Treatment of Pediatric Glioblastoma with Combination Olaparib and Temozolomide Demonstrates 2-Year Durable Response.

For pediatric patients with high-grade gliomas, standard-of-care treatment includes surgery, chemotherapy, and radiation therapy; however, most patients ultimately succumb to their disease. With advances in genomic characterization of pediatric high-grade gliomas, the use of targeted therapies in combination with current treatment modalities offer the potential to improve survival in this patient population. In this report, we present the case of a 3-year-old girl with glioblastoma who continues to experience an exceptional and durable response (>2 years) to the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib. Our patient presented with persistent and progressive seizure activity that upon workup was the result of a large heterogeneously enhancing, mixed cystic and solid mass in the left frontal-parietal-temporal region. Histopathologic analysis of resected tumor tissue confirmed the diagnosis of glioblastoma, and comprehensive genomic profiling demonstrated absence of any BRAF or H3F3A mutations. Genomic profiling, however, did reveal a probable germline heterozygous BRCA2 Lys3326Ter (K3226*) nonsense variant. After debulking surgery, the patient received standard-of-care treatment with radiation and temozolomide. Nine months later the PARP inhibitor olaparib was administered in combination with temozolomide for 16 cycles. This regimen was well tolerated by the patient and serial imaging showed reduction in tumor size. Since completion of the regimen, the patient remains neurologically intact with no evidence of tumor recurrence. To our knowledge, this represents the first case of a pediatric glioblastoma that maintains a durable response to a therapeutic strategy that included the PARP inhibitor olaparib and more generally highlights the potential clinical utility of incorporating these agents into the treatment of pediatric high-grade gliomas. KEY POINTS: Germline mutations detected in pediatric gliomas may represent a cancer predisposition syndrome. Integrating molecular testing into routine clinical care for pediatric patients with glioma is critical to identify therapeutic targets and patients with a cancer predisposition syndrome. Patients with glioma with defects in DNA repair pathway components (e.g., BRCA1/2) may show increased responsiveness to poly (ADP-ribose) polymerase (PARP) inhibitors. Combining PARP inhibitors with temozolomide (standard-of-care treatment) revealed no adverse events or toxicities over the course of 18 months.

The Pan-Cancer Landscape of Coamplification of the Tyrosine Kinases KIT, KDR, and PDGFRA.

PURPOSE: Amplifications of receptor tyrosine kinases (RTKS) are therapeutic targets in multiple tumor types (e.g. HER2 in breast cancer), and amplification of the chromosome 4 segment harboring the three RTKs KIT, PDGFRA, and KDR (4q12amp) may be similarly targetable. The presence of 4q12amp has been sporadically reported in small tumor specific series but a large-scale analysis is lacking. We assess the pan-cancer landscape of 4q12amp and provide early clinical support for the feasibility of targeting this amplicon. EXPERIMENTAL DESIGN: Tumor specimens from 132,872 patients with advanced cancer were assayed with hybrid capture based comprehensive genomic profiling which assays 186-315 genes for all classes of genomic alterations, including amplifications. Baseline demographic data were abstracted, and presence of 4q12amp was defined as 6 or more copies of KIT/KDR/PDGFRA. Concurrent alterations and treatment outcomes with matched therapies were explored in a subset of cases. RESULTS: Overall 0.65% of cases harbored 4q12amp at a median copy number of 10 (range 6-344). Among cancers with >100 cases in this series, glioblastomas, angiosarcomas, and osteosarcomas were enriched for 4q12amp at 4.7%, 4.8%, and 6.4%, respectively (all p < 0.001), giving an overall sarcoma (n = 6,885) incidence of 1.9%. Among 99 pulmonary adenocarcinoma cases harboring 4q12amp, 50 (50%) lacked any other known driver of NSLCC. Four index cases plus a previously reported case on treatment with empirical TKIs monotherapy had stable disease on average exceeding 20 months. CONCLUSION: We define 4q12amp as a significant event across the pan-cancer landscape, comparable to known pan-cancer targets such as NTRK and microsatellite instability, with notable enrichment in several cancers such as osteosarcoma where standard treatment is limited. The responses to available TKIs observed in index cases strongly suggest 4q12amp is a druggable oncogenic target across cancers that warrants a focused drug development strategy. IMPLICATIONS FOR PRACTICE: Coamplification of the receptor tyrosine kinases (rtks) KIT/KDR/PDGFRA (4q12amp) is present broadly across cancers (0.65%), with enrichment in osteosarcoma and gliomas. Evidence for this amplicon having an oncogenic role is the mutual exclusivity of 4q12amp to other known drivers in 50% of pulmonary adenocarcinoma cases. Furthermore, preliminary clinical evidence for driver status comes from four index cases of patients empirically treated with commercially available tyrosine kinase inhibitors with activity against KIT/KDR/PDGFRA who had stable disease for 20 months on average. The sum of these lines of evidence suggests further clinical and preclinical investigation of 4q12amp is warranted as the possible basis for a pan-cancer drug development strategy.

The sleeping ugly: Tumour microenvironment's act to make or break the spell of dormancy.

Metastasis is the main cause of death for most cancer patients. It appears clear from clinical observations that the majority of cancers, particularly carcinoma do not follow a linear model of metastatic progression, where cancer cells shed from the primary tumour, disseminate to a distant organ and immediately outgrow to form clinical metastasis. Certainly, while cancer spreading is an early event, metastasis occurs much later during tumour progression and frequently arises several years after primary tumour resection. The time spent by disseminated cancer cells (DTCs) in a distant organ before their outgrowth is termed metastatic latency. We will examine here the current knowledge of the mechanisms allowing metastatic latency and discuss the crucial role of the DTCs' tissue microenvironment in this process.

Genomic Features for Therapeutic Insights of Chemotherapy-Resistant, Primary Mediastinal Nonseminomatous Germ Cell Tumors and Comparison with Gonadal Counterpart.

Primary mediastinal nonseminomatous germ cell tumors (PMNSGCT) frequently become refractory to chemotherapy, and no effective salvage therapy exists. We performed genomic profiling on a series of 44 PMNSGCT and compared the results with those from chemorefractory, metastatic pure seminomatous (Sem, n = 22) and nonseminomatous (NS, n = 86) testicular germ cell tumors. Archival tissues were sequenced by a hybrid capture-based technology (FoundationONE; Foundation Medicine, Inc., Cambridge, MA). Microsatellite instability (MSI) and tumor mutational burden (TMB, mutations [mut]/Mb) were determined.Statistically significant differences in genomic alterations (GA) of PMNSGCT versus NS included higher TP53 pathway GA (p < .0001), PIK3CA pathway GA (p < .0001), and lower cell-cycle pathway GA (p = .0004). There were no MSI-high PMNSGCT cases. Mean TMB was similar between the groups, but there were more ≥10 mut/Mb in the PMNSGCT group versus NS (11.4% vs. 4.6%).The GA identified in PMNSGCT were similar to the findings from NS, with differential opportunities for targeted therapies and immunotherapies. Further study of precision treatments appears warranted.

Genomic Landscape of Adult and Pediatric BCR-ABL1-Like B-Lymphoblastic Leukemia Using Parallel DNA and RNA Sequencing.

BCR-ABL1-like B-Acute Lymphoblastic Leukemia (B-ALL) is a subset of B-ALL with a poor prognosis that is found in all age groups. Definitive identification of these patients is difficult in routine clinical practice as gene expression profiling, the gold standard test, is not widely available. Comprehensive genomic profiling performed on 450 patients with extensive fusion profiling revealed a wide range of genomic alterations which were consistent with a classification of BCR-ABL1-like B-ALL in 29% of cases. This manuscript highlights a clinically available alternative method for identifying a large subset of patients with BCR-ABL1-like B-ALL.

Comparative Genomic Profiling of Refractory and Metastatic Penile and Nonpenile Cutaneous Squamous Cell Carcinoma: Implications for Selection of Systemic Therapy.

PURPOSE: Metastatic penile squamous cell carcinoma is an aggressive malignancy with limited treatment options. We compared the potential therapy impacting genomic alterations between metastatic penile squamous cell carcinoma and nonpenile metastatic cutaneous squamous cell carcinoma. MATERIALS AND METHODS: DNA was extracted from 40 µ of formalin fixed, paraffin embedded samples from 78 cases of metastatic penile squamous cell carcinoma and 338 of metastatic cutaneous squamous cell carcinoma. Comprehensive genomic profiling was performed using a hybrid capture, adaptor ligation based, next generation sequencing assay to a mean coverage depth of greater than 500×. The tumor mutational burden was determined on 1.1 Mbp of sequenced DNA and microsatellite instability was determined on 114 loci. RESULTS: Potential targeted therapy opportunities in metastatic penile squamous cell carcinoma cases included alterations in the MTOR pathway ( NF1 genomic alterations in 7% and PTEN genomic alterations in 4%) and in the DNA repair pathway ( BRCA2 and ATM genomic alterations in 7% each) and tyrosine kinase ( EGFR genomic alterations in 6%, and FGFR3 and ERBB2 genomic alterations in 4% each). The tumor mutational burden was significantly higher in predominantly ultraviolet light exposed metastatic squamous cell carcinoma than in metastatic penile squamous cell carcinoma, making metastatic squamous cell carcinoma potentially more responsive to immunotherapies than metastatic penile squamous cell carcinoma. Microsatellite high status was extremely rare for metastatic penile and metastatic cutaneous squamous cell carcinoma. CD274 ( PD-L1) amplification was also rare in both tumor types. CONCLUSIONS: Metastatic penile squamous cell carcinoma is a unique subtype of squamous cell carcinoma with distinctive genomic features which contrast with those identified in metastatic cutaneous squamous cell carcinoma of nonpenile ultraviolet light exposed skin. Although not rich in predictors of the response to immunotherapy (the tumor mutational burden and microsatellite instability are low), more than a quarter of metastatic penile squamous cell carcinoma cases may potentially benefit from existing and available therapies targeting MTOR, DNA repair and tyrosine kinase pathways.

Constitutively active ESR1 mutations in gynecologic malignancies and clinical response to estrogen-receptor directed therapies.

OBJECTIVE: Endocrine therapy is often considered as a treatment for hormone-responsive gynecologic malignancies. In breast cancer, activating mutations in the estrogen receptor (mutESR1) contribute to therapeutic resistance to endocrine therapy, especially aromatase inhibitors (AIs). The purpose of this study was to evaluate the frequency and clinical relevance of ESR1 genomic alterations in gynecologic malignancies. METHODS: DNA from FFPE tumor tissue obtained during routine clinical care for 9645 gynecologic malignancies (ovary, fallopian tube, uterus, cervix, vagina, vulvar, and placenta) was analyzed for all classes of genomic alterations (base substitutions (muts), insertions, deletions, rearrangements, and amplifications) in ESR1 by hybrid capture next generation sequencing. A subset of alterations was characterized in laboratory-based transcription assays for response to endocrine therapies. RESULTS: A total of 295 ESR1 genomic alterations were identified in 285 (3.0%) cases. mutESR1 were present in 86 (0.9%) cases and were more common in uterine compared to other cancers (2.0% vs <1%, respectively p < 0.001). mutESR1 were enriched in carcinomas with endometrioid versus serous histology (4.4% vs 0.2% respectively, p < 0.0001 in uterine and 3.5% vs 0.3% respectively, p = 0.0004 in ovarian carcinomas). In three of four patients with serial sampling, mutESR1 emerged under the selective pressure of AI therapy. Despite decreased potency of estrogen receptor (ER) antagonists in transcriptional assays, clinical benefit was observed following treatment with selective ER-targeted therapy, in one case lasting >48 months. CONCLUSIONS: While the prevalence of ESR1 mutations in gynecologic malignancies is low, there are significant clinical implications useful in guiding therapeutic approaches for these cancers.

Targeting HER2 in colorectal cancer: The landscape of amplification and short variant mutations in ERBB2 and ERBB3.

BACKGROUND: In contrast to lung cancer, few precision treatments are available for colorectal cancer (CRC). One rapidly emerging treatment target in CRC is ERBB2 (human epidermal growth factor receptor 2 [HER2]). Oncogenic alterations in HER2, or its dimerization partner HER3, can underlie sensitivity to HER2-targeted therapies. METHODS: In this study, 8887 CRC cases were evaluated by comprehensive genomic profiling for genomic alterations in 315 cancer-related genes, tumor mutational burden, and microsatellite instability. This cohort included both colonic (7599 cases; 85.5%) and rectal (1288 cases; 14.5%) adenocarcinomas. RESULTS: A total of 569 mCRCs were positive for ERBB2 (429 cases; 4.8%) and/or ERBB3 (148 cases; 1.7%) and featured ERBB amplification, short variant alterations, or a combination of the 2. High tumor mutational burden (≥20 mutations/Mb) was significantly more common in ERBB-mutated samples, and ERBB3-mutated CRCs were significantly more likely to have high microsatellite instability (P<.002). Alterations affecting KRAS (27.3%) were significantly underrepresented in ERBB2-amplified samples compared with wild-type CRC samples (51.8%), and ERBB2- or ERBB3-mutated samples (49.0% and 60.8%, respectively) (P<.01). Other significant differences in mutation frequency were observed for genes in the PI3K/MTOR and mismatch repair pathways. CONCLUSIONS: Although observed less often than in breast or upper gastrointestinal carcinomas, indications for which anti-HER2 therapies are approved, the percentage of CRC with ERBB genomic alterations is significant. Importantly, 32% of ERBB2-positive CRCs harbor short variant alterations that are undetectable by routine immunohistochemistry or fluorescence in situ hybridization testing. The success of anti-HER2 therapies in ongoing clinical trials is a promising development for patients with CRC. Cancer 2018;124:1358-73. © 2018 Foundation Medicine, Inc. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

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.

Precision Neuro-oncology: the Role of Genomic Testing in the Management of Adult and Pediatric Gliomas.

OPINION STATEMENT: In recent years, large-scale genomic studies have expanded our knowledge regarding genomic drivers in tumors of the central nervous system. While histopathologic analysis of brain tumors remains the primary method for tumor classification, the clinical utility of molecular and genomic testing to support and/or complement tumor classification continues to expand. This approach enhances diagnostic accuracy and provides clinicians with objective data to facilitate discussions regarding prognosis and treatment decisions, including selection of clinical trials. Ensuring accurate diagnoses is fundamental to the management of brain tumor patients. However, given the morphologic overlap among primary brain tumors, genomic data can be used to help distinguish tumor lineage. In its clearest form, we have embraced the concept of an integrated diagnosis, which combines traditional histopathology findings with molecular and genomic data. Patient prognosis varies significantly based on a tumor's genomic profile. For neuro-oncology patients, outcome studies linking diagnoses with genomic profiles show significant differences based on tumor biomarkers such as IDH1/2, H3F3A, BRAF, and CDKN2A and TERT status. Therefore, easy access to reliable genomic data is important in understanding a patient's disease and developing a clinical strategy wherein targeted molecular or immune therapies can be incorporated into the discussion.

Comprehensive genomic profiling of different subtypes of nasopharyngeal carcinoma reveals similarities and differences to guide targeted therapy.

BACKGROUND: To date, no targeted therapy has been approved for nasopharyngeal carcinoma (NPC), and this underscores the need for an in-depth understanding of clinically relevant genomic alterations (CRGAs). METHODS: Comprehensive genomic profiling was performed for 190 NPC patients, including 20 patients with nasopharyngeal adenocarcinoma (NPAC), 62 patients with nasopharyngeal squamous cell carcinoma (NPSCC), and 108 patients with nasopharyngeal undifferentiated carcinoma (NPUC). The associations of genes and pathways with subtypes, Epstein-Barr virus (EBV) infections, and the tumor mutation burden (TMB) were statistically evaluated. RESULTS: Although the overall rates of genomic alterations were similar, the 3 NPC subtypes exhibited different mutational landscapes. Notably, mutations in a proven-treatable target gene, isocitrate dehydrogenase 2 (IDH2), were significantly associated with NPUC but not with NPAC or NPSCC. The top 5 ranked CRGAs included CDKN2A (29%), IDH2 (16%), SMARCB1 (7%), PIK3CA (6%), and NF1 (5%) in NPUC; CDKN2A (27%), PIK3CA (23%), FBXW7 (11%), PTEN (11%), and EGFR (8%) in NPSCC; and CDKN2A (20%), KRAS (15%), CCND1 (10%), MAP3K1 (10%), and NOTCH1 (10%) in NPAC. The incidence of EBV infections significantly correlated with the subtypes and with TP53, CDKN2A, and CDKN2B. The TMB status correlated with the subtypes and with LRP1B, FBXW7, and PIK3CA mutations as well as DNA repair, phosphoinositide 3-kinase/mammalian target of rapamycin, and mitogen-activated protein kinase pathways. CONCLUSIONS: These results indicate that different NPC subtypes harbor different CRGAs. Both EBV infections and the TMB are associated with the NPC subtypes as well as the alterations of individual genes and pathways. The high frequency of IDH2 mutations in NPUC may facilitate potential targeted therapy and will ultimately point to new therapeutic strategies. Cancer 2017;123:3628-37. © 2017 American Cancer Society.

Comprehensive Genomic Profiling of Esthesioneuroblastoma Reveals Additional Treatment Options.

BACKGROUND: Esthesioneuroblastoma (ENB), also known as olfactory neuroblastoma, is a rare malignant neoplasm of the olfactory mucosa. Despite surgical resection combined with radiotherapy and adjuvant chemotherapy, ENB often relapses with rapid progression. Current multimodality, nontargeted therapy for relapsed ENB is of limited clinical benefit. MATERIALS AND METHODS: We queried whether comprehensive genomic profiling (CGP) of relapsed or refractory ENB can uncover genomic alterations (GA) that could identify potential targeted therapies for these patients. CGP was performed on formalin-fixed, paraffin-embedded sections from 41 consecutive clinical cases of ENBs using a hybrid-capture, adaptor ligation based next-generation sequencing assay to a mean coverage depth of 593X. The results were analyzed for base substitutions, insertions and deletions, select rearrangements, and copy number changes (amplifications and homozygous deletions). RESULTS: Clinically relevant GA (CRGA) were defined as GA linked to drugs on the market or under evaluation in clinical trials. A total of 28 ENBs harbored GA, with a mean of 1.5 GA per sample. Approximately half of the ENBs (21, 51%) featured at least one CRGA, with an average of 1 CRGA per sample. The most commonly altered gene was TP53 (17%), with GA in PIK3CA, NF1, CDKN2A, and CDKN2C occurring in 7% of samples. CONCLUSION: We report comprehensive genomic profiles for 41 ENB tumors. CGP revealed potential new therapeutic targets, including targetable GA in the mTOR, CDK and growth factor signaling pathways, highlighting the clinical value of genomic profiling in ENB. IMPLICATIONS FOR PRACTICE: Comprehensive genomic profiling of 41 relapsed or refractory ENBs reveals recurrent alterations or classes of mutation, including amplification of tyrosine kinases encoded on chromosome 5q and mutations affecting genes in the mTOR/PI3K pathway. Approximately half of the ENBs (21, 51%) featured at least one clinically relevant genomic alteration (CRGA), with an average of 1 CRGA per sample. The most commonly altered gene was TP53 (17%), and alterations in PIK3CA, NF1, CDKN2A, or CDKN2C were identified in 7% of samples. Responses to treatment with the kinase inhibitors sunitinib, everolimus, and pazopanib are presented in conjunction with tumor genomics.

Clinical Benefit in Response to Palbociclib Treatment in Refractory Uterine Leiomyosarcomas with a Common CDKN2A Alteration.

BACKGROUND: Uterine leiomyosarcoma (uLMS) responds poorly to conventional chemotherapeutic agents, and personalized therapies have yet to be systematically explored. Comprehensive genomic profiling (CGP) can identify therapeutic targets and provide insight into the biology of this highly aggressive tumor. We report a case of uLMS treated with the CGP-matched therapy palbociclib, a CDK4/6 inhibitor, with sustained clinical benefit in this rare and deadly malignancy. MATERIALS AND METHODS: This study analyzed 279 clinically advanced/recurrent uLMS samples. Median patient age was 54 years (range, 23-83 years). DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections, and CGP was performed on hybridization-captured, adaptor ligation-based libraries for up to 405 cancer-related genes plus introns from up to 31 genes frequently rearranged in cancer. Sequencing data were analyzed for base pair substitutions, insertions/deletions, copy number alterations, and rearrangements. RESULTS: CGP shows that 97.1% of uLMS harbor at least one alteration, and approximately 57% harbor alterations in one or more therapeutically targetable pathways. CDKN2A mutations that inactivate p16INK4a were identified in 11% of uLMS. We report the first demonstration of clinical benefit in response to palbociclib treatment for a uLMS patient with a CDKN2A mutation, resulting in disease stabilization and significant symptom reduction. CONCLUSION: A patient with uLMS harboring a CDKN2A mutation experienced clinical benefit from treatment with palbociclib, and genomic analysis of 279 uLMS samples revealed that 19% of patients had mutations affecting the cyclin-dependent kinase (CDK) pathway. These observations provide a rationale for a clinical trial investigating treatment with CDK pathway inhibitors for uLMS harboring relevant genomic alterations. The Oncologist 2017;22:416-421Implications for Practice: Comprehensive genomic profiling (CGP) of individuals with uterine leiomyosarcoma (uLMS) indicates that nearly 20% of patients may harbor a mutation affecting the cyclin-dependent kinase (CDK) pathway. The case presented demonstrates that a CDK inhibitory drug may provide clinical benefit to such individuals. Given the lack of curative therapies for uLMS, CGP could be performed on all cases of advanced uLMS and a CDK inhibitor could be recommended (preferably as part of a clinical trial) for individuals harboring a mutation in the CDK pathway.

ALK Fusions in a Wide Variety of Tumor Types Respond to Anti-ALK Targeted Therapy.

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 < .0001). Patients with non-NSCLC tumors harboring fALK were significantly younger (p < .0001) and more often female (p < .0001) than patients with fALK-positive NSCLC. EML4 was more often the fusion partner in NSCLC (83.5%) versus non-NSCLC tumors (30.9%; p < .0001). 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. IMPLICATIONS FOR PRACTICE: Rearrangements involving the ALK gene have been detected in dozens of cancer types using next-generation sequencing. Patients whose tumors harbor ALK rearrangements or fusions respond to treatment with crizotinib and alectinib, including tumors not normally associated with ALK mutations, such as non-Langerhans cell histiocytosis or renal cell carcinoma. Comprehensive genomic profiling using next-generation sequencing can detect targetable ALK fusions irrespective of tumor type or fusions partner.

Comprehensive Genomic Profiling of 282 Pediatric Low- and High-Grade Gliomas Reveals Genomic Drivers, Tumor Mutational Burden, and Hypermutation Signatures.

BACKGROUND: Pediatric brain tumors are the leading cause of death for children with cancer in the U.S. Incorporating next-generation sequencing data for both pediatric low-grade (pLGGs) and high-grade gliomas (pHGGs) can inform diagnostic, prognostic, and therapeutic decision-making. MATERIALS AND METHODS: We performed comprehensive genomic profiling on 282 pediatric gliomas (157 pHGGs, 125 pLGGs), sequencing 315 cancer-related genes and calculating the tumor mutational burden (TMB; mutations per megabase [Mb]). RESULTS: In pLGGs, we detected genomic alterations (GA) in 95.2% (119/125) of tumors. BRAF was most frequently altered (48%; 60/125), and FGFR1 missense (17.6%; 22/125), NF1 loss of function (8.8%; 11/125), and TP53 (5.6%; 7/125) mutations were also detected. Rearrangements were identified in 35% of pLGGs, including KIAA1549-BRAF, QKI-RAF1, FGFR3-TACC3, CEP85L-ROS1, and GOPC-ROS1 fusions. Among pHGGs, GA were identified in 96.8% (152/157). The genes most frequently mutated were TP53 (49%; 77/157), H3F3A (37.6%; 59/157), ATRX (24.2%; 38/157), NF1 (22.2%; 35/157), and PDGFRA (21.7%; 34/157). Interestingly, most H3F3A mutations (81.4%; 35/43) were the variant K28M. Midline tumor analysis revealed H3F3A mutations (40%; 40/100) consisted solely of the K28M variant. Pediatric high-grade gliomas harbored oncogenic EML4-ALK, DGKB-ETV1, ATG7-RAF1, and EWSR1-PATZ1 fusions. Six percent (9/157) of pHGGs were hypermutated (TMB >20 mutations per Mb; range 43-581 mutations per Mb), harboring mutations deleterious for DNA repair in MSH6, MSH2, MLH1, PMS2, POLE, and POLD1 genes (78% of cases). CONCLUSION: Comprehensive genomic profiling of pediatric gliomas provides objective data that promote diagnostic accuracy and enhance clinical decision-making. Additionally, TMB could be a biomarker to identify pediatric glioblastoma (GBM) patients who may benefit from immunotherapy. IMPLICATIONS FOR PRACTICE: By providing objective data to support diagnostic, prognostic, and therapeutic decision-making, comprehensive genomic profiling is necessary for advancing care for pediatric neuro-oncology patients. This article presents the largest cohort of pediatric low- and high-grade gliomas profiled by next-generation sequencing. Reportable alterations were detected in 95% of patients, including diagnostically relevant lesions as well as novel oncogenic fusions and mutations. Additionally, tumor mutational burden (TMB) is reported, which identifies a subpopulation of hypermutated glioblastomas that harbor deleterious mutations in DNA repair genes. This provides support for TMB as a potential biomarker to identify patients who may preferentially benefit from immune checkpoint inhibitors.

Comprehensive genomic profiling of malignant phyllodes tumors of the breast.

PURPOSE: Malignant phyllodes tumors (MPT) are exceptionally rare, and the genomic drivers of these tumors are still being elucidated. We performed comprehensive genomic profiling (CGP) of MPT to identify genomic alterations that will inform approaches to targeted therapy for patients with MPT, including relapsed, refractory, and metastatic disease. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded samples from 24 consecutive patient cases of MPT. CGP was performed using a hybrid capture, adaptor ligation-based next generation sequencing assay to a high, uniform coverage (mean, 582×). Tumor mutational burden (TMB) was calculated from a minimum of 1.14 Mb of sequenced DNA as previously described and reported as mutations/Mb. The results were analyzed for all classes of genomic alterations, including short variants (SV; base substitutions, small insertions, and deletions), rearrangements, and copy number changes, including amplifications and homozygous deletions. RESULTS: The 24 cases of MPT included 15 patients with localized and 9 with metastatic disease. The median TMB was 2.7 mut/Mb, and no cases had a TMB > 10 mut/Mb. 20 out of 24 cases were evaluable for microsatellite status, and all were microsatellite stable. The most commonly mutated genes were TP53 (58.3%), TERT-promoter (57.9%), NF1 (45.8%), MED12 (45.8%), CDKN2A/B (33.3%), and MLL2 (33.3%). Targetable kinase fusions including KIAA1549-BRAF or FGFR3-TACC3 were identified in 2/24 (8.3%) tumors. CONCLUSIONS: This study identifies clinically relevant genomic alterations that suggest novel targeted therapy approaches for patients with MPT.

Comprehensive genomic profiling reveals inactivating SMARCA4 mutations and low tumor mutational burden in small cell carcinoma of the ovary, hypercalcemic-type.

OBJECTIVE: Small cell carcinoma of the ovary, hypercalcemic-type (SCCOHT) is a rare, extremely aggressive neoplasm that usually occurs in young women and is characterized by deleterious germline or somatic SMARCA4 mutations. We performed comprehensive genomic profiling (CGP) to potentially identify additional clinically and pathophysiologically relevant genomic alterations in SCCOHT. METHODS: CGP assessment of all classes of coding alterations in up to 406 genes commonly altered in cancer and intronic regions for up to 31 genes commonly rearranged in cancer was performed on 18 SCCOHT cases (16 exhibiting classic morphology and 2 cases exhibiting exclusive a large cell variant morphology). In addition, a retrospective database search for clinically advanced ovarian tumors with genomic profiles similar to SCCOHT yielded 3 additional cases originally diagnosed as non-SCCOHT. RESULTS: CGP revealed inactivating SMARCA4 alterations and low tumor mutational burden (TMB) (<6mutations/Mb) in 94% (15/16) of SCCOHT with classic morphology. In contrast, both (2/2) cases exhibiting only large cell variant morphology were hypermutated (TMB scores of 90 and 360mut/Mb) and were wildtype for SMARCA4. In our retrospective search, an index ovarian cancer patient harboring inactivating SMARCA4 alterations, initially diagnosed as endometrioid carcinoma, was re-classified as SCCOHT and responded to an SCCOHT chemotherapy regimen. CONCLUSION: The vast majority of SCCOHT demonstrate genomic SMARCA4 loss with only rare co-occurring alterations. Our data support a role for CGP in the diagnosis and management of SCCOHT and of other lesions with overlapping histological and clinical features, since identifying the former by genomic profile suggests benefit from an appropriate regimen and treatment decisions, as illustrated by an index patient.

Next-Generation Sequencing Reveals Pathway Activations and New Routes to Targeted Therapies in Cutaneous Metastatic Melanoma.

BACKGROUND: Comprehensive genomic profiling of clinical samples by next-generation sequencing (NGS) can identify one or more therapy targets for the treatment of metastatic melanoma (MM) with a single diagnostic test. METHODS: NGS was performed on hybridization-captured, adaptor ligation-based libraries using DNA extracted from 4 formalin-fixed paraffin-embedded sections cut at 10 microns from 30 MM cases. The exons of 182 cancer-related genes were fully sequenced using the Illumina HiSeq 2000 at an average sequencing depth of 1098X and evaluated for genomic alterations (GAs) including point mutations, insertions, deletions, copy number alterations, and select gene fusions/rearrangements. Clinically relevant GAs (CRGAs) were defined as those identifying commercially available targeted therapeutics or therapies in registered clinical trials. RESULTS: The 30 American Joint Committee on Cancer Stage IV MM included 17 (57%) male and 13 (43%) female patients with a mean age of 59.5 years (range 41-83 years). All MM samples had at least 1 GA, and an average of 2.7 GA/sample (range 1-7) was identified. The mean number of GA did not differ based on age or sex; however, on average, significantly more GAs were identified in amelanotic and poorly differentiated MM. GAs were most commonly identified in BRAF (12 cases, 40%), CDKN2A (6 cases, 20%), NF1 (8 cases, 26.7%), and NRAS (6 cases, 20%). CRGAs were identified in all patients, and represented 77% of the GA (64/83) detected. The median and mean CRGAs per tumor were 2 and 2.1, respectively (range 1-7). CONCLUSION: Comprehensive genomic profiling of MM, using a single diagnostic test, uncovers an unexpectedly high number of CRGA that would not be identified by standard of care testing. Moreover, NGS has the potential to influence therapy selection and can direct patients to enter relevant clinical trials evaluating promising targeted therapies.

Comprehensive Genomic Profiling of Central Giant Cell Lesions Identifies Clinically Relevant Genomic Alterations.

PURPOSE: Comprehensive genomic profiling (CGP) can simultaneously detect clinically relevant genomic alterations (CRGAs) in hundreds of cancer-related genes and direct treatment toward patient-specific therapy options for many tumors. This pilot study aimed to use CGP to describe CRGAs present in central giant cell lesions (CGCLs) to characterize any possible underlying genomic drivers of CGCLs. MATERIALS AND METHODS: With institutional review board approval, electronic medical records were searched for patients with histologically confirmed CGCLs who underwent biopsy at Mayo Clinic from 2000 through 2014. Clinical characteristics were recorded from the medical records. At least 50 ng of DNA was extracted from formalin-fixed paraffin-embedded archival CGCL specimens by use of hybridization-capture, adaptor ligation-based libraries targeting all exons from 315 cancer-related genes plus select introns from 28 genes commonly rearranged in cancer. Samples were sequenced to high, uniform coverage and assessed for all 4 classes of genomic alterations: base substitutions, small insertions and deletions, rearrangements, and copy number alterations. RESULTS: Of 8 CGCL specimens, 3 (37.5%) harbored CRGAs, including base substitutions in BRAF, GNAS, and KRAS that are predicted to be oncogenic. In 1 sample, focal high-level amplification of the MITF gene was detected. Rearrangement in the PDGFRB gene was identified in a fourth sample, although the significance of this alteration is uncertain. CONCLUSIONS: This pilot study shows that a relatively high frequency of CRGAs (37.5%) can be identified in CGCLs by use of CGP. Furthermore, 25% of CGCLs analyzed had somatic mutations predicted to activate the mitogen-activated protein kinase signaling pathway, suggesting it may be a driver of the aggressive behavior of these lesions. On the basis of this study, genomic profiling of a larger cohort of CGCLs to validate these observations, as well as correlate mutations with aggressive versus nonaggressive biological behavior and therapeutic responses, appears warranted.

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.