ALK monoclonal antibody 5A4. IHC positive cases were confirmed by FISH, nCounter assays, and NGS-based comprehensive genomic profiling (CGP). ALK IHC was further applied to CRC patients enrolled in a pathway-directed therapeutic trial. Results: Four hundred thirty-two GC and 172 CRC cases were screened by IHC. No GC sample was ALK IHC positive. One CRC (0.6%) was ALK IHC positive (3+) that was confirmed by ALK FISH and a novel CAD-ALK (C35; A20) fusion variant that resulted from a paracentric inversion event inv(2)(p22–21p23) was identified by CGP. One out of 50 CRC patients enrolled in a pathway-directed therapeutic trial was ALK IHC positive (3+) confirmed by ALK FISH and found to harbor the EML4-ALK (E21, A20) fusion variant by CGP. Growth of a tumor cell line derived from this EML4-ALK CRC patient was inhibited by ALK inhibitors crizotinib and entrectinib. Conclusions: ALK IHC is a viable screening strategy for identifying ALK rearrangement in CRC. ALK rearrangement is a potential actionable driver mutation in CRC based on survival inhibition of patient tumor-derived cell line by potent ALK inhibitors.

Cervical cancer epitomizes the success of cancer prevention through the human papillomavirus (HPV) vaccine, but significant challenges remain in the treatment of advanced disease. We report the first three cases of cervical carcinoma harboring an FGFR3–TACC3 fusion, which serves as a novel therapeutic target. The fusion, identified by comprehensive genomic profiling, activates the FGFR pathway that has been implicated in HPV-driven carcinogenesis. One of the patients whose tumor contained the FGFR3–TACC3 fusion was treated with an investigational FGFR tyrosine kinase inhibitor. Concomitant molecular alterations involving the PI3K/AKT/mTOR and RAF/MEK pathways were also identified and suggest other treatment strategies that deserve investigation. This case series highlights the role of comprehensive genomic profiling in the identification of new therapeutic targets and in targeted therapy selection for patients with cervical cancer., Highlights • The first cases of cervical carcinoma harboring an FGFR3–TACC3 fusion • One of patients whose tumor contained the FGFR3–TACC3 fusion received an investigational FGFR tyrosine kinase inhibitor • Manuscript highlights the role of genomic profiling identifying new therapeutic targets and targeted therapies

Background Thyroid carcinoma, which is rare in pediatric patients (age 0–18 years) but more common in adolescent and young adult (AYA) patients (age 15–39 years), carries the potential for morbidity and mortality. Methods Hybrid-capture-based comprehensive genomic profiling (CGP) was performed prospectively on 512 consecutively submitted thyroid carcinomas, including 58 from pediatric and AYA (PAYA) patients, to identify genomic alterations (GAs), including base substitutions, insertions/deletions, copy number alterations, and rearrangements. This PAYA data series includes 41 patients with papillary thyroid carcinoma (PTC), 3 with anaplastic thyroid carcinoma (ATC), and 14 with medullary thyroid carcinoma (MTC). Results GAs were detected in 93% (54/58) of PAYA cases, with a mean of 1.4 GAs per case. In addition to BRAF V600E mutations, detected in 46% (19/41) of PAYA PTC cases and in 1 of 3 AYA ATC cases, oncogenic fusions involving RET, NTRK1, NTRK3, and ALK were detected in 37% (15/41) of PAYA PTC and 33% (1/3) of AYA ATC cases. Ninety-three percent (13/14) of MTC patients harbored RET alterations, including 3 novel insertions/deletions in exons 6 and 11. Two of these MTC patients with novel alterations in RET experienced clinical benefit from vandetanib treatment. Conclusion CGP identified diverse clinically relevant GAs in PAYA patients with thyroid carcinoma, including 83% (34/41) of PTC cases harboring activating kinase mutations or activating kinase rearrangements. These genomic observations and index cases exhibiting clinical benefit from targeted therapy suggest that young patients with advanced thyroid carcinoma can benefit from CGP and rationally matched targeted therapy.

Background Metastatic recurrence after treatment for locoregional cancer is a major cause of morbidity and cancer-specific mortality. Distinguishing metastatic recurrence from the development of a second primary cancer has important prognostic and therapeutic value and represents a difficult clinical scenario. Advances beyond histopathological comparison are needed. We sought to interrogate the ability of comprehensive genomic profiling (CGP) to aid in distinguishing between these clinical scenarios. Materials and methods We identified three prospective cases of recurrent tumors in patients previously treated for localized cancers in which histologic analyses suggested subsequent development of a distinct second primary. Paired samples from the original primary and recurrent tumor were subjected to hybrid capture next-generation sequencing-based CGP to identify base pair substitutions, insertions, deletions, copy number alterations (CNA), and chromosomal rearrangements. Genomic profiles between paired samples were compared using previously established statistical clonality assessment software to gauge relatedness beyond global CGP similarities. Results A high degree of similarity was observed among genomic profiles from morphologically distinct primary and recurrent tumors. Genomic information suggested reclassification as recurrent metastatic disease, and patients received therapy for metastatic disease based on the molecular determination. Conclusions Our cases demonstrate an important adjunct role for CGP technologies in separating metastatic recurrence from development of a second primary cancer. Larger series are needed to confirm our observations, but comparative CGP may be considered in patients for whom distinguishing metastatic recurrence from a second primary would alter the therapeutic approach. The Oncologist 2017;22:152-157Implications for Practice: Distinguishing a metastatic recurrence from a second primary cancer can represent a difficult clinicopathologic problem but has important prognostic and therapeutic implications. Approaches to aid histologic analysis may improve clinician and pathologist confidence in this increasingly common clinical scenario. Our series provides early support for incorporating paired comprehensive genomic profiling in clinical situations in which determination of metastatic recurrence versus a distinct second primary cancer would influence patient management.

Pediatric cancers are generally characterized by low mutational burden and few recurrently mutated genes. Recent studies suggest that genomic alterations may help guide treatment decisions and clinical trial selection. Here, we describe genomic profiles from 1,215 pediatric tumors representing sarcomas, extracranial embryonal tumors, brain tumors, hematologic malignancies, carcinomas, and gonadal tumors. Comparable published datasets identified similar frequencies of clinically relevant alterations, validating this dataset as biologically relevant. We identified novel ALK fusions in a neuroblastoma (BEND5–ALK) and an astrocytoma (PPP1CB–ALK), novel BRAF fusions in an astrocytoma (BCAS1–BRAF) and a ganglioglioma (TMEM106B–BRAF), and a novel PAX3–GLI2 fusion in a rhabdomyosarcoma. Previously characterized ALK, NTRK1, and PAX3 fusions were observed in unexpected malignancies, challenging the "disease-specific" alterations paradigm. Finally, we identified recurrent variants of unknown significance in MLL3 and PRSS1 predicted to have functional impact. Data from these 1,215 tumors are publicly available for discovery and validation. Cancer Res; 77(2); 509–19. ©2017 AACR.

Purpose TMPRSS2-ERG gene fusions are frequently found in prostate cancer and are pathognomonic for prostatic origin. In a series of cancer cases assayed with comprehensive genomic profiling (CGP) in the course of clinical care, we reviewed the frequency of TMPRSS2-ERG fusions in patient tumors of various histologic subtypes. Methods Frequency of TMPRSS2-ERG fusions was determined in CGPs from 64,263 cancer cases submitted to Foundation Medicine to assess genomic alterations suggesting benefit from targeted therapy. Genomic results are presented from an index case of prostate cancer that underwent evolution from adenocarcinoma to pure squamous cell carcinoma. Results TMPRSS2-ERG fusions were identified for 0.86% of male patients (250 of 29,030) and not found for female patients (none of 35,233). TMPRSS2-ERG fusions were detected in six tumors classified as squamous carcinoma, five of which were of unknown primary site. The index case is a patient with a large, left retrovesical mass diagnosed as squamous carcinoma by morphologic examination and a history of Gleason score 9 prostate cancer with prior prostatectomy and salvage radiation therapy. TMPRSS2-ERG was detected by genomic profiling in the squamous cell tumor, the primary adenocarcinoma of the prostate, and in a metachronous prostatic adenocarcinoma metastasis. On the basis of these results, the patient received androgen deprivation therapy. A phylogenetic tree demonstrating clonal and histopathologic evolution of prostate cancer in the index patient was constructed. Conclusion In this large CGP dataset, TMPRSS2-ERG fusion was seen in approximately 30% of prostate cancers regardless of histologic type; on occasion, the fusion was detected in advanced cancers not initially carrying a diagnosis of prostate carcinoma. CGP of advanced cancers in men may reveal prostatic origin by detection of the pathognomonic TMPRSS2-ERG fusion gene.

Purpose An association between mutational burden and response to immune checkpoint therapy has been documented in several cancer types. The potential for such a mutational burden threshold to predict response to immune checkpoint therapy was evaluated in several clinical datasets, where mutational burden was measured either by whole-exome sequencing or by using commercially available sequencing panels. Methods Whole-exome sequencing and RNA sequencing data of 33 solid cancer types from The Cancer Genome Atlas were analyzed to determine whether a robust immune checkpoint–activating mutation (iCAM) burden threshold associated with evidence of immune checkpoint activation exists in these cancers that may serve as a biomarker of response to immune checkpoint blockade therapy. Results We found that a robust iCAM threshold, associated with signatures of immune checkpoint activation, exists in eight of 33 solid cancers: melanoma, lung adenocarcinoma, colon adenocarcinoma, endometrial cancer, stomach adenocarcinoma, cervical cancer, estrogen receptor–positive/human epidermal growth factor receptor 2–negative breast cancer, and bladder-urothelial cancer. Tumors with a mutational burden higher than the threshold (iCAM positive) also had clear histologic evidence of lymphocytic infiltration. In published datasets of melanoma, lung adenocarcinoma, and colon cancer, patients with iCAM-positive tumors had significantly better response to immune checkpoint therapy compared with those with iCAM-negative tumors. Receiver operating characteristic analysis using The Cancer Genome Atlas predictions as the gold standard showed that iCAM-positive tumors are accurately identifiable using clinical sequencing assays, such as FoundationOne (Foundation Medicine, Cambridge, MA) or StrandAdvantage (Strand Life Sciences, Bangalore, India). Using the FoundationOne-derived threshold, an analysis of 113 melanoma tumors showed that patients with iCAM-positive disease have significantly better response to immune checkpoint therapy. iCAM-positive and iCAM-negative tumors have distinct mutation patterns and different immune microenvironments. Conclusion In eight solid cancers, a mutational burden threshold exists that may predict response to immune checkpoint blockade. This threshold is identifiable using available clinical sequencing assays.