Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq.

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.

R269C variant of ESR1: high prevalence and differential function in a subset of pancreatic cancers.

BACKGROUND: Estrogen receptor α (ESR1) plays a critical role in promoting growth of various cancers. Yet, its role in the development of pancreatic cancer is not well-defined. A less studied region of ESR1 is the hinge region, connecting the ligand binding and DNA domains. rs142712646 is a rare SNP in ESR1, which leads to a substitution of arginine to cysteine at amino acid 269 (R269C). The mutation is positioned in the hinge region of ESR1, hence may affect the receptor structure and function. We aimed to characterize the activity of R269C-ESR1 and study its role in the development of pancreatic cancer. METHODS: Transcriptional activity was evaluated by E2-response element (ERE) and AP1 -luciferase reporter assays and qRT-PCR. Proliferation and migration were assessed using MTT and wound healing assays. Gene-expression analysis was performed using RNAseq. RESULTS: We examined the presence of this SNP in various malignancies, using the entire database of FoundationOne and noted enrichment of it in a subset of pancreatic non-ductal adenocarcinoma (n = 2800) compared to pancreatic ductal adenocarcinoma (PDAC) as well as other tumor types (0.53% vs 0.29%, p = 0.02). Studies in breast and pancreatic cancer cells indicated cell type-dependent activity of ESR1 harboring R269C. Thus, expression of R269C-ESR1 enhanced proliferation and migration of PANC-1 and COLO-357 pancreatic cancer cells but not of MCF-7 breast cancer cells. Moreover, R269C-ESR1 enhanced E2-response elements (ERE) and AP1-dependent transcriptional activity and increased mRNA levels of ERE and AP1-regulated genes in pancreatic cancer cell lines, but had a modest effect on MCF-7 breast cancer cells. Accordingly, whole transcriptome analysis indicated alterations of genes associated with tumorigenicity in pancreatic cancer cells and upregulation of genes associated with cell metabolism and hormone biosynthesis in breast cancer cells. CONCLUSIONS: Our study shed new light on the role of the hinge region in regulating transcriptional activity of the ER and indicates cell-type specific activity, namely increased activity in pancreatic cancer cells but reduced activity in breast cancer cells. While rare, the presence of rs142712646 may serve as a novel genetic risk factor, and a possible target for therapy in a subset of non-ductal pancreatic cancers.

A computational approach to distinguish somatic vs. germline origin of genomic alterations from deep sequencing of cancer specimens without a matched normal.

A key constraint in genomic testing in oncology is that matched normal specimens are not commonly obtained in clinical practice. Thus, while well-characterized genomic alterations do not require normal tissue for interpretation, a significant number of alterations will be unknown in whether they are germline or somatic, in the absence of a matched normal control. We introduce SGZ (somatic-germline-zygosity), a computational method for predicting somatic vs. germline origin and homozygous vs. heterozygous or sub-clonal state of variants identified from deep massively parallel sequencing (MPS) of cancer specimens. The method does not require a patient matched normal control, enabling broad application in clinical research. SGZ predicts the somatic vs. germline status of each alteration identified by modeling the alteration's allele frequency (AF), taking into account the tumor content, tumor ploidy, and the local copy number. Accuracy of the prediction depends on the depth of sequencing and copy number model fit, which are achieved in our clinical assay by sequencing to high depth (>500x) using MPS, covering 394 cancer-related genes and over 3,500 genome-wide single nucleotide polymorphisms (SNPs). Calls are made using a statistic based on read depth and local variability of SNP AF. To validate the method, we first evaluated performance on samples from 30 lung and colon cancer patients, where we sequenced tumors and matched normal tissue. We examined predictions for 17 somatic hotspot mutations and 20 common germline SNPs in 20,182 clinical cancer specimens. To assess the impact of stromal admixture, we examined three cell lines, which were titrated with their matched normal to six levels (10-75%). Overall, predictions were made in 85% of cases, with 95-99% of variants predicted correctly, a significantly superior performance compared to a basic approach based on AF alone. We then applied the SGZ method to the COSMIC database of known somatic variants in cancer and found >50 that are in fact more likely to be germline.

Hybrid Capture-Based Comprehensive Genomic Profiling Identifies Lung Cancer Patients with Well-Characterized Sensitizing Epidermal Growth Factor Receptor Point Mutations That Were Not Detected by Standard of Care Testing.

BACKGROUND: In our recent study, of cases positive for epidermal growth factor receptor (EGFR) exon 19 deletions using comprehensive genomic profiling (CGP), 17/77 (22%) patients with prior standard of care (SOC) EGFR testing results available were previously negative for exon 19 deletion. Our aim was to compare the detection rates of CGP versus SOC testing for well-characterized sensitizing EGFR point mutations (pm) in our 6,832-patient cohort. MATERIALS AND METHODS: DNA was extracted from 40 microns of formalin-fixed paraffin-embedded sections from 6,832 consecutive cases of non-small cell lung cancer (NSCLC) of various histologies (2012-2015). CGP was performed using a hybrid capture, adaptor ligation-based next-generation sequencing assay to a mean coverage depth of 576×. Genomic alterations (pm, small indels, copy number changes and rearrangements) involving EGFR were recorded for each case and compared with prior testing results if available. RESULTS: Overall, there were 482 instances of EGFR exon 21 L858R (359) and L861Q (20), exon 18 G719X (73) and exon 20 S768I (30) pm, of which 103 unique cases had prior EGFR testing results that were available for review. Of these 103 cases, CGP identified 22 patients (21%) with sensitizing EGFR pm that were not detected by SOC testing, including 9/75 (12%) patients with L858R, 4/7 (57%) patients with L861Q, 8/20 (40%) patients with G719X, and 4/7 (57%) patients with S768I pm (some patients had multiple EGFR pm). In cases with available clinical data, benefit from small molecule inhibitor therapy was observed. CONCLUSION: CGP, even when applied to low tumor purity clinical-grade specimens, can detect well-known EGFR pm in NSCLC patients that would otherwise not be detected by SOC testing. Taken together with EGFR exon 19 deletions, over 20% of patients who are positive for EGFR-activating mutations using CGP are previously negative by SOC EGFR mutation testing, suggesting that thousands of such patients per year in the U.S. alone could experience improved clinical outcomes when hybrid capture-based CGP is used to inform therapeutic decisions. IMPLICATIONS FOR PRACTICE: This study points out that genomic profiling, as based on hybrid capture next-generation sequencing, can identify lung cancer patients with point mutation in epidermal growth factor receptor (EGFR) missed by standard molecular testing who can likely benefit from anti-EGFR targeted therapy. Beyond the specific findings regarding false-negative point mutation testing for EGFR, this study highlights the need for oncologists and pathologists to be cognizant of the performance characteristics of testing deployed and the importance of clinical intuition in questioning the results of laboratory testing.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

Tumor-specific HSP90 inhibition as a therapeutic approach in JAK-mutant acute lymphoblastic leukemias.

The development of the dual Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib for the treatment of myeloproliferative neoplasms (MPNs) has led to studies of ruxolitinib in other clinical contexts, including JAK-mutated acute lymphoblastic leukemia (ALL). However, the limited ability of JAK inhibition to induce molecular or clinicopathological responses in MPNs suggests a need for development of better therapies for JAK kinase-dependent malignancies. Here, we demonstrate that heat shock protein 90 (HSP90) inhibition using a purine-scaffold HSP90 inhibitor in early clinical development is an effective therapeutic approach in JAK-dependent ALL and can overcome persistence to JAK-inhibitor therapy in ALL cells.

Genomic and functional analysis of leukemic transformation of myeloproliferative neoplasms.

Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.

The distribution of BRAF gene fusions in solid tumors and response to targeted therapy.

Although the BRAF V600E base substitution is an approved target for the BRAF inhibitors in melanoma, BRAF gene fusions have not been investigated as anticancer drug targets. In our study, a wide variety of tumors underwent comprehensive genomic profiling for hundreds of known cancer genes using the FoundationOne™ or FoundationOne Heme™ comprehensive genomic profiling assays. BRAF fusions involving the intact in-frame BRAF kinase domain were observed in 55 (0.3%) of 20,573 tumors, across 12 distinct tumor types, including 20 novel BRAF fusions. These comprised 29 unique 5' fusion partners, of which 31% (9) were known and 69% (20) were novel. BRAF fusions included 3% (14/531) of melanomas; 2% (15/701) of gliomas; 1.0% (3/294) of thyroid cancers; 0.3% (3/1,062) pancreatic carcinomas; 0.2% (8/4,013) nonsmall-cell lung cancers and 0.2% (4/2,154) of colorectal cancers, and were enriched in pilocytic (30%) vs. nonpilocytic gliomas (1%; p < 0.0001), Spitzoid (75%) vs. nonSpitzoid melanomas (1%; p = 0.0001), acinar (67%) vs. nonacinar pancreatic cancers (<1%; p < 0.0001) and papillary (3%) vs. nonpapillary thyroid cancers (0%; p < 0.03). Clinical responses to trametinib and sorafenib are presented. In conclusion, BRAF fusions are rare driver alterations in a wide variety of malignant neoplasms, but enriched in Spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers.

Nonamplification ERBB2 genomic alterations in 5605 cases of recurrent and metastatic breast cancer: An emerging opportunity for anti-HER2 targeted therapies.

BACKGROUND: Activating, nonamplification ERBB2 mutations (ERBB2mut) are not detected by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), but are detected by DNA sequencing and may predict clinical responses to human epidermal growth factor receptor (HER2)-targeted therapy. The authors queried 5605 advanced/metastatic breast cancers (mBC) to uncover the frequency of ERBB2mut genomic alterations. Clinical responses to anti-HER2 therapeutics were identified. METHODS: DNA was extracted from 40 µm of formalin-fixed paraffin-embedded (FFPE) sections. Comprehensive genomic profiling (CGP) was used to evaluate up to 315 genes (592× mean coverage depth). Results were analyzed for base substitutions, short indels, copy number changes, and selected rearrangements. RESULTS: Of 5605 cases, 698 (12.5%) featured ERBB2 alterations, including 596 (10.6%) ERBB2 amplifications (ERBB2amp) and 138 (2.4%) ERBB2mut; 38 cases (0.7%) had co-occurring ERBB2amp and ERBB2mut. ERBB2mut predominantly affected the kinase (124 cases; 90%) or extracellular (15 cases; 11%) domains. Both primary BC (52 cases; 38%) and metastatic site biopsies (86 cases; 62%) were found to harbor ERBB2mut, which were distributed across carcinoma not otherwise specified (NOS) (69 cases; 50%), invasive ductal carcinoma (IDC) (40 cases; 29%), invasive lobular carcinoma (ILC) (27 cases; 20%), and mucinous mBC (2 cases; 1%). Genes commonly coaltered with ERBB2 were tumor protein 53 (TP53) (49%); phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) (42%); cadherin 1, type 1 (CDH1) (37%); MYC (17%); and cyclin D1 protein (CCND1) (16%). CDH1 mutations were enriched in ERBB2mut mBC (P<0.0006) and associated with recurrent mBC. Selected patients with ERBB2mut, without ERBB2amp, who responded to anti-HER2 targeted therapies are presented herein. CONCLUSIONS: Within this large series, 1.8% of cases harbored ERBB2mut, which are undetectable by standard-of-care IHC or FISH tests. Metastatic BC driven by ERBB2mut respond to anti-HER2 targeted therapies, and expanding clinical trials designed to detect ERBB2mut by CGP and optimize targeted treatments are warranted. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2654-2662. © 2016 American Cancer Society.

Comprehensive genomic profiling of 295 cases of clinically advanced urothelial carcinoma of the urinary bladder reveals a high frequency of clinically relevant genomic alterations.

BACKGROUND: In the current study, the authors present a comprehensive genomic profile (CGP)-based study of advanced urothelial carcinoma (UC) designed to detect clinically relevant genomic alterations (CRGAs). METHODS: DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections from 295 consecutive cases of recurrent/metastatic UC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 688X for all coding exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer, using process-matched normal control samples as a reference. CRGAs were defined as GAs linked to drugs on the market or currently under evaluation in mechanism-driven clinical trials. RESULTS: All 295 patients assessed were classified with high-grade (International Society of Urological Pathology classification) and advanced stage (stage III/IV American Joint Committee on Cancer) disease, and 294 of 295 patients (99.7%) had at least 1 GA on CGP with a mean of 6.4 GAs per UC (61% substitutions/insertions/deletions, 37% copy number alterations, and 2% fusions). Furthermore, 275 patients (93%) had at least 1 CRGA involving 75 individual genes with a mean of 2.6 CRGAs per UC. The most common CRGAs involved cyclin-dependent kinase inhibitor 2A (CDKN2A) (34%), fibroblast growth factor receptor 3 (FGFR3) (21%), phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) (20%), and ERBB2 (17%). FGFR3 GAs were diverse types and included 10% fusions. ERBB2 GAs were equally divided between amplifications and substitutions. ERBB2 substitutions were predominantly within the extracellular domain and were highly enriched in patients with micropapillary UC (38% of 32 cases vs 5% of 263 nonmicropapillary UC cases; P<.0001). CONCLUSIONS: Using a CGP assay capable of detecting all classes of GA simultaneously, an extraordinarily high frequency of CRGA was identified in a large series of patients with advanced UC. Cancer 2016;122:702-711. © 2015 American Cancer Society.

Evaluation of 122 advanced-stage cutaneous squamous cell carcinomas by comprehensive genomic profiling opens the door for new routes to targeted therapies.

BACKGROUND: The authors hypothesized that comprehensive genomic profiling of advanced-stage cutaneous squamous cell carcinoma (cSCC) could identify genomic-derived drug targets of therapy for patients with conventional therapy-resistant disease. METHODS: Comprehensive genomic profiling of 315 cancer genes was applied to 50 ng of DNA from 122 cSCC cases for the evaluation of all classes of genomic alterations (GAs). Clinically relevant genomic alterations (CRGAs) were defined as those identifying anticancer drugs on the market or in registered clinical trials. RESULTS: There were 21 women (17%) and 101 men (83%) with a median age of 64.9 years (range, 21-87 years). Eleven cSCC cases (9%) were histologic AJCC grade 1, 69 (57%) were grade 2, and 42 (34%) were grade 3. The primary cSCC was used for sequencing in 77 cases (63%). Metastatic lesions were sequenced in 37% of cases. There were 1120 total GAs identified (average of 9.2 GAs per tumor), with 100% of cases harboring at least 1 alteration. Of the 122 cSCCs, 107 (88%) harbored at least 1 CRGA (2.5 CRGAs per cSCC) includingNOTCH1 (43%); patched 1 (PTCH1) (11%); BRCA2 (10%); HRAS (8%); ataxia telangiectasia mutated (ATM) (7%); erb-B2 receptor tyrosine kinase 4 (ERBB4) (7%); neurofibromatosis type 1 (NF1) (7%); erb-B2 receptor tyrosine kinase 2 (ERBB2) (6%); phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) (6%); cyclin D1 (CCND1) (6%); epidermal growth factor receptor (EGFR) (5%); and F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase (FBXW7) (5%). CONCLUSIONS: In the current study, approximately 88% of patients with cSCC were found to harbor clinically relevant GAs that have the potential to guide the treatment of patients with advanced-stage tumors with targeted therapeutic agents. Cancer 2016;122:249-257. © 2015 American Cancer Society.

Comprehensive Genomic Profiling of Advanced Penile Carcinoma Suggests a High Frequency of Clinically Relevant Genomic Alterations.

BACKGROUND: Advanced penile squamous cell carcinoma (PSCC) is associated with poor survival due to the aggressiveness of the disease and lack of effective systemic therapies. Comprehensive genomic profiling (CGP) was performed to identify clinically relevant genomic alterations (CRGAs). MATERIALS AND METHODS: DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections in patients with advanced PSCC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 692× for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. CRGAs were defined as genomic alterations (GAs) linked to targeted therapies on the market or under evaluation in mechanism-driven clinical trials. RESULTS: Twenty male patients with a median age of 60 years (range, 46-87 years) were assessed. Seventeen (85%) cases were stage IV and three cases (15%) were stage III. CGP revealed 109 GAs (5.45 per tumor), 44 of which were CRGAs (2.2 per tumor). At least one CRGA was detected in 19 (95%) cases, and the most common CRGAs were CDKN2A point mutations and homozygous deletion (40%), NOTCH1 point mutations and rearrangements (25%), PIK3CA point mutations and amplification (25%), EGFR amplification (20%), CCND1 amplification (20%), BRCA2 insertions/deletions (10%), RICTOR amplifications (10%), and FBXW7 point mutations (10%). CONCLUSION: CGP identified CRGAs in patients with advanced PSCC, including EGFR amplification and PIK3CA alterations, which can lead to the rational administration of targeted therapy and subsequent benefit for these patients. IMPLICATIONS FOR PRACTICE: Few treatment options exist for patients with advanced penile squamous cell carcinoma (PSCC). Outcomes are dismal with platinum-based chemotherapy, with median survival estimated at 1 year or less across multiple series. Biological studies of patients with PSCC to date have principally focused on human papillomavirus status, but few studies have elucidated molecular drivers of the disease. To this end, comprehensive genomic profiling was performed in a cohort of 20 patients with advanced PSCC. Findings of frequent mutations in CDKN2A, NOTCH1, PIK3CA, and EGFR (all in excess of 20%) point to potential therapeutic avenues. Trials of targeted therapies directed toward these mutations should be explored.

Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization.

INTRODUCTION: For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. MATERIALS AND METHODS: Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. RESULTS: A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. CONCLUSION: Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. IMPLICATIONS FOR PRACTICE: Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.

Comprehensive Genomic Profiling of Clinically Advanced Medullary Thyroid Carcinoma.

OBJECTIVE: The aim of this study was to determine the genomic alterations of cancer-related genes in advanced medullary thyroid carcinoma during the course of clinical care. METHODS: Hybrid-capture-based comprehensive genomic profiling was performed on 34 consecutive medullary thyroid carcinoma cases to identify all four classes of genomic alterations, and outcome for an index patient was collected. RESULTS: RET was mutated in 88% (30/34) of cases, with RET M918T being responsible for 70% (21/30) of the RET alterations. The other RET alterations were RET E632_L633del, C634R, C620R, C618G/R/S, V804M, and RET amplification. Two of the four RET wild-type patients harbored mutations in KRAS or HRAS (1/34 each). The next most frequent genomic alterations were amplifications of CCND1, FGF3, and FGF19 and alterations in CDKN2A (3/34 each). One case with a RET M918T mutation developed acquired resistance to progressively dose-escalated vandetanib. When the mTOR inhibitor everolimus was added to continued vandetanib treatment, the patient achieved a second 25% reduction of tumor volume (RECIST 1.1) for 8 months. CONCLUSIONS: Comprehensive genomic profiling identified the full breadth of RET alterations in metastatic medullary thyroid carcinoma and possible cooperating oncogenic driver alterations. This approach may refine the use of targeted therapy for these patients.

New class of gene-termini-associated human RNAs suggests a novel RNA copying mechanism.

Small (<200 nucleotide) RNA (sRNA) profiling of human cells using various technologies demonstrates unexpected complexity of sRNAs with hundreds of thousands of sRNA species present. Genetic and in vitro studies show that these RNAs are not merely degradation products of longer transcripts but could indeed have a function. Furthermore, profiling of RNAs, including the sRNAs, can reveal not only novel transcripts, but also make clear predictions about the existence and properties of novel biochemical pathways operating in a cell. For example, sRNA profiling in human cells indicated the existence of an unknown capping mechanism operating on cleaved RNA, a biochemical component of which was later identified. Here we show that human cells contain a novel type of sRNA that has non-genomically encoded 5' poly(U) tails. The presence of these RNAs at the termini of genes, specifically at the very 3' ends of known mRNAs, strongly argues for the presence of a yet uncharacterized endogenous biochemical pathway in cells that can copy RNA. We show that this pathway can operate on multiple genes, with specific enrichment towards transcript-encoding components of the translational machinery. Finally, we show that genes are also flanked by sense, 3' polyadenylated sRNAs that are likely to be capped.

Comprehensive Genomic Profiling of Advanced Esophageal Squamous Cell Carcinomas and Esophageal Adenocarcinomas Reveals Similarities and Differences.

BACKGROUND: Esophageal squamous cell carcinomas (ESCCs) and esophageal adenocarcinomas (EACs) account for >95% of esophageal malignancies and represent a major global health burden. ESCC is the dominant histology globally but represents a minority of U.S. cases, with EAC accounting for the majority of U.S. CASES: The patient outcomes for advanced ESCC and EAC are poor, and new therapeutic options are needed. Using a sensitive sequencing assay, we compared the genomic profiles of ESCC and EAC with attention to identification of therapeutically relevant genomic alterations. METHODS: Next-generation sequencing-based comprehensive genomic profiling was performed on hybridization-captured, adaptor ligation-based libraries to a median coverage depth of >650× for all coding exons of 315 cancer-related genes plus selected introns from 28 genes frequently rearranged in cancer. Results from a single sample were evaluated for all classes of genomic alterations (GAs) including point mutations, short insertions and deletions, gene amplifications, homozygous deletions, and fusions/rearrangements. Clinically relevant genomic alterations (CRGAs) were defined as alterations linked to approved drugs and those under evaluation in mechanism-driven clinical trials. RESULTS: There were no significant differences by sex for either tumor type, and the median age for all patients was 63 years. All ESCCs and EACs were at an advanced stage at the time of sequencing. All 71 ESCCs and 231 EACs featured GAs on profiling, with 522 GAs in ESCC (7.4 per sample) and 1,303 GAs in EAC (5.6 per sample). The frequency of clinically relevant GAs in ESCC was 94% (2.6 per sample) and 93% in EAC (2.7 per sample). CRGAs occurring more frequently in EAC included KRAS (23% EAC vs. 6% ESCC) and ERBB2 (23% EAC vs. 3% ESCC). ESCC samples were enriched for CRGA in PIK3CA (24% ESCC vs. 10% EAC), PTEN (11% ESCC vs. 4% EAC), and NOTCH1 (17% ESCC vs. 3% EAC). Other GAs that differed significantly between histologic tumor types included SMAD4 (14% EAC vs. 1% ESCC), RB1 (14% ESCC vs. 2% EAC), SOX2 (18% ESCC vs. 1% EAC), and NFE2L2 (24% ESCC vs. 1% EAC). CONCLUSION: ESCC and EAC share similarly high frequencies of overall and clinically relevant genomic alterations; however, the profiles of genomic alterations in the two diseases differ widely, with KRAS and ERBB2 far more frequently altered in EAC compared with ESCC and with mammalian target of rapamycin (MTOR) pathway genes (PIK3CA and PTEN) and NOTCH1 more frequently altered in ESCC compared with EAC. Comprehensive genomic profiling highlights the promise of identifying clinically relevant genomic alterations in both ESCC and EAC and suggests new avenues for molecularly directed therapies in esophageal cancer.

Oncogenic alterations in ERBB2/HER2 represent potential therapeutic targets across tumors from diverse anatomic sites of origin.

BACKGROUND: Targeted ERBB2/HER2 inhibitors are approved by the U.S. Food and Drug Administration for the treatment of breast, gastric, and esophageal cancers that overexpress or amplify HER2/ERBB2, as measured by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. Activating mutations in ERBB2 have also been reported and are predicted to confer sensitivity to these targeted agents. Testing for these mutations is not performed routinely, and FISH and IHC are not applied outside of these approved indications. MATERIALS AND METHODS: We explored the spectrum of activating ERBB2 alterations across a collection of ∼ 7,300 solid tumor specimens that underwent comprehensive genomic profiling using next-generation sequencing. Results were analyzed for base substitutions, insertions and deletions, select rearrangements, and copy number changes. RESULTS: Known oncogenic ERBB2 alterations were identified in tumors derived from 27 tissues, and ERBB2 amplification in breast, gastric, and gastroesophageal cancers accounted for only 30% of these alterations. Activating mutations in ERBB2 were identified in 131 samples (32.5%); amplification was observed in 246 samples (61%). Two samples (0.5%) harbored an ERBB2 rearrangement. Ten samples (2.5%) harbored multiple ERBB2 mutations, yet mutations and amplifications were mutually exclusive in 91% of mutated cases. CONCLUSION: Standard slide-based tests for overexpression or amplification of ERBB2 would fail to detect the majority of activating mutations that occur overwhelmingly in the absence of copy number changes. Compared with current clinical standards, comprehensive genomic profiling of a more diverse set of tumor types may identify ∼ 3.5 times the number of patients who may benefit from ERBB2-targeted therapy.

Fluorescence in situ hybridization, immunohistochemistry, and next-generation sequencing for detection of EML4-ALK rearrangement in lung cancer.

BACKGROUND: The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis. MATERIALS AND METHODS: We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance. RESULTS: Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC. CONCLUSION: The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.

Prospective comprehensive genomic profiling of advanced gastric carcinoma cases reveals frequent clinically relevant genomic alterations and new routes for targeted therapies.

BACKGROUND: Gastric cancer (GC) is a major global cancer burden and the second most common cause of global cancer-related deaths. The addition of anti-ERBB2 (HER2) targeted therapy to chemotherapy improves survival for ERBB2-amplified advanced GC patients; however, the majority of GC patients do not harbor this alteration and thus cannot benefit from targeted therapy under current practice paradigms. MATERIALS AND METHODS: Prospective comprehensive genomic profiling of 116 predominantly locally advanced or metastatic (90.0%) gastric cancer cases was performed to identify genomic alterations (GAs) associated with a potential response to targeted therapies approved by the U.S. Food and Drug Administration or targeted therapy-based clinical trials. RESULTS: Overall, 78% of GC cases harbored one clinically relevant GA or more, with the most frequent alterations being found in TP53 (50%), ARID1A (24%), KRAS (16%), CDH1 (15%), CDKN2A (14%), CCND1 (9.5%), ERBB2 (8.5%), PIK3CA (8.6%), MLL2 (6.9%), FGFR2 (6.0%), and MET (6.0%). Receptor tyrosine kinase genomic alterations were detected in 20.6% of cases, primarily ERBB2, FGFR2, and MET amplification, with ERBB2 alterations evenly split between amplifications and base substitutions. Rare BRAF mutations (2.6%) were also observed. One MET-amplified GC patient responded for 5 months to crizotinib, a multitargeted ALK/ROS1/MET inhibitor. CONCLUSION: Comprehensive genomic profiling of GC identifies clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions.

Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations.

Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.