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IntroductionIn 2020, the world experienced the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the coronavirus disease 2019 (COVID-19) pandemic. Mounting evidence indicates that the gut microbiome plays a role in host immune response to infections and, in turn, may have an impact on the disease trajectory of SARS-CoV2 infection. However, it remains to be established whether modulation of the microbiome can impact COVID-19–related symptomatology and patient outcomes. Therefore, we conducted a study designed to modulate the microbiome evaluating the safety and physiologic effects of KB109 combined with self-supportive care (SSC) vs SSC alone in non-hospitalized patients with mild to moderate COVID-19. KB109 is a novel synthetic glycan developed to increase the production of gut microbial metabolites that support immune system homeostasis through gut microbiome modulation. Our goal was to gain a better understanding of the safety of KB109, the natural course of COVID-19 symptomatology, and the possible role of the gut microbiome in patients with mild to moderate COVID-19.MethodsAdult patients who tested positive for COVID-19 were randomized 1:1 to receive KB109 combined with SSC or SSC alone for 14 days and were then followed for an additional 21 days (35 days in total). Patients self-assessed their COVID-19–related symptoms (8 cardinal symptoms plus 5 additional symptoms) and self-reported comorbidities. The primary and secondary objectives were to evaluate the safety of KB109 plus SSC compared with that of SSC alone and to evaluate selected measures of health, respectively.ResultsBetween July 2, 2020 and December 23, 2020, 350 patients were randomized to receive KB109 and SSC (n=174) or SSC alone (n=176). Overall, the most common comorbidities reported were hypertension (18.0% [63/350 patients]) followed by chronic lung disease (8.6% 30/350 patients). KB109 was well tolerated with most treatment-emergent adverse events being mild to moderate in severity. The administration of KB109 plus SSC reduced medically-attended visits (ie, hospitalization, emergency room visits, or urgent care visits) by 50.0% in the overall population and by 61.7% in patients with ≥1 comorbidity; in patients aged ≥45 years or with ≥1 comorbidity, medically-attended visits were reduced by 52.8%, In the SSC group, patients reporting ≥1 comorbidity had a longer median time to resolution of symptoms than those who reported no comorbidities at baseline (13 overall symptoms: 30 vs 21 days, respectively; hazard ratio [HR]=1.163 [95% CI, 0.723-1.872]; 8 cardinal symptoms: 21 vs 15 days, respectively; HR=1.283 [95% CI, 0.809-2.035]). In patients reporting ≥1 comorbidity, median time to resolution of symptoms was shorter in the KB109 plus SSC group compared with the SSC alone group (13 overall symptoms: 30 vs 21 days, respectively; HR=1.422 [95% CI, 0.898-2.250]; 8 cardinal symptoms: 17 vs 21 days, respectively; HR=1.574 [95% CI, 0.997-2.485]). In the KB109 plus SSC group, patients aged ≥45 years or with ≥1 comorbidity had a shorter median time to resolution of symptoms compared with SSC alone (overall 13 symptoms: 21 vs 31 days; HR=1.597 [95% CI, 1.064-2.398]).ConclusionsResults from our study show that KB109 is well tolerated among patients with mild to moderate COVID-19. Patients with ≥1 comorbidity had a longer duration of COVID-19 symptoms than those without comorbidities. Moreover, in patients reporting ≥1 comorbidity or aged ≥45 years (at-risk population), administration of KB109 plus SSC improved median time to resolution of COVID-19–related symptoms and reduced the rate of medically-attended visits compared with SSC alone.

Objectives These 2 parallel studies (K031 and K032) aim to evaluate the safety of KB109 in addition to supportive self-care (SSC) compared with SSC alone in outpatients with mild to moderate coronavirus disease 2019 (COVID-19). KB109 is a novel synthetic glycan that was formulated to modulate the gut microbiome composition and metabolic output in order to increase beneficial short-chain fatty acid (SCFA) production in the gut. The K031 study is designed to evaluate the safety of KB109 and characterize its impact on the natural progression of COVID-19 in patients with mild to moderate disease. The K032 study is evaluating the effect of KB109 on the gut microbiota structure and function in this same patient population. Additionally, both studies are evaluating measures of health care utilization, quality of life (QOL), laboratory indices, biomarkers of inflammation, and serological measures of immunity in patients who received SSC alone or with KB109. Noteworthy aspects of these outpatient studies include study design measures aimed at limiting in-person interactions to minimize the risk of infection spread, such as use of online diaries, telemedicine, and at-home sample collection. Study design K031 and K032 are randomized, controlled, open-label, clinical food studies. Participants Inclusion Criteria: • Adults ≥18 years of age • Patients willing and able to give informed consent • Screening/randomization telemedicine visit within 2 days of testing positive test for COVID-19 In K031 study, symptomatic patients at COVID-19 testing must report new or worsening symptoms at baseline that have not been present for more than 5 days ▪ Cardinal COVID-19 symptoms include fever, chills/repeated shaking with chills, cough, shortness of breath, headache, muscle pain, anosmia/ageusia, and sore throat. The 5 additional symptoms include gastrointestinal (GI) disturbance/symptoms (other than diarrhea), diarrhea, fatigue, nasal congestion, and chest tightness In K031, at COVID-19 testing, pre-symptomatic patients must report new cardinal COVID-19 symptoms within 7 days of a positive test and they must be screened and randomized within 5 days of developing symptoms • Mild to moderate COVID-19 and self-reported outpatient management In K032, mild to moderate COVID-19 was defined as having the following symptoms for no more than 72 hours before COVID-19 testing: a self- reported fever or cough (new or exacerbated) or presence of at least 2 of the following: anosmia, sore throat, or nasal congestion • Ability to adhere to the study visit schedule and other protocol requirements • Consistent internet or cell phone access with a data plan and access to a smartphone, tablet, or computer • The K031 and K032 studies are currently being conducted at 17 clinical institutions throughout the United States. Exclusion Criteria: • In the primary investigator’s (PI) judgement, patients likely to require hospitalization for COVID-19 • Patients who are hospitalized for in-patient treatment or currently being evaluated for potential hospitalization at the time of informed consent for conditions other than COVID-19 • History of chronic lung disease with chronic hypoxia • History of documented cirrhosis or end-stage liver disease • Ongoing requirement for oxygen therapy • Shortness of breath in resting position • Diagnosis of sleep apnea requiring bilevel positive airway pressure (BIPAP)/continuous positive airway pressure (CPAP) • Female patients who are pregnant, trying to become pregnant, or lactating • Concurrent use of immunomodulatory agent within 12 months; systemic antibiotics, antifungals, or antivirals for treatment of active infection within 28 days; systemic immunosuppressive therapy within 3 months; or drugs or other compounds that modulate GI motility (eg, stool softeners, laxatives, or fiber supplements) taken currently, or within 7 days. Antacid (histamine 2 blockers and proton pump inhibitors) and antidiarrheal agents are not prohibited • History of GI surgery (6 months prior to randomization), including but not limited to bariatric surgery and bowel resection, or history of, or active GI disease(s) that may affect assessment of tolerability, including but not limited to inflammatory bowel disease, irritable bowel syndrome, autoimmune disease, or GI malignancy • Participation in an interventional clinical trial or use of any investigational agent within 30 days before randomization • Clinically significant or uncontrolled concomitant medical condition that would put the patient at risk or jeopardize the objectives of the study in the opinion of the PI • In the opinion of the PI, patient unlikely for any reason to be able to comply with study procedures • Contraindications, sensitivities, or known allergy to the use of the study product or its components Intervention and comparator Patients will be randomized (1,1) to receive either SSC and KB109 or SSC alone. During SSC, patients should follow the steps as instructed by their healthcare provider to care for themselves and protect other people in the home and community from potentially contracting COVID-19. Management of COVID-19-related symptoms with over-the-counter cough, cold, and anti-pyretic medications by patients is permitted in accordance with the medications’ respective drug facts label or as instructed by the patient’s healthcare provider. Following randomization, patients assigned to receive KB109 and SSC will receive a Kaleido Biosciences, Inc at-home study kit including a thermometer, pulse oximeter, and KB109. During the Intake Period (days 1–14), KB109 will be reconstituted in water by the patient and consumed by the patient twice daily (at least 8 hours apart), following an up-titration dosing schedule: Days 1 to 2: 9 g twice daily for a total daily dose of 18 g Days 3 to 4: 18 g twice daily for a total daily dose of 36 g Days 5 to 14: 36 g twice daily for a total daily dose of 72 g During the intake period, patients will record their daily COVID-19–related symptoms, selected COVID-19 signs (as self-measured using the provided thermometer and pulse oximeter), responses to questions related to QOL measures, health care use measures, and concomitant medications taken in the previous 24 hours. Wellness visits by telephone will be conducted between days 1 and 14 to follow up on patient’s health status and to ascertain compliance with KB109 and completion of questions. On day 14, all patients will undergo a telemedicine visit where the following will be conducted: abbreviated physical examination, assessment of safety and other protocol-specified measures of health, and an evaluation of whether follow-up treatment is recommended owing to a progression of COVID-19 symptoms. If feasible, blood samples for clinical chemistries, biomarkers and serological measure of immunity, and nasal/oropharyngeal swabs for quantitative viral load assessments will be collected. Beginning on day 15, patients in both groups will enter the follow-up period (days 15–35) where COVID-19 signs, symptoms, and health care use indices will be collected. Wellness visits by telephone will be conducted on days 21, 28, and 35 to follow-up on the patient’s health status. On day 35, all patients will undergo a telemedicine visit where the same information as the day 14 telemedicine visit will be collected, including any blood samples. Main outcomes The primary outcome for the K031 and K032 studies is to evaluate the safety of KB109 in addition to SSC compared with SSC alone in outpatients with mild to moderate COVID-19 by assessing the number of patients experiencing KB109-related treatment-emergent adverse events (TEAEs) during the study. K031 will also evaluate duration of symptoms among outpatients with mild to moderate COVID-19. This will be as an assessment made during the intake and/or follow-up periods of the following: • Time to resolution of the 13 overall and the 8 cardinal COVID-19–related symptoms from day 1 until the day at which the composite score of the 13 overall and 8 cardinal COVID-19–related symptoms becomes 0 or 1 and remains at 0 or 1 for the rest of the intake period and for the follow-up period • Proportion of patients with a reduction from baseline in each of the 13 overall COVID-19–related symptoms • Proportion of patients in whom symptoms (present at baseline) become absent for each of the 13 overall COVID-19–related symptoms • Change from baseline in the overall composite score of the 13 overall COVID-19–related symptoms and the 8 cardinal COVID-19–related symptoms • Time to resolution of fever (defined as from day 1 until the day at which a patient’s daily maximum temperature achieves and remains below 100.4°F without antipyretic medication) • Proportion of patients with oxygen saturation • Measures collected from the health care provider wellness visits • Proportion of patients experiencing hospital admissions (all cause and COVID-19–related) • Health care use K032 will evaluate the effect of KB109 in addition to SSC compared with SSC alone on the gut microbiota structure and function in outpatients with mild to moderate COVID-19. Before days 1, 14, and 35, microbiota structure (eg, magnitude of change in gut microbiome structure, composition of gut microbiome) will be analysed by methods such as nucleic acid sequencing and gut microbiome function will be analysed via levels of stool inflammatory biomarkers (eg, lipocalin) and gut microbiome metabolites (eg, SCFA). The health of outpatients with mild to moderate COVID-19 will be evaluated during the intake and follow- up periods by: measures of QOL; measures collected from the healthcare provider wellness visits; the proportion of patients experiencing hospital admissions; health care use, the proportions of patients with oxygen saturation Randomisation All patients deemed eligible for the studies will be randomized in a 1:1 ratio to KB109 in addition to SSC or SSC alone group using an interactive response technology system. Randomization will be stratified by study site/center, age groups (≥18– Blinding (masking) These studies are open-label; therefore, no blinding is necessary. Numbers to be randomised (sample size) K031 will enroll approximately 350 to 400 (175–200 patients per group) whereas K032 will enroll approximately 50 patients (25 per group). Study status K031 protocol version 4, December 9, 2020; recruitment started in August, 2020, and the study is estimated to be completed in March 2021. This study is active and enrollment was completed in January, 2021. K032 protocol version 2, June 30, 2020; recruitment is estimated to start in July, 2020. This study is recruiting and the study is estimated to be completed in March 2021. Study registration K031 is registered with the US National Library of Medicine, Identifier NCT04414124 as of June 4, 2020. K032 is registered with the US National Library of Medicine, Identifier NCT04486482 as of July 24, 2020. Full protocol The full protocols are attached as additional files (Additional files 1 and 2), accessible from the ClinicalTrials.gov website. In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocols. The study protocols have been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional files 3 and 4).

11574 Background: Malignant perivascular epithelioid cell tumor (PEComa) is a rare and aggressive sarcoma. nab-Sirolimus is an albumin-bound intravenous (IV) mTOR inhibitor (mTORi) approved for the treatment of adult patients with locally advanced unresectable or metastatic malignant PEComa. The AMPECT trial (NCT02494570) was the first prospective study in advanced malignant PEComa. In exploratory biomarker analyses, TSC1 or TSC2 alterations were associated with response. We report data from the final analysis of AMPECT patients, who were naïve to mTORi, and in patients with malignant PEComa with prior mTORi exposure treated with nab-sirolimus in an expanded access program (EAP) (NCT03817515). Methods: In AMPECT, patients with malignant PEComa naïve to mTORi received nab-sirolimus (100 mg/m2 IV days 1 and 8 of every 21-day cycle) until progression or unacceptable toxicity. The primary endpoint was ORR by independent radiology review. Other endpoints included duration of response (DOR) and disease control rate (DCR), defined as complete response (CR), partial response (PR), or stable disease (SD) at ≥12 weeks. In the EAP, patients with malignant PEComa and prior mTORi exposure received the same dose of nab-sirolimus as in AMPECT. Responses and DCR were evaluated post hoc via electronic medical record review. Genetic profiling, including TSC1 or TSC2 status, was assessed in both protocols, but no specific mutation criteria were required for enrollment. Results: Data include 47 total efficacy-evaluable patients, 31 in AMPECT and 16 with malignant PEComa and prior mTORi exposure treated in the EAP from July 2019–July 2021. Prior mTORi on the EAP included sirolimus, everolimus, temsirolimus, or sapanisertib; 12 patients had exposure to 1 prior mTORi and 4 to ≥2 prior mTORi, and 50% had had progressive disease as best response on mTORi. In AMPECT, ORR was 39% (12/31 patients), and DCR was 71%. Median DOR was not reached after 3 years of follow-up. On the EAP, 4/16 patients (25%) achieved PR (DOR range: 1.3+–25.2+ months, 3 ongoing), and 8/16 (50%) had SD as best response; the DCR was 63% (10/16). Of patients with known TSC1 or TSC2 inactivating alterations in the combined datasets (n = 23), 57% had a response (AMPECT, 64%; EAP, 44%). There were no Grade 4 or 5 treatment-related adverse events on either protocol Conclusions: nab-Sirolimus provided durable responses in mTORi-naïve patients with malignant PEComa and clinical benefit in an expanded access protocol for patients with malignant PEComa with prior mTORi therapy. Although AMPECT and the EAP cannot be directly compared, response rates showed similar trends regardless of prior mTORi exposure and in patients with TSC1 or TSC2 alterations. Based on the emerging biomarker results, a tissue-agnostic study in patients with TSC1 and TSC2 alterations has been initiated (NCT05103358). Clinical trial information: NCT02494570, NCT03817515.

Purpose The promise of precision oncology is that identification of genomic alterations will direct the rational use of molecularly targeted therapy. This approach is particularly applicable to neoplasms that are resistant to standard cytotoxic chemotherapy, like T-cell leukemias and lymphomas. In this study, we tested the feasibility of targeted next-generation sequencing in profiles of diverse T-cell neoplasms and focused on the therapeutic utility of targeting activated JAK1 and JAK3 in an index case. Patients and Methods Using Foundation One and Foundation One Heme assays, we performed genomic profiling on 91 consecutive T-cell neoplasms for alterations in 405 genes. The samples were sequenced to high uniform coverage with an Illumina HiSeq and averaged a coverage depth of greater than 500× for DNA and more than 8M total pairs for RNA. An index case of T-cell prolymphocytic leukemia (T-PLL), which was analyzed by targeted next-generation sequencing, is presented. T-PLL cells were analyzed by RNA-seq, in vitro drug testing, mass cytometry, and phospho-flow. Results One third of the samples had genomic aberrations in the JAK-STAT pathway, most often composed of JAK1 and JAK3 gain-of-function mutations. We present an index case of a patient with T-PLL with a clonal JAK1 V658F mutation that responded to ruxolitinib therapy. After relapse developed, an expanded clone that harbored mutant JAK3 M511I and downregulation of the phosphatase, CD45, was identified. We demonstrate that the JAK missense mutations were activating, caused pathway hyperactivation, and conferred cytokine hypersensitivity. Conclusion These results underscore the utility of profiling occurrences of resistance to standard regimens and support JAK enzymes as rational therapeutic targets for T-cell leukemias and lymphomas.

Background: Most COVID-19 cases in the US are managed via outpatient care, yet the outpatient natural history of COVID-19 is not well understood. We assessed outpatient natural history of COVID-19 as part of a clinical food study evaluating KB109 safety and its effects on select health measures when combined with supportive self-care (SSC) vs SSC alone. KB109 is a novel synthetic glycan developed to support immune system homeostasis through gut microbiome modulation. The SSC-alone arm permitted COVID-19 natural history exploration, the focus of this abstract. Methods: Adult patients were randomized 1:1 to SSC alone or SSC+KB109. Randomization was stratified by site, age subgroup, and comorbidity status. Signs/symptoms of COVID-19 and other end points were evaluated over a 35-d study period. Patients self-assessed 8 COVID-19–related cardinal symptoms; 5 additional symptoms were collected (13 overall symptoms). Results: Overall, 350 patients were randomized (SSC alone, n=176). Interim results are presented; full analysis expected in March 2021. In the SSC-alone interim results (n=89), 36.0% of patients self-reported ≥1 comorbidity. Median time to resolution of the 8 cardinal symptoms was 12d in patients with no comorbidities vs 27d in patients with ≥1 comorbidity (HR=1.708 [0.875, 3.332]). Median time to resolution of the 13 overall symptoms was 14d in patients with no comorbidities vs 27d in patients reporting ≥1 comorbidity (HR=1.800 [0.953, 3.400]). The most common comorbidities were hypertension (19.1%) and chronic lung disease (9.0%). Conclusions: This is the first clinical study to show ≥1 comorbidity is associated with a protracted time to resolution of symptoms in non-hospitalized patients with mild to moderate COVID-19.

Background Gangliogliomas are slow-growing, low-grade central nervous system tumors affecting children and young adults. However, some patients will experience tumor recurrence and/or malignant progression. This article reports on the clinical history, molecular findings, and treatment response in a patient with BRAF V600-mutated high-grade glioma arising from ganglioglioma. Methods Hematoxylin-eosin staining and comprehensive genomic profiling via Foundation One were performed on the tumor sample from a male patient undergoing treatment at the Department of Neuro-Oncology at Baylor University Medical Center. Results The patient was eligible for participation in a clinical trial (ClinicalTrials.gov identifier: NCT00916409) of a tumor treatment fields (TTFields) device, NovoTTF-100A, with concurrent radiation and chemotherapy (CCRT). His disease relapsed 4 months after completion of his CCRT, with MRI showing areas of enhancement. Temozolomide was discontinued and he was offered dabrafenib, an oral selective inhibitor of BRAF V600E, with continued use of NovoTTF. At the time of this report, after 2 years of treatment with dabrafenib and TTFields, the patient shows a durable complete response in all areas with no active lesions or new areas of enhancement. Conclusions This report suggests that TTFields delivered in combination with targeted therapy dabrafenib yielded a remarkable clinical and radiologic response in this recurrent high-grade glioma. Targeted therapy matched to genomic alterations combined with TTFields treatment could provide clinical benefit and should be prospectively explored in the near future.

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.

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

Cisplatin-based neoadjuvant chemotherapy (NAC) before cystectomy is the standard of care for muscle-invasive bladder cancer (MIBC), with 25-50% of patients expected to achieve a pathologic response. Validated biomarkers predictive of response are currently lacking.To discover and validate biomarkers predictive of response to NAC for MIBC.Pretreatment MIBC samples prospectively collected from patients treated in two separate clinical trials of cisplatin-based NAC provided the discovery and validation sets. DNA from pretreatment tumor tissue was sequenced for all coding exons of 287 cancer-related genes and was analyzed for base substitutions, indels, copy number alterations, and selected rearrangements in a Clinical Laboratory Improvements Amendments-certified laboratory.The mean number of variants and variant status for each gene were correlated with response. Variant data from the discovery cohort were used to create a classification tree to discriminate responders from nonresponders. The resulting decision rule was then tested in the independent validation set.Patients with a pathologic complete response had more alterations than those with residual tumor in both the discovery (p=0.024) and validation (p=0.018) sets. In the discovery set, alteration in one or more of the three DNA repair genes ATM, RB1, and FANCC predicted pathologic response (p0.001; 87% sensitivity, 100% specificity) and better overall survival (p=0.007). This test remained predictive for pathologic response in the validation set (p=0.033), with a trend towards better overall survival (p=0.055). These results require further validation in additional sample sets.Genomic alterations in the DNA repair-associated genes ATM, RB1, and FANCC predict response and clinical benefit after cisplatin-based chemotherapy for MIBC. The results suggest that defective DNA repair renders tumors sensitive to cisplatin.Chemotherapy given before bladder removal (cystectomy) improves the chance of cure for some but not all patients with muscle-invasive bladder cancer. We found a set of genetic mutations that when present in tumor tissue predict benefit from neoadjuvant chemotherapy, suggesting that testing before chemotherapy may help in selecting patients for whom this approach is recommended.

Inflammatory breast cancer (IBC) is a distinct clinicopathologic entity that carries a worse prognosis relative to non-IBC breast cancer even when matched for standard biomarkers (ER/PR/HER2). The objective of this study was to identify opportunities for benefit from targeted therapy, which are not currently identifiable in the standard workup for advanced breast cancer. Comprehensive genomic profiling on 53 IBC formalin-fixed paraffin-embedded specimens (mean, 800× + coverage) using the hybrid capture-based FoundationOne assay. Academic and community oncology clinics. From a series of 2208 clinical cases of advanced/refractory invasive breast cancers, 53 cases with IBC were identified. The presence of clinically relevant genomic alterations (CRGA) in IBC and responses to targeted therapies. CRGA were defined as genomic alterations (GA) associated with on label targeted therapies and targeted therapies in mechanism-driven clinical trials. For the 44 IBCs with available biomarker data, 19 (39 %) were ER-/PR-/HER2- (triple-negative breast cancer, TNBC). For patients in which the clinical HER2 status was known, 11 (25 %) were HER2+ with complete (100 %) concordance with ERBB2 (HER2) amplification detected by the CGP assay. The 53 sequenced IBC cases harbored a total of 266 GA with an average of 5.0 GA/tumor (range 1-15). At least one alteration associated with an FDA approved therapy or clinical trial was identified in 51/53 (96 %) of cases with an average of 2.6 CRGA/case. The most frequently altered genes were TP53 (62 %), MYC (32 %), PIK3CA (28 %), ERBB2 (26 %), FGFR1 (17 %), BRCA2 (15 %), and PTEN (15 %). In the TNBC subset of IBC, 8/19 (42 %) showed MYC amplification (median copy number 8X, range 7-20) as compared to 9/32 (28 %) in non-TNBC IBC (median copy number 7X, range 6-21). Comprehensive genomic profiling uncovered a high frequency of GA in IBC with 96 % of cases harboring at least 1 CRGA. The clinical benefit of selected targeted therapies in individual IBC cases suggests that a further study of CGP in IBC is warranted.

Background: Inflammatory breast cancer (IBC) is a distinct clinicopathologic entity that carries a worse prognosis relative to non-IBC breast cancer even when matched for biomarkers (ER/PR/HER2). Genomic profiling of IBC cases may identify alterations that suggest response to targeted therapies, but is best implemented by an integrated NGS assay capable of detecting all classes of genomic alterations (GA). Methods: Hybridization capture of 3769 exons of 236 cancer-related genes and 47 introns from 19 genes that are frequently rearranged in cancer were fully sequenced to high, uniform coverage from a commercial CLIA-certified laboratory (Foundation Medicine). Results: Of 2,208 clinical breast cancer cases assayed, 55 IBC cases were identified, and of the 50 cases for which hormone receptor and HER2/neu status were know, 34% were ER-/PR-/HER2- (TNBC). IBC cases harbored 274 GA with an average of 5.0 GA/tumor (range 1-15). At least one alteration associated with an FDA approved therapy or clinical trial was identified in 53/55 (96%) of cases, yielding an average of 2.6 clinically relevant GA/case. Genes most frequently altered were TP53 (60%), MYC (31%), PIK3CA (25%), ERBB2 (20%), FGFR1 (18%) and PTEN (16%). MYC amplifications were present in 24% of the 2,208 clinical breast carcinoma cases. In the TNBC subset of IBC, 8/19 (42%) pt samples showed MYC amplification (median copy number 8X, range 7-20) as compared to 9/36 (25%) in non-TNBC pt samples (median copy number 7X, range 6-21). Within this prospective series, treatment decisions were made based on FoundationOne results. A58-year old pt with likely secondary IBC harbored two ERBB2 activating base substitutions (V777L and S310F), but without amplification of ERBB2), and had durable response to lapatinib (Ali et al. JCO 2014). In another case, a 53 year old pt presented with ERBB2-amplified IBC now refractory to HER2-targeted therapy. FoundationOne testing revealed an activating EGFR mutation (L858R) as well as the previously described ERBB2 amplification, suggesting that the EGFR alteration may underlie the acquired resistance. The pt responded to erlotinib monotherapy for 8 months. (Ali et al. Clin Br Ca, 2013). Clinical follow-up for additional patients is ongoing. Conclusions: 96% of IBC cases harbored at least one alteration that suggests responsiveness to agents that are FDA approved or being studied in clinical trials. IBC cases also frequently had MYC amplifications (31%), but this may reflect a high percentage of TNBC-IBC cases with MYC amplifications (42%) in this series. Given the limited treatment options and poor prognosis of patients with metastatic IBC, the FoundationOne assay with comprehensive NGS-based genomic profiling has the potential to identify new treatment paradigms and address an unmet clinical need for this disease. Citation Format: Norma A Palma, Siraj M Ali, Kai Wang, Juliann Chmeleiki, Gary Palmer, Deborah Morosini, Jeffrey S Ross, Vincent A Miller, Phil J Stephens, Massimo Cristofannilli. Genomic profiling by FoundationOne® analysis of inflammatory breast cancer cases reveals a high frequency of clinically relevant genomic alterations (GA) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-02.

Background: Targeted ERBB2/HER2 inhibitors are FDA-approved for the treatment of breast and gastric cancers. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to test for overexpression and amplification of ERBB2/HER2, respectively, are performed as part of routine clinical care. Recently, activating mutations in ERBB2 have been reported and may confer sensitivity to targeted agents. Testing for these mutations is not routine, and testing for amplifications is not done outside of approved indications. We explored the complete spectrum of activating ERBB2 mutations and amplifications across a collection of ∼7,300 solid tumor specimens, including a large number of breast cancer specimens, to determine a)how many breast cancer patients could benefit from HER2-targeted therapy due to activating mutations in ERBB2 and b)how widespread ERBB2 alterations are across the solid tumor spectrum. Methods: Extracted DNA from clinical tumor samples underwent comprehensive genomic profiling using FoundationOne . 3769 exons of 236 cancer-related genes and 47 introns from 19 genes that are frequently rearranged in cancer were fully sequenced to high, uniform coverage and results were analyzed for base substitutions, insertions and deletions, amplifications/deletions, and rearrangements. Results: Known oncogenic ERBB2 alterations were identified in approximately 6% of all solid tumors across 27 different histologies. Of all the ERBB2 alterations, activating mutations in ERBB2 were identified in 131 samples and amplifications were observed in 246 samples. Two samples harbored an ERBB2 rearrangement. Ten samples harbored multiple ERBB2 mutations, yet mutations and amplifications were mutually exclusive in 91% of mutated cases. ERBB2 amplification in breast and gastric cancers accounted for only 30% of these alterations. Breast cancer accounted for 37% of all the ERBB2 alterations detected, and included primarily amplifications. 25% of the alterations found in breast cancers were activating base substitutions. Standard tests for overexpression or amplification of ERBB2 would fail to detect these potentially treatable mutations. Non-amplification ERBB2 alterations were enriched in cases of relapsed lobular breast cancer. Multiple breast cancer patients with non-amplification ERBB2 alterations have responded to combinations of anti-HER2 targeted therapies. Conclusions: Comprehensive genomic profiling through FoundationOne identifies 25% more breast cancers that may be susceptible to HER2 targeted therapy due to the presence of activating mutations in ERBB2. Also, many more solid tumor cases could potentially benefit as well from ERBB2 testing. The current policy of testing only breast and gastric tumors for only HER2 amplifications is greatly limiting the potential value of the ERBB2/HER2 inhibitors. Citation Format: Gary A Palmer, Jeffrey S Ross, Kai Wang, Garrett M Frampton, Siraj M Ali, Norma Palma, Deborah Morosini, Vincent A Miller, Roman Yelensky, Doron Lipson, Philip J Stephens, Juliann Chmielecki. Activating mutations in ERBB2/HER2 as found by FoundationOneTM represent potential therapeutic targets in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-03.

Background: Carcinoma of unknown primary (CUP) accounts for 3-5% of all adult solid tumors. An extensive search for the anatomic site of origin is often undertaken in an attempt to tailor systemic treatment, but the latter often has limited efficacy - especially in the setting of an initial treatment failure. Molecularly targeted therapy is an emerging approach that may offer greater efficacy and less toxicity but is most likely to be effective when pairing a tumor harboring a sensitizing genomic alteration with an agent directed at the altered gene product. We report a patient with a CUP harboring a MET amplification with a complete metabolic response to crizotinib despite also harboring a KRAS mutation. Methods: Genomic profiling was performed using a clinical next-generation-sequencing-based assay, FoundationOne®, in a CAP-accredited laboratory certified by Clinical Laboratory Improvement Amendments (Foundation Medicine, Cambridge, Mass., USA). Results: The CUP harbored both MET amplification (16 copies) and a KRAS G12V mutation. The patient was treated with crizotinib, a MET inhibitor, and has experienced a complete normalization of tumor metabolic activity for more than 19 months. Conclusions: Genomic profiling of CUP may reveal clinically meaningful genomic alterations that can guide targeted therapy decision-making. The use of this approach should be studied prospectively as a strategy for the effective treatment of CUP patients and for avoiding resource-intensive workups to identify the tumor site of origin.

To study the frequency with which targeted tumor sequencing results will lead to implemented change in care, this study assessed tumors from 100 patients for utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. Comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations., Background. The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. Methods. A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. Results. Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0–10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. Conclusion. Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability. Implications for Practice: Identification of key factors that facilitate use of genomic tumor testing results and implementation of genomically guided therapy may lead to enhanced benefit for patients with rare or difficult to treat cancers. Clinical use of a targeted next-generation sequencing assay in the setting of an institutional molecular tumor board led to implementable clinical action in over one third of patients with rare and poor prognosis cancers. The major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access both on trial and off label. Approaches to increase actionability include early and serial sequencing in the clinical course and expanded access to genomically guided early phase clinical trials and targeted agents.

Background Squamous cell cancers of the anal canal (ASCC) are increasing in frequency and lack effective therapies for advanced disease. Although an association with human papillomavirus (HPV) has been established, little is known about the molecular characterization of ASCC. A comprehensive genomic analysis of ASCC was undertaken to identify novel genomic alterations (GAs) that will inform therapeutic choices for patients with advanced disease. Patients and methods Hybrid-capture-based next-generation sequencing of exons from 236 cancer-related genes and intronic regions from 19 genes commonly rearranged in cancer was performed on 70 patients with ASCC. HPV status was assessed by aligning tumor sequencing reads to HPV viral genomes. GAs were identified using an established algorithm and correlated with HPV status. Results Sixty-one samples (87%) were HPV-positive. A mean of 3.5 GAs per sample was identified. Recurrent alterations in phosphoinositol-3-kinase pathway (PI3K/AKT/mTOR) genes including amplifications and homozygous deletions were present in 63% of cases. Clinically relevant GAs in genes involved in DNA repair, chromatin remodeling, or receptor tyrosine kinase signaling were observed in 30% of cases. Loss-of-function mutations in TP53 and CDKN2A were significantly enhanced in HPV-negative cases (P Conclusions This is the first comprehensive genomic analysis of ASCC, and the results suggest new therapeutic approaches. Differing genomic profiles between HPV-associated and HPV-negative ASCC warrants further investigation and may require novel therapeutic and preventive strategies.

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

BACKGROUND: Primary CNS tumors, which include both low-grade and high-grade gliomas, are the most common pediatric solid malignancies. We queried whether CGP could uncover a significant frequency of CRGA that could inform treatment decisions and improve clinical outcome for these aggressive tumors. METHODS: DNA was extracted from 75 pediatric glioma formalin-fixed paraffin-embedded clinical specimens. Hybridization captured libraries of 236 (FoundationOne, n = 43) or 405 (FoundationOne Heme, n = 32) genes, plus select introns frequently rearranged in cancer were sequenced to high (>500x), uniform coverage. All classes of genomic alterations including base substitutions, small insertions and deletions, rearrangements, and copy number alterations, were evaluated and reported. CRGA were defined as GA associated with FDA approved therapies or targeted therapies in mechanism-driven clinical trials. RESULTS: The median age of the patients was 9 years (range 0 to 18 years). There were 41 male (57%) and 34 female (43%) patients. The study included glioblastomas (n = 30, 42%), astrocytomas not otherwise specified (NOS) (n = 17, 24%), gliomas NOS (n = 10, 14%), pilocytic astrocytomas and anaplastic astrocytomas (9 each, 13%). 89% of cases harbored at least one CRGA. Pediatric gliomas frequently harbored short variants or copy number alterations in TP53 (39%); BRAF (15%) for which 82% were the V600E base substitution; CDKN2A/B (15%), NF1 (14%), PIK3CA (14%), ATRX (13%) and EGFR (11%). Ten cases (14%) harbored a BRAF fusion, including 44% of pilocytic astrocytomas. BRAF fusions included the known KIAA1549-BRAF and the novel CCDC6-BRAF and BCAS1-BRAF. ATG7-RAF1, ALK-PPCB1 and QK1-RAF1 rearrangements were also identified. Outcomes from cases where targeted therapy was utilized will be presented. CONCLUSIONS: CRGA are frequently identified in pediatric glial tumors of both low-grade and high-grade histology and can be identified by CGP in the course of clinical care. CGP has the potential to identify opportunities for targeted therapies or enrollment in clinical trials for these patients.

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-amplifiedadvancedGCpatients;however,themajority 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)associatedwithapotentialresponsetotargeted therapiesapprovedbytheU.S.FoodandDrug Administrationor targeted therapy-based clinical trials. Results. Overall, 78% of GC cases harbored one clinically relevant GAormore,withthemostfrequentalterationsbeingfoundinTP53 (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, andMETamplification,withERBB2alterationsevenlysplitbetween 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. ComprehensivegenomicprofilingofGCidentifies clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions. The Oncologist 2015;20:1–9 ImplicationsforPractice:Despitedescriptionofmanypotentiallyclinicallyrelevantgenomicalterationsinretrospectiveresearch studies, these alterations are not regularly assessed in a comprehensive manner in clinical practice.This study demonstratesthe feasibility of prospective comprehensive genomic profiling (CGP) for advanced gastric carcinoma. We demonstrated a high frequency of genomic alterations associated with potential benefit from targeted therapies. CGP in this setting may inform therapeutic options beyond standard of care testing by identifying genomic alterations such as point mutations in the kinase domain of ERBB2 and MET amplification. Genotype-directed management is highlighted by the response of a MET-amplified gastric carcinoma patient to crizotinib.

Background Collecting duct carcinoma (CDC) is a rare type of renal cell carcinoma (RCC) originating from the renal medulla. Clinical outcomes are poor, and there are no consensus guidelines to guide therapy. Objective To determine genomic alterations (GAs) in a series of patients with locally advanced or metastatic CDC for whom genomic profiling was performed during the course of clinical care. Design, setting, and participants Formalin-fixed, paraffin-embedded blocks or slides were obtained for 17 patients with CDC. DNA was extracted and comprehensive genomic profiling was performed in a laboratory certified under the Clinical Laboratory Improvement Amendments. Outcome measurements and statistical analysis Bayesian algorithms and local alignment algorithms were used to detect substitutions and insertions/deletions, respectively. A comparison to normal control samples was used to detect copy number alterations. Clinically relevant GAs (CRGAs) were defined as those linked to approved or investigational targeted therapies. Results and limitations The median age in the cohort was 53 yr (range 26–73), and 14 primary tumors and three metastatic sites assessed. A total of 36 GAs were detected in this series of patients, with an average of 2.1 GAs per case. The most common GAs were in NF2 (5/17, 29%), SETD2 (4/17, 24%), SMARCB1 (3/17, 18%), and CDKN2A (2/17, 12%). Of nine cases assessed for FH GAs, two patients had FH homozygous loss. A limitation is that targeted interrogation of genes known to be implicated in other cancers was performed, so mutations outside of these cannot be excluded. Conclusions Recurrent CRGAs were detected in this series of CDC cases and suggest a possible benefit from targeted therapy. In particular, mTOR inhibitors may be of interest in patients with NF2 alterations. Alterations in FH and SMARCB1 also occurred in a mutually exclusive manner to NF2 alterations. Patient summary This report provides important genomic insights into collecting duct carcinoma, a rare type of renal cell carcinoma with a very aggressive course. These insights could further rationalize the use of targeted therapies for rare tumors according to the individual genomic alterations harbored.

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.

We analyzed the genomic and phosphoproteomic profiles of breast cancer tissue obtained from six patients with estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer who had highly durable (≥5 years) and, in some cases, ongoing clinical responses with capecitabine. Formalin-fixed, paraffin-embedded tissue samples from patients’ primary (n = 4) or metastatic (n = 2) breast cancers were utilized for targeted next-generation sequencing and reversed phase protein microarray. Two patients received capecitabine monotherapy. Four patients received capecitabine in combination with paclitaxel; three of these continued single-agent capecitabine after stopping paclitaxel. Capecitabine was discontinued for progressive disease after a mean of 66 months in four patients (range 54–86 months), and two patients remain on therapy, having received capecitabine for >91 months and >122 months, respectively. Three patients’ cancers (50%) had likely functional alterations in DNA repair and chromatin remodeling genes, while three other patients’ cancers had variants of unknown significance in these pathways. Mutations in PIK3CA, amplifications of FGFR1 or ZNF703, or phosphorylation of HER family receptors and their downstream proteins did not preclude exceptional responses to capecitabine. None of the patients’ tumors harbored TP53 or PTEN mutations. Four of the patients had breast cancer tissue available for PTEN immunohistochemistry, and all four patients’ cancers were positive for PTEN. These surprising findings in a group of phenotypically similar patients with ER-positive, endocrine therapy-pretreated, HER2-negative metastases, are supported by preclinical data showing that sensitivity to 5-fluorouracil is enhanced by deficiencies in chromatin remodeling and homologous recombination genes. Our findings suggest that mutations that inactivate homologous recombination and/or chromatin remodeling genes within ER-positive, HER2-negative breast cancers may predict for highly durable responses to capecitabine.

Purpose Neoadjuvant cisplatin-based chemotherapy is standard of care for muscle-invasive bladder cancer (MIBC); however, it is infrequently adopted in practice because of concerns regarding toxicity and delay to cystectomy. We hypothesized that three cycles of neoadjuvant accelerated methotrexate, vinblastine, doxorubicin, and cisplatin (AMVAC) would be safe, shorten the time to surgery, and yield similar pathologic complete response (pT0) rates compared with historical controls. Patients and Methods Patients with cT2-T4a and N0-N1 MIBC were eligible and received three cycles of AMVAC with pegfilgrastim followed by radical cystectomy with lymph node dissection. The primary end point was pT0 rate. Telomere length (TL) and p53 mutation status were correlated with response and toxicity. Results Forty-four patients were accrued; 60% had stage III to IV disease; median age was 64 years. Forty patients were evaluable for response, with 15 (38%; 95% CI, 23% to 53%) showing pT0 at cystectomy, meeting the primary end point of the study. Another six patients (14%) were downstaged to non–muscle invasive disease. Most (82%) experienced only grade 1 to 2 treatment-related toxicities. There were no grade 3 or 4 renal toxicities and no treatment-related deaths. One patient developed metastases and thus did not undergo cystectomy; all others (n = 43) proceeded to cystectomy within 8 weeks after last chemotherapy administration. Median time from start of chemotherapy to cystectomy was 9.7 weeks. TL and p53 mutation did not predict response or toxicity. Conclusion AMVAC is well tolerated and results in similar pT0 rates with 6 weeks of treatment compared with standard 12-week regimens. Further analysis is ongoing to ascertain whether molecular alterations in tumor samples can predict response to chemotherapy.

Background: The TSC1/TSC2 complex inhibits the mTOR pathway. Loss of function of this complex leads to hyperperactivation of the mTOR pathway which promotes growth and survival. Few studies have examined the role of TSC1 and TSC2 mutations in breast cancer and as a clinically relevant target for molecularly targeted therapy, such as everolimus, a rapamycin analog. Methods: Hybridization capture of 3769 exons of 236 cancer-related genes and 47 introns from 19 genes that are frequently rearranged in cancer were fully sequenced to high, uniform coverage from a commercial CLIA-certified laboratory (Foundation Medicine). Results: 30 of 2,208 breast cancer pt samples (1.3%) harbored either TSC1 (13/30, 43%) or TSC2 (17/30, 56%) genomic alterations (GAs). The samples with TSC1/2 consisted of breast carcinoma n.o.s. (17/30) and invasive ductal carcinoma (12/30); one invasive lobular carcinoma (ILC) harbored a TSC2 alteration. Sequencing was performed on metastatic (18/30, 60%) and primary (12/30, 40%) tumors. All TSC1 alterations resulted in truncation of the protein product; TSC2 GA consisted of truncations (14/17) and point mutations (3/17). TSC1/2 GAs frequently co-occurred with GAs in TP53 (19/30; 63%) and amplifications of MYC (9/30; 30%) and FGFR1 (7/30; 23%). Of the TSC1/2-mutant cases where hormone receptor and HER2/neu status was known (23/30), 65% were ER-/PR-/HER2- (TNBC), 26% were ER+/HER2- and 9% were ER-/HER2+. Patient case: We describe a postmenopausal node positive ILC (ER+/PR+/HER2-) pt who received adjuvant chemotherapy followed by 9 yrs adjuvant anastrazole then with disease recurrence. On FoundationOne testing a TSC2 A1141T missense mutation, along with GAs in CDH1, ETV6, and CSF1R, were found in her metastatic disease in the soft tissues of her left orbit associated with substantial swelling resulting in loss of vision. While TSC2 A1141T has not been functionally characterized, in vitro and in vivo data suggest sensitivity to mTOR inhibitors. Her disease stabilized but did not decrease in size with vinorelbine + capecitabine. She was treated with letrozole + everolimus and had a near clinical complete response with restoration of vision for 5 + months. Collection of clinical data on additional pts is currently ongoing. Conclusion: 1.3% of breast cancer cases harbored GAs in either TSC1 orTSC2. The majority of TSC1/2 GAs cause inactivation of these negative regulatory proteins leading to activation of the mTOR signaling pathway. TSC1/2 GAs were enriched for TNBC (65%) versus non-TNBC (35%) (p= 0.077; Fisher’s exact test). Truncation of TSC1/2 occurred at multiple codons, underscoring the importance of examining the entire coding region of tumor suppressor genes for these inactivating events. Comprehensive genomic profiling has the potential to identify the broad spectrum of these inactivating events, a subset of which may be clinically relevant. Citation Format: Norma A Palma, Juliann Chmielecki, Garrett Frampton, Siraj Ali, Maren Levin, Jeffrey S Ross, Deborah Morosini, Gary Palmer, Vincent A Miller, Phil Stephens, Joyce O'Shaughnessy. FoundationOne profiling of TSC1 and TSC2-mutated advanced breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-06.

151 Background: 10 to 17% of TNBCs harbor PIK3CA mutations (TCGA Nature 490:61, 2012; Khambata-Ford S. ASCO 2010, abst 1056). Here we report clinical history and molecular findings for a TNBC patient with loss of mutant PIK3CA in a C-refractory metastasis that was present in her primary BC, who has had an ExRx to C. Methods: Following IRB-approved informed consent, targeted NGS was performed on the pt’s FFPE primary TNBC and on a C-refractory recurrent lung metastasis at a CLIA-certified laboratory (Foundation Medicine) to characterize all classes of genomic alterations across 287 cancer-related genes. RPPA was performed at a CLIA-certified laboratory (Theranostics Health) where immunostaining with 24 antibodies was directed against HER1/2/3 pathway proteins and AR. Results: 37 yo woman presented in 2006 with grade 3 primary TNBC, infraclavicular LNs and lung metastases at 33 weeks gestation. There was no response to preop doxorubicin/cyclophosphamide and following delivery of a healthy baby, she was treated with irinotecan, carboplatin, and C (#NCT00248287) and had near complete response (CR) in her lungs and pathologic CR in breast. In 2008, after 20 mos on C, chest CT showed a new lung met which was resected and she remains disease-free on C alone. NGS: Primary BC: PIK3CA C420R, TP53 mutations, MCL1 amplification (amp), and RAD51D germline mutation; C-refractory lung met: TP53, MLL2 mutations, MCL1, MYC, KDM5A, CCNE1 amp and RAD51D germline mutation (loss of PIK3CAmutation confirmed). RPPA: Primary BC: p-AR S650 (3+); p-ERK (2+), and HER1, p-HER1, p-HER3, p-AKT, p-S6, p-4EBP1 (1+); C-refractory lung met: loss of p-ERK; p-HER1, p-4EBP1 (2+), and p-AR, p-AKT, p-mTOR (1+). PTEN: Primary BC: 60% cells positive by IHC MAb 6H2.1 (Cascade Biosciences). Conclusions: The pt’s ExRx to C was dependent on presence of PIK3CA mutation which was lost in the C-refractory lung met. Loss of RAD51D function may also have contributed (Liping L. Ca Res 68:9141, 2008). High p-AR expression did not preclude response to C. Activating PIK3CA mutations induce an EGFR/ERK paracrine signaling axis in TNBCs (Young CD. Mol Cell Proteomics 2015). A prospective trial of EGFR inhibition in PIK3CA-mutant TNBC is warranted.

e22068 Background: The management challenges of advanced gynecologic malignant neoplasms are often intensified in a community setting. We utilized comprehensive genomic profiling (CGP) of advanced gynecologic malignancies to identify routes of possible benefit from targeted therapy. Methods: DNA was extracted from 40 microns of FFPE sections of advanced gynecologic malignancies. CGP was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of > 500x for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. All classes of genomic alterations (GA) were evaluated, and clinically relevant genomic alterations (CRGA) were defined as GA linked to drugs on the market or under evaluation in mechanism driven clinical trials. Results: The 30 female patients had a median age of 62 years (range 32-88). There were 16 (56%) ovarian carcinoma, 9 (30%) endometrial carcinoma, and 1 (3.3%) case each of uterine sarcoma, uterine carcinos...

5602 Background: High risk HPV initiates neoplasia in cSCC and progression requires additional genomic alterations (GA). Treatment of localized cSCC is effective but the response to systemic therap...

3522 Background: Anal squamous cell carcinoma (ASCC) is an HPV-associated rare tumor that has nearly doubled in incidence since 1973, with cisplatin-based chemotherapy as the only treatment shown t...

621 Background: Pretreatment (preRx) biopsies of HER2-amplified breast cancers (BCs) that did not achieve pCR with preop chemotherapy plus H vs L vs HL had PI3K pathway activation with high p-FOXO ...

e22183 Background: Comprehensive genomic profiling is increasingly incorporated in cancer treatment decision-making in an effort to identify druggable genomic events. FoundationOne (F1) is next-gen...

e15628 Background: Penile squamous cell carcinoma (PSCC) is difficult to manage due to the physiological and psychological implications of local treatment with surgery and radiation but minimal evidence is available to guide systemic therapy. We performed comprehensive genomic profiling (CGP) on 20 patients with relapsed/metastatic PSCC in the course of clinical care to identify clinically relevant genomic alterations (CRGA). Methods: DNA was extracted from 40 microns of FFPE sections of advanced PSCC cases. CGP was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of 692X for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. All classes of genomic alterations (GA) were identified. Clinically relevant genomic alterations (CRGA) were defined as GA linked to drugs on the market or under evaluation in mechanism driven clinical trials. Results: There were 20 male patients with a mean age of 60 years (range 46-87)...

e22054 Background: Comprehensive genomic profiling (CGP) of routinely fixed tissue from pts with advanced solid tumors can uncover tumor specific gene alterations. Foundation Medicine has developed a CLIA-certified, CAP-accredited test to simultaneously characterize all 4 classes of genomic alterations in 236 (originally 182) cancer genes. Primary study objective was to determine how often physician treatment decisions for pts with advanced cancer would be modified after reviewing results of this test. Methods: This was a prospective, single-center investigator initiated trial in pts with metastatic solid tumors. Pts with disease progression after at least one treatment regimen, determined to be likely to fail current treatment (2ndline or more) within 6 months and an expected survival of ≥ 3 months were eligible. Pts signed protocol informed consent and tumor tissue was sent for CGP. Physicians completed a questionnaire indicating their choice of next appropriate treatment for pts progressing on current ...

Background: Estrogen receptor (ER) inhibition is an important treatment option for advanced breast cancer (BC) pts. Recent studies describe recurring somatic mutations in codons 537 and 538 within the ligand-binding domain (LBD) of ∼ 20% of ER+ metastatic disease, but not treatment-naïve ER+ cancer or ER-negative disease, that render the ER constitutively active and confer partial resistance to endocrine agents. Few studies have described the clinicopathologic and genomic covariates that accompany the ESR1 genomic alterations (GAs). Correlating genomic events as captured by FoundationOne in pts with ESR1-mutated BCs with clinical history may provide clinical insight into these alterations. Methods: Hybridization capture of 3769 exons of 236 cancer-related genes and 47 introns from 19 genes that are frequently rearranged in cancer were fully sequenced to high, uniform coverage using FoundationOne (Foundation Medicine, Cambridge, MA). Results: 176 of the 2,208 (7.9%) BC pt cases harbored ESR1 alterations. 1127 short variants (SV) were detected in 176 patient samples for an average of 6.4 GA/sample. 16.5% SV (186/1127) were ESR1 GAs with an average of 1.0 ESR1 GAs/sample. ESR1 GAs consisted of base substitutions (77%) and amplifications (20%, median copy number 9X, range 6-28). Base substitutions occurred at codons 538 (71/145) and 537 (70/145) and at two other novel sites, 341 (2/145) and 563 (1/145). A patient-derived xenograft study suggested that tumors refractory to ER inhibitors and harboring ESR1 amplification could be responsive to higher doses of estradiol. (Li et al, Cell Reports, 2013). The most frequently co-occurring GAs were PIK3CA (37.5%), GATA3 (22.7%), TP53 (24.4%), MAP3K1 (10.2%) and CDH1 (7.9). Collection of data on tissue sites sequenced, treatments and outcomes on these pts with ESR1 GAs are ongoing. Of note, one pt had recurrent disease following adjuvant tamoxifen and letrozole. FoundationOne testing of her recurrent bony disease revealed an ESR1 Y537 alteration along with mutations in PIK3CA, APC, and RAD51. She has been on fulvestrant monotherapy with stable metastatic disease 13 + months. Conclusions: We found through FoundationOne testing that ESR1 GAs are reasonably common in advanced BCs, with base substitutions accounting for 77% of ESR1 GAs and amplifications for 20%. Identification and characterization of ESR1-mutated advanced BC pts by comprehensive genomic profiling capable of detecting both base substitutions and copy number changes may identify clinically relevant GAs. Clinical correlation is pending. Citation Format: Norma A Palma, Siraj Ali, Garrett Frampton, Kai Wang, Hannah Gilmore, Julio Peguero, Lyndsay N Harris, Massimo Cristofanilli, Juliann Chmielecki, Jeffrey S Ross, Deborah Morosini, Vincent A Miller, Phil J Stephens, Gary Palmer, Joyce O'Shaughnessy. Profiling of ESR1-mutated metastatic breast cancers by FoundationOne® allows a broad genomic understanding for potential clinical implications [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-5.

Author(s): Chalmers, ZR; Ali, SM; Ohgami, RS; Campregher, PV; Frampton, GM; Yelensky, R; Elvin, JA; Palma, NA; Erlich, R; Vergilio, J-A; Chmielecki, J; Ross, JS; Stephens, PJ; Hermann, R; Miller, VA; Miles, CR

Background: Non-Hodgkin lymphoma (NHL) is a large group of hematolymphoid malignant neoplasms that can occur at any age. Genomic alterations of histone modifying genes and the NF-kB signaling pathway are characteristic of NHLs. Histone deacetylase and methyltransferase inhibitors including the EZH2 inhibitors E7438, GSK126 and El1 have shown anti-tumor activity in NHLs (PMID: 24217204). Cancers arising in adolescent and young adults (age 15-39, AYA) often exhibit different clinical and biological features than older adults. However, there is very limited information on age-related genomic profiles of NHLs to facilitate clinical investigation of targeted therapies in the AYA population. Methods: Nucleic acid was extracted from 149 cases of NHLs including diffuse large B-cell lymphoma (DLBCL, 45%), chronic lymphocytic leukemia (CLL, 18%), follicular lymphoma (FL, 9%), mantle cell lymphoma (MCL, 7%), NK/T-cell lymphoma (NKTCL, 5%), anaplastic large cell lymphoma (ALCL, 3%), cutaneous T-cell lymphoma (CTCL, 3%), B-cell lymphoma (3%), Burkitt lymphoma (BL, 2%), T-cell large granular lymphocytic leukemia (T-LGL, 2%) and lymphoblastic lymphoma as well as not otherwise specified NHL (3% in total), and captured using custom baits targeting all exons up to 405 cancer-related genes and 265 frequently rearranged genes (FoundationOne HemeTM). All captured libraries were sequenced to high uniform depth in a CLIA-certified, CAP-accredited laboratory with an average depth of >500x for DNA and >6M unique pairs for RNA. There were 29 AYA and 120 older adult (>39 years) NHL cases in this study. DLBCL was the most common diagnosis across groups in 34% of AYAs and 47% of older adults, respectively. Results: The most frequent genomic alterations (GAs) in AYAs were IGH rearrangement (31%), TP53 (28%), MYC (21%), CIITA (16%), DDX3X (16%), ALK (14%), CDKN2A (10%), and TNFAIP3 (10%). Older adults often harbored IGH rearrangements (33%), TP53 (26%), CDKN2A (24%), MLL2 (21%), CREBBP (15%), TNFAIP3 (14%), and MYD88(13%) alterations. Only 1 MLL2 mutation was identified in our AYA samples (3%). In contrast, MLL2 mutations were found in 25/120 (21%) of adult NHLs, including 30% of DLBCLs and 58% of FLs (two tailed Fisher's exact test p-value=0.03). Ninety-seven percent of the MLL2 mutations were reading frame disruptions or truncations, supporting the concept of MLL2 functioning as a tumor suppressor in NHLs. Patients whose tumors harbored mutant MLL2 frequently contained 2 mutations simultaneously (35%). No additional alteration of histone modification genes was found in AYAs. But 39% of adult NHLs carried alterations of those genes (ASXL1, CREBBP, EP300, EZH2, KDM5A, MLL2, MLL3, SETD2, and WHSC1) across different subtypes, including 92% of FL cases, 46% of DLBCLs, 50% of ALCLs, 33% of CTCLs, 33% of NKTCLs, 27% of MCLs, and 12% of CLLs. The difference in the alteration frequency of histone modification genes between AYA (1/29) and older adult (47/120) is significant (p=0.0001). This observation was also seen when excluding the FL cases (1/27 versus 36/108, p=0.001). Conclusions: The unique genomic features of AYA NHLs and guarded clinical outcome strongly favor that AYA NHLs exhibit distinct molecular characteristics compared with older adults. Using comprehensive genomic sequencing (FoundationOne Heme), we discovered that the alterations of histone modifying genes are extremely rare in our AYA samples. Further study of the unique genomic profile of AYA NHLs to expand our knowledge of their biology and discover potential new therapeutic targets for AYA patients appears warranted. Disclosures Wang: Foundation Medicine, Inc. : Employment, Equity Ownership. Morosini:Foundation Medicine, Inc. : Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc. : Employment, Equity Ownership. Palma:Foundation Medicine, Inc.: Employment, Equity Ownership. Johnson:Foundation Medicine, Inc. : Employment, Equity Ownership. Lipson:Foundation Medicine, Inc. : Employment, Equity Ownership. Chmielecki:Foundation Medicine, Inc. : Employment, Equity Ownership. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine, Inc. : Employment, Equity Ownership. Stephens:Foundation Medicine, Inc. : Employment, Equity Ownership. Miller:Foundation Medicine, Inc. : Employment, Equity Ownership.

Background: T-cell leukemias and lymphomas (TL&Ls) are rare when compared with other hematologic malignancies, and pose a challenge for clinical management. TL&Ls are characteristically resistant to chemotherapeutic regimens used to treat other hematologic malignancies (PMID 17369126, 22649104, 25042790). Recently, activating JAK1 and JAK3 mutations have been described in a subset of mature TL&Ls including adult T-cell leukemias/lymphoma (ATLL), NK/T-cell lymphoma, and T-cell prolymphocytic leukemia (T-PLL). We used a comprehensive hybrid capture-based sequencing assay to identify genomic alterations that might suggest benefit from targeted therapy. Methods and Results. Using FoundationOne® and FoundationOne® Heme assays, we performed genomic profiling on 28 consecutive TL&Ls (leukemias, n=10; lymphomas, n=18) for all alterations in 370 or 405 cancer-related genes and select introns of 19 or 31 genes commonly rearranged in cancer, or both DNA and RNA were extracted and captured using custom baits for 405 genes and 265 frequently rearranged genes, respectively. The samples were sequenced to high uniform coverage, averaging >500X for DNA and >8M total pairs for RNA (PMID 24142049). JAK3 alterations were the most common at 25% (7/28) with an enrichment in T-cell derived leukemias (2 T-PLL, 3 T-ALL, and 1 cutaneous T-cell lymphoma (CTCL)). Other altered genes in the 28 cases included CDKN2A/B (21%), TP53 (18%), and TET2 , DNMT3A , and STAT3 (11% each). A complex rearrangement involving FCHO1 and JAK3 resulting from a ~70kb genomic segment deletion was also identified in a CTCL. The most frequently co-altered genes identified in the JAK3 mutated cases were relevant to the chromatin remodeling pathway. Focal STAT3, STAT5A, and STAT5B amplifications were identified in 2 cases (CTCL and ALCL) as well as one STAT3 D661Y in a T-cell large granular lymphocytic leukemia (T-LGL). All 3 cases with STAT gene alterations were mutually exclusive with JAK3 alterations. Additionally, 2 patients with T-PLL and T-ALL respectively were both found to harbor multiple co-existing JAK1 and JAK3mutations (2/28; 7%). One heavily pre-treated T-PLL patient harbored a major JAK1 V658F clone (40%) and a minor JAK3 M5111 clone (5%), and loss of heterozygosity for mutant ATM and TP53. Based on assay results, the patient was treated with ruxolitinib, a potent and specific inhibitor of JAK1 and JAK2. The patient had an impressive clinical response of duration of 4 months after which her T-PLL cell count rose markedly to pre-treatment levels and she developed worsening thrombocytopenia, indicating acquired resistance to therapy. The resistant T-PLL was assayed and was found to harbor an increased frequency of JAK3 M5111 (28%), decrease of JAK1 V658F (18%) and a new EZH2mutation. The patient’s disease progressed through another line of cytotoxic chemotherapy and she succumbed to her disease. Signal transduction analysis was conducted which showed in T-PLL, the JAK1 and JAK3 mutations were functionally significant as they induced constitutive phosphorylation of downstream STAT proteins. Importantly, these phosphorylations could be inhibited by JAK inhibitors. Collection of clinical outcome on additional cases is ongoing. Conclusion. In this study, we discovered a high frequency of JAK-STAT alterations in 28 T-cell neoplasms with 25% of cases harboring JAK3 missense mutations and two cases with concordant JAK1 and JAK3 mutations. In one case, treatment with the JAK1/2 inhibitor, ruxolitinib, resulted in an impressive clinical response. Our data argue for oncogene dependence upon the JAK-STAT pathway in diverse T-cell neoplasms and underscore the importance of genomic profiling by FoundationOne Heme® of rare and difficult to treat cases by revealing alterations that may be targeted by specific inhibitors, such as ruxolitinib, and may also be predictive of treatment resistance. Disclosures Off Label Use: Ruxolitinib is FDA approved for myeloproliferative disorders.. Palma:Foundation Medicine, Inc.: Employment, Equity Ownership. Wang:Foundation Medicine Inc: Employment. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership.

Based on our current understanding of ARID1A function, it is difficult to predict a therapeutic strategy for these tumours. Therefore we adopted an unbiased approach using integrated functional and molecular profiling of ARID1A mutant and wild-type tumour cell models. Methods: We profiled two parallel systems; a panel of ARID1A mutant and wild-type OCCC cell lines models and an isogenic pair of ARID1A proficient and deficient HCT116 cell lines, using high throughput screening with chemical and siRNA libraries. This has been integrated with molecular profiling data generated from exome and RNA sequencing to aid the discovery of novel targets. Results: Using functional screens we have identified critical genetic dependencies and potential therapeutics in a series of clear cell ovarian cancer models. Specifically, we have established that dasatinib is selective for OCCC models with loss of function ARID1A mutations both in vitro and in vivo. Dasatinib is not only selective for ARID1A mutant OCCC models, but experimental induction of ARID1A deficiency drives dasatinib sensitivity in ARID1A wild-type OCCC models as well as in isogenic cell systems (both mouse and human) where ARID1A has been rendered dysfunctional by gene targeting. Conclusions: These results provide a framework for the discovery of therapeutic targets for clear cell ovarian cancer characterised by ARID1A mutations. Using an unbiased drug and siRNA sensitivity screening approach, we have identified dasatinib as a candidate therapeutic approach for OCCC. This Dasatinib/ARID1A synthetic lethal approach can be observed both in in vitro and in vivo and suggests that dasatinib should be assessed in clinical trials in OCCC patients.

625 Background: Understanding the molecular alterations in residual, treatment-refractory HER2-positive breast cancer (BC) following preoperative trastuzumab (H), lapatinib (L) or both (HL) in combination with chemotherapy may help identify patients at risk for not achieving pathologic complete responses as well as genomic alterations (GA) to target to overcome therapy resistance. Here we summarize the GA present in 21 pts’ residual BC following preoperative anti-HER2 therapy. Methods: 100 pts with stage 2/3 HER2+ BC were randomized to treatment with H vs L 1250mg vs HL (L 750-1000mg) along with preoperative 5-fluoruracil, epirubicin, cyclophosphamide followed by weekly paclitaxel (Holmes F. BMC Res Notes 6:507, 2013). Following IRB-approved informed consent, targeted NGS was performed on 21 pts’ FFPE residual disease (RD) after preoperative therapy at a CLIA-certified laboratory to characterize all classes of genomic alterations across 287 cancer-related genes. Results: 3 of 21 pts with RD were HER2-nega...

4142 Background: Metastatic gallbladder adenocarcinoma (GBCA) has a poor prognosis and systemic therapies are commonly extrapolated from those used in other gastrointestinal malignancies. In this s...

555 Background: We sought to understand the genomic alterations in tumors of MBC pts who had extreme durations of response to capecitabine (C). Methods: Following IRB-approved informed consent, tar...

4538 Background: Neoadjuvant cisplatin based chemotherapy prior to cystectomy is standard of care for MIBC, with 30-40% expected to achieve a pT0. Biomarkers predictive of pT0 are lacking. Methods:...

8049 Background: Genomic rearrangements of ALK are the defining feature of a subset of lung non-small cell carcinomas (NSCLC), and predict response to ALK-targeted therapies. We reviewed our experi...

8020 Background: Cancer genomic profiling via NGS in a clinical setting can reveal additional actionable genomic alterations (GA) in patients with lung cancer (LC) previously tested only by hotspot analysis and leading to unanticipated avenues of targeted treatment. Methods: We performed an NGS-based diagnostic test (FoundationOne) to characterize all classes of GA across 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on 386 LC FFPE specimens in a CLIA-certified lab (Foundation Medicine). Specimens included fine needle aspirates, core needle biopsies, and malignant effusions. 95% of cases were NSCLC (367/386). Actionable GAs are defined as those linked to targeted anti-cancer therapies approved or being evaluated in clinical trials. NGS confirmed known hotspot results for EGFR, KRAS and EML4:ALK in 100% of cases. Results: Genomic profiles were generated from 364/386 (94%) of lung cancer cases, identifying 1205 GA, averaging 3.31 alterations per tumor (range 0 to 10). 85% of tumors (310) harbored at least one actionable GA, with a mean of 1.79 GA per tumor (range 0 to 6). In 68% of tumors (248), at least one GA was detected that would be missed by current ‘hotspot assays’. ERBB2 harbored base substitutions or indels in 1.3% of cases. BRAF and C-Kit were altered at frequencies of 2% and 1% respectively. The mTOR/PI3K pathway is likely to be activated via alterations in tumor suppressors STK11 (11%), NF1 (6%) and PTEN (4%), as well as by alterations of PIK3CA (10%) and in AKT1/2/3 (4%), suggesting possible benefit from mTOR/PI3K inhibitors. The Hedgehog pathway (PTCH1/SMO/SUFU) was altered in 2% of cases. ALK and RET were rearranged in 4% and 2% of cases, respectively, with several cases initially diagnosed negative by FISH testing. Conclusions: Profiling the tumor genomes of 364 LC patients led to the identification of a series of GA not detectable by hotspot testing that could significantly inform targeted treatment decisions. Moreover, actionable GA appeared in unexpected tumor type, i.e. an EGFR mutation in a SCLC, reinforcing the likely utility of clinical cancer genomic profiling for the personalized treatment of LC patients.

11020 Background: ST oncology has been transformed by the linkage of GA with targeted therapeutics. Unfortunately, most STs still have no target detected by clinically available assays. More comprehensive testing platforms are needed to determine GA in ST and thus broaden treatment options. We developed a ST NGS diagnostic assay, optimized for routine clinical FFPE specimens including core and fine needle biopsies and malignant effusions, and analyzed > 2,200 patients’ tumors in a CLIA-certified lab (Foundation Medicine). Methods: Hybridization capture of 3,320 exons from 182 cancer-related genes and 37 introns of 14 genes commonly rearranged in cancer was applied to ≥ 50ng of DNA extracted from 2,200+ consecutive FFPE tumor specimens and sequenced to high unique coverage. GA (base substitutions, small indels, rearrangements, copy number alterations) were categorized as “actionable” if directly linked to a clinically available targeted treatment option or a mechanism-driven clinical trial. Results: 2,112/2,221 (95%) of specimens (most common 1° sites: lung 18%, breast 14%, colon 7%, other 34%) were successfully profiled (mean coverage 1134X). Alterations were reported in 155/182 (85%) of genes. Seventy-six percent of cases harbored ≥1 actionable GA, mean 1.6 (range 0-16); sixty-two percent harbored at least one actionable GA not assayed by available tumor-type specific tests or hotspot panels. This approach has led to novel insights into advanced cancer including: 13 novel, potentially druggable kinase gene fusions; alterations in known drug targets (e.g. ALK, EGFR, ERRB2, KIT, MET, PDGFR α and β, RAF1 and RET) in novel tumor types and new mechanisms of resistance to approved targeted therapies. Several patients demonstrated dramatic responses to treatment with targeted therapies directed against these alterations. Conclusions: Comprehensive NGS genomic profiling was successful in profiling >2,200 unselected clinical cases, identified actionable alterations in 76% of cases and provided additional treatment options for 62% of patients targeting alterations in genes not assayed by available hotspot panels.

11068 Background: MET expression has been shown to be prognostic and possibly predictive in several tumor types and both small molecule inhibitors of MET kinase as well as monoclonal antibodies are under study as therapies for this subset of patients. However, the methods to assess MET overexpression are not well standardized. In the clinic, NGS is an advanced diagnostic method for identifying known and unknown targeted-therapy options. We therefore sought to explore the ability of NGS to detect high level MET amplifications in a general oncology population. Methods: We review here the results from analysis of 2,221 FFPE tissue samples across a range of tumor types by an NGS assay in a CLIA-certified laboratory (Foundation Medicine). We specifically report base pair substitutions, small insertions/homozygous deletions (indels), high level (> 6 copies/nucleus) amplification (amp) and select rearrangements in 189 genes, including MET. Results: MET was altered in 28/2,223 (1.2%) of patient samples. MET alterations were limited to amplifications (median 15X, range 6X-40X); no base pair substitutions, indels, or rearrangements were found. MET amp was present in 3/48 (6.2%) primary bone sarcomas, 1/20 (5.0%) myogenic sarcomas, 2/58 (3.4%) gastric, 1/38 (2.6%) kidney, 2/78 (2.5%) hepatocellular, 1/41 (2.4%) uterine, 4/188 (2.1%) unknown primary, 2/99 (2.0%) ovarian, 8/386 (2.0%) lung, 3/157 (1.9%) colorectal, and 1/109 (0.9%) pancreatic carcinomas. Conclusions: Use of NGS to characterize MET alterations in a 2,000+ patient population provides evidence for the role of NGS in identifying the future use of MET-targeted therapy in a variety of common and uncommon solid tumors. Clinical follow-up of the subset of cases treated with a MET inhibitor is ongoing.