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Molecular genetic pathology (MGP) is a subspecialty of pathology and medical genetics and genomics. Genomic testing, which is defined as that which generates large data sets and interrogates large segments of the genome in a single assay, is increasingly recognized as essential for optimal patient care through precision medicine. The most common genomic testing technologies in clinical laboratories are next-generation sequencing and microarray. It is essential to train in these methods and to consider the data generated in the context of the diagnosis, medical history, and other clinical findings of individual patients. Accordingly, updating the MGP fellowship curriculum to include genomics is timely, important, and challenging. At the completion of training, an MGP fellow should be capable of independently interpreting and signing out results of a wide range of genomic assays and, given the appropriate context and institutional support, of developing and validating new assays in compliance with applicable regulations. The Genomics Task Force of the MGP Program Directors, a working group of the Association for Molecular Pathology Training and Education Committee, has developed a genomics curriculum framework and recommendations specific to the MGP fellowship. These recommendations are presented for consideration and implementation by MGP fellowship programs with the understanding that MGP programs exist in a diversity of clinical practice environments with a spectrum of available resources.

The spectrum of neoplasms associated with DICER1 variants continues to expand, with the recent addition of primary “DICER1-associated central nervous system sarcoma” (DCS). DCS is a high-grade malignancy predominantly affecting pediatric patients. Six pediatric DCS were identified through a combination of clinical diagnostic studies, archival inquiry, and interinstitutional collaboration. Clinical, histologic, immunohistologic, and molecular features were examined. Genomic findings in the 6 DCS were compared with those in 14 additional DICER1-associated tumors sequenced with the same assay. The six patients presented at ages 3–15 years with CNS tumors located in the temporal (n = 2), parietal (n = 1), fronto-parietal (n = 1), and frontal (n = 2) lobes. All underwent surgical resection. Histologic examination demonstrated high-grade malignant spindle cell tumors with pleuropulmonary blastoma-like embryonic “organoid” features and focal rhabdomyoblastic differentiation; immature cartilage was seen in one case. Immunohistochemically, there was patchy desmin and myogenin staining, and patchy loss of H3K27me3, and within eosinophilic cytoplasmic globules, alfa-fetoprotein staining. Biallelic DICER1 variants were identified in all cases, with germline variants in two of five patients tested. DCS demonstrated genomic alterations enriched for Ras pathway activation and TP53 inactivation. Tumor mutational burden was significantly higher in the 6 DCS tumors than in 14 other DICER1-associated tumors examined (mean 12.9 vs. 6.8 mutations/Mb, p = 0.035). Postoperative care included radiation (n = 5) and chemotherapy (n = 3); at the last follow-up, three patients were alive without DCS, and three had died of disease. Our analysis expands the clinical, histologic, immunohistological, and molecular spectrum of DCS, identifying distinctive features that can aid in the diagnosis, multidisciplinary evaluation, and treatment of DCS.

Aims The classification of paediatric spindle mesenchymal tumours is evolving, and the spectrum of so-called 'infantile fibrosarcoma' has expanded to include tumours with NTRK, BRAF and MET gene fusions. RET-rearranged paediatric spindle cell neoplasms are an emerging group; there is sparse literature on their clinical, pathological and genetic features, and their nosological place in the canon of soft tissue tumours is uncertain. In this study, we report five RET-rearranged paediatric spindle cell tumours with fusion partners MYH10, KIAA1217 and CLIP2. Methods and results The tumours occurred in the pelvic region, paraspinal region, kidney and subcutaneous tissue of hand and abdomen. The patients' ages ranged from 6 months to 13 years (median 1 year). The tumours were composed of monomorphic spindle cells arranged in a fascicular pattern. Lesional cells had minimally atypical ovoid or tapered nuclei and pale cytoplasm with indistinct borders. Necrosis was not identified. Mitoses numbered three to 12 per 10 high-power field. Cases showed inconsistent and variable expression of S100, CD34 and SMA. Clinical behaviour ranged from small lesions potentially cured by simple resection to large lesions exhibiting metastasis, but responsive to kinase inhibitor therapy. Conclusions Our findings help to define RET-rearranged spindle cell tumours. Although it is likely that these tumours comprise part of the morphological and clinical spectrum of infantile fibrosarcoma (IFS), identification of RET gene alteration is important for its unique therapeutic implications.

Cranial fasciitis is a benign myofibroproliferative lesion of the scalp and underlying bones typically occurring in the pediatric population. Histologically, it is characterized by loose fascicles of stellate cells in a fibromyxoid background, findings similar to those described in the closely related variant nodular fasciitis. Previously characterized as a reactive process, the identification of USP6 translocations in over 90% of nodular fasciitis cases prompted their reclassification as a clonal neoplastic process. Unlike nodular fasciitis, the molecular underpinnings of cranial fasciitis are less clear. While a subset of cranial fasciitis has been associated with Wnt/β-catenin pathway dysregulation, recent case reports suggest that this entity may also harbor USP6 fusions, a finding we sought to further investigate. We identified fifteen archival cases of cranial fasciitis, five females and ten males ranging in age from 3 months to 9 years (median 11 months), composed of formalin-fixed paraffin-embedded and fresh frozen tissues (11 and 4 cases respectively). Samples were evaluated on an RNA-based targeted sequencing panel targeting genes recurrently rearranged in neoplasia, including USP6. Five of fifteen cases (33%) were positive for USP6 rearrangements predicted to result in the fusion of the entire USP6 coding region to the promoter of the 5′ partner, (three of which were novel): two SERPINH1-USP6 (novel) and one each of COL3A1-USP6 (novel), SPARC-USP6, and MYH9-USP6. These results demonstrate the recurrent nature of USP6 rearrangements in cranial fasciitis, and highlight the success of targeted RNA sequencing in identifying known and novel fusion partners. The identification of USP6 promoter-swapping rearrangements is helpful in understanding the underlying biology of cranial fasciitis, and reinforces its biologic relationship to nodular fasciitis. Targeted RNA sequencing is a helpful tool in diagnosing this pseudosarcomatous lesion.

PURPOSE Given regulatory approval of immune checkpoint inhibitors in patients with mismatch repair–deficient (MMR-D) cancers agnostic to tumor type, it has become important to characterize occurrence of MMR-D and develop cost-effective screening approaches. Using a next-generation sequencing (NGS) panel (OncoPanel), we developed an algorithm to identify MMR-D frequency in tumor samples and applied it in a clinical setting with pathologist review. METHODS To predict MMR-D, we adapted methods described previously for use in NGS panels, which assess patterns of single base-pair insertion or deletion events occurring in homopolymer regions. Tumors assayed with OncoPanel between July 2013 and July 2018 were included. For tumors tested after June 2017, sequencing results were presented to pathologists in real time for clinical MMR determination, in the context of tumor mutation burden, other mutational signatures, and clinical data. RESULTS Of 20,301 tumors sequenced, 2.7% (553) were retrospectively classified as MMR-D by the algorithm. Of 4,404 samples with pathologist sign-out of MMR status, the algorithm classified 147 (3.3%) as MMR-D: in 116 cases, MMR-D was confirmed by a pathologist, five cases were overruled by the pathologist, and 26 were assessed as indeterminate. Overall, the highest frequencies of OncoPanel-inferred MMR-D were in endometrial (21%; 152/723), colorectal (9.7%; 169/1,744), and small bowel (9.3%; 9/97) cancers. When algorithm predictions were compared with historical MMR immunohistochemistry or polymerase chain reaction results in a set of 325 tumors sequenced before initiation of pathologist assessment, the overall sensitivity and specificity of the algorithm were 91.1% and 98.2%, respectively. CONCLUSION We show that targeted, tumor-only NGS can be leveraged to determine MMR signatures across tumor types, suggesting that broader biomarker screening approaches may have clinical value.

Pediatric tumors can be divided into hematologic malignancies, central nervous system tumors, and extracranial solid tumors of bone, soft tissue, or other organ systems. Molecular alterations that impact diagnosis, prognosis, treatment, and familial cancer risk have been described in many pediatric solid tumors. In addition to providing a concise summary of clinically relevant molecular alterations in extracranial pediatric solid tumors, this review discusses conventional and next-generation sequencing-based molecular techniques, relevant tumor predisposition syndromes, and the increasing integration of molecular data into the practice of diagnostic pathology for children with solid tumors.

Inflammatory myofibroblastic tumor (IMT) is a rare, mesenchymal tumor that has an increased incidence in childhood. Tumors are usually isolated to the chest, abdomen, and retroperitoneum, but metastatic presentations can be seen. Presenting symptoms are nonspecific and include fever, weight loss, pain, shortness of breath, and cough. Approximately 85% of IMTs harbor actionable kinase fusions. The diagnosis can be delayed because of overlapping features with inflammatory disorders, such as elevated inflammatory markers, increased immunoglobin G levels, fever, weight loss, and morphologic similarity with nonmalignant conditions. We present a girl aged 11 years with a TFG-ROS1 fusion–positive tumor of the lung that was initially diagnosed as an immunoglobin G4–related inflammatory pseudotumor. She underwent complete left-sided pneumonectomy and later recurred with widely metastatic disease. We then report the case of a boy aged 9 years with widely metastatic TFG-ROS1 fusion–positive IMT with rapid molecular diagnosis. In both children, there was an excellent response to oral targeted therapy. These cases reveal that rapid molecular testing of inflammatory tumors is not only important for diagnosis but also reveals therapeutic opportunities. Targeted inhibitors produce significant radiologic responses, enabling potentially curative treatment approaches for metastatic ROS1 fusion IMT with previously limited treatment options. Primary care pediatricians and pediatric subspecialists have a crucial role in the early consultation of a pediatric oncology center experienced in molecular diagnostics to facilitate a comprehensive evaluation for children with inflammatory tumors.

Medullary thyroid carcinoma (MTC) is most commonly associated with RET gene mutations. ALK fusions have rarely been described, although not previously in pediatrics and not previously partnered wit...

Introduction. Myofibromas are rare tumors of pericytic lineage, typically affecting children, and are sometimes aggressive. A subset of sporadic and familial myofibromas have activating variants in PDGFRB. The relationship of myofibroma and PDGFRB to the NOTCH pathway has not yet been described. Methods. Ten myofibroma cases were sequenced with a targeted panel of 447 genes, including copy number variation and selected fusions. Immunohistochemical analysis of total NOTCH3 and activated NOTCH3 was assessed for all 10 myofibroma cases, and a series of histologic mimics (n = 20). Results. Alterations identified by next-generation sequencing included PDGFRB sequence variants in 8/10 cases (80%), a NOTCH3 variant in 1/10 cases (10%), and a NOTCH2 variant in 1/10 cases (10%). All 10 cases also showed a pattern of low-amplitude (1.5- to 2-fold) copy number alterations including gains in PDGFRB and NOTCH3. Ten of 10 myofibromas (100%) showed cytoplasmic staining for total NOTCH3 and 9 of 10 cases (90%) showed nuclear staining for activated NOTCH3. Within the control cohort of histologic mimics, 3 of 3 nodular fasciitis cases (100%) were positive for activated and total NOTCH3, and the remaining 17 cases were negative for pan NOTCH3, while 3 of 3 desmoid-type fibromatosis cases (100%) showed patchy weak nuclear staining for activated NOTCH3. Discussion. Our findings suggest a common pathway of PDGFRB/NOTCH3 activation in myofibromas, even in cases that lack PDGFRB sequence variants. These results support the pericytic lineage of myofibroma. Identification of the characteristic genomic alterations or immunohistochemical staining pattern may facilitate a difficult pathologic diagnosis, and support the use of targeted treatments.

Purpose: Kaposiform lymphangiomatosis (KLA) is a rare, frequently aggressive, systemic disorder of the lymphatic vasculature, occurring primarily in children. Even with multimodal treatments, KLA has a poor prognosis and high mortality rate secondary to coagulopathy, effusions and systemic involvement. We hypothesized that, as has recently been found for other vascular anomalies, KLA may be caused by somatic mosaic variants affecting vascular development. Methods: We performed exome sequencing of tumor samples from five individuals with KLA, along with samples from uninvolved control tissue in three of the five. We used digital PCR (dPCR) to validate the exome findings and to screen KLA samples from six other individuals. Results: We identified a somatic activating NRAS variant (c.182A>G, p.Q61R) in lesional tissue from 10/11 individuals, at levels ranging from 1–28%, that was absent from the tested control tissues. Conclusion: The activating NRAS p.Q61R variant is a known ‘hotspot’ variant, frequently identified in several types of human cancer, especially melanoma. KLA, therefore, joins a growing group of vascular malformations and tumors caused by somatic activating variants in the RAS/PI3K/mTOR signalling pathways. This discovery will expand treatment options for these high risk patients as there is potential for use of targeted RAS pathway inhibitors.

Undifferentiated embryonal sarcoma of the liver (UESL) is a rare aggressive neoplasm that occurs predominantly in children. Like mesenchymal hamartoma of the liver (MHL), UESL harbors recurrent rearrangements involving 19q13.3 and 19q13.4, a region of the genome that contains a primate-specific cluster of micro-RNAs. Here, we present a case of a high-grade neoplasm that arose in the left hepatic lobe of a 5-year-old male and gave rise to widespread lymph node, visceral, and soft tissue metastases. The tumor was composed of sheets, tubules, and papillae of epithelioid cells with rhabdoid morphology. INI1 and BRG1 expression were retained. Tumor cells diffusely expressed epithelial markers, including multiple keratins. While the morphologic and immunophenotypic features were suggestive of poorly differentiated carcinoma with rhabdoid features, the tumor was found to harbor the t(11;19)(q13;q13.3) translocation characteristic of UESL, as well as a TP53 mutation. Given the clinical presentation, imaging, clinical course, the tumor was classified as UESL with unusual, carcinoma-like histopathologic features. In the context of an unclassified high-grade hepatic tumor in a young child, molecular or cytogenetic testing for chromosome 19q13 alterations should be considered.

Alterations in the BCOR gene, including internal tandem duplications (ITDs) of exon 15 have emerged as important oncogenic changes that define several diagnostic entities. In pediatric cancers, BCOR ITDs have recurrently been described in clear cell sarcoma of kidney (CCSK), primitive myxoid mesenchymal tumor of infancy (PMMTI), and central nervous system high-grade neuroepithelial tumor with BCOR ITD in exon 15 (HGNET-BCOR ITDex15). In adults, BCOR ITDs are also reported in endometrial and other sarcomas. The utility of multiplex targeted RNA sequencing for the identification of BCOR ITD in pediatric cancers was investigated. All available archival cases of CCSK, PMMTI, and HGNET-BCOR ITDex15 were collected. Each case underwent anchored multiplex PCR library preparation with a custom-designed panel, with BCOR targeted for both fusions and ITDs. BCOR ITD was detected in all cases across three histologic subtypes using the RNA panel, with no other fusions identified in any of the cases. All BCOR ITDs occurred in the final exon, within 16 codons from the stop sequence. Multiplex targeted RNA sequencing from formalin-fixed, paraffin-embedded tissue is successful at identifying BCOR internal tandem duplications. This analysis supports the use of anchored multiplex PCR targeted RNA next-generation sequencing panels for identification of BCOR ITDs in pediatric tumors. The use of post-analytic algorithms to improve the detection of BCOR ITD using DNA panels was also explored.

Cancer of unknown primary (CUP) is a common diagnosis in adult oncology, but is extremely rare in pediatrics, with few published reports and none in the era of molecular profiling. Here, we describe a 13-year old boy with metastatic adenocarcinoma of unknown primary that was refractory to conventional chemotherapy. Molecular profiling revealed an activating ERBB2 D769Y variant. He subsequently achieved disease control on a lapatinib-containing regimen, and upon progression, he transiently responded to neratinib prior to dying from disease. Our case reports a rare example of pediatric CUP and highlights the utility of molecular profiling in these cases.

Context Thyroid ultrasound screening is recommended in children with PTEN hamartoma tumor syndrome (PHTS) due to increased risk of thyroid neoplasia, but the natural history of thyroid disease in children with PHTS is unclear. Objective Determine the prevalence and natural history of thyroid disease in children with PHTS. Methods Retrospective cohort study (1998-2019) in an academic pediatric hospital of individuals with genetically confirmed PHTS diagnosed before age 19 years. Clinical, thyroid ultrasound, and laboratory characteristics are described. Primary outcomes were the prevalence of thyroid nodules ≥10 mm diameter and time course and risk factors for nodule development assessed by Cox regression analysis. Secondary outcomes included thyroid nodule requiring biopsy, other ultrasound findings, and prevalence of autoimmune thyroid disease. Results Among 64 subjects with PHTS, 50 underwent thyroid ultrasound. A thyroid nodule ≥10 mm was diagnosed in 22/50 (44%) subjects at median (range) age 13.3 (7.0-22.9) years. Nodules were diagnosed earlier in females than in males (10.8 [7.0-17.9] vs 14.2 [9.9-22.9] years, P = .009). In multivariate analysis, risk of thyroid nodules was significantly associated with female sex (hazard ratio 2.90, 95% CI 1.16-7.27, P = .02) and inversely associated with the presence of neurologic findings of PHTS (HR 0.27, 95% CI 0.10-0.69, P = .007). Abnormal-appearing lymph nodes with echogenic foci were observed by ultrasound in 20% of subjects, but these were not associated with malignancy. Autoimmune thyroid disease was present in 10/33 (30.3%) of subjects in whom it was assessed. Conclusion Thyroid disease is common in children with PHTS. This study supports current consensus recommendations for ultrasound screening.

Introduction Tissue from pediatric solid tumors is in high demand for use in high-impact research studies, making the allocation of tissue from an anatomic pathology laboratory challenging. We designed, implemented, and assessed an interdepartmental process to optimize tissue allocation of pediatric solid tumors for both clinical care and research. Methods Oncologists, pathologists, surgeons, interventional radiologists, pathology technical staff, and clinical research coordinators participated in the workflow design. Procedures were created to address patient identification and consent, prioritization of protocols, electronic communication of requests, tissue preparation, and distribution. Pathologists were surveyed about the value of the new workflow. Results Over a 5-year period, 644 pediatric solid tumor patients consented to one or more studies requesting archival or fresh tissue. Patients had a variety of tumor types, with many rare and singular diagnoses. Sixty-seven percent of 1768 research requests were fulfilled. Requests for archival tissue were fulfilled at a significantly higher rate than those for fresh tissue (P > .001), and requests from resection specimens were fulfilled at a significantly higher rate than those from biopsies (P > .0001). In an anonymous survey, seven of seven pathologists reported that the process had improved since the introduction of the electronic communication model. Conclusions A collaborative and informed model for tissue allocation is successful in distributing archival and fresh tissue for clinical research studies. Our workflows and policies have gained pathologists' approval and streamlined our processes. As clinical and research programs evolve, a thoughtful tissue allocation process will facilitate ongoing research.

Summary Lipoblastomas can occasionally require further molecular confirmation when occurring outside of the usual age groups or demonstrating unusual morphology. We reviewed 28 lipoblastomas with 16 controls. Lipoblastomas were subdivided into myxoid (n = 7), classic (n = 9), or lipoma-like (n = 12) subtypes. PLAG1 immunohistochemistry, PLAG1 fluorescence in situ hybridization (FISH), and targeted RNA sequencing were performed on formalin-fixed paraffin-embedded tissue. Karyotypes were available in a subset of lipoblastomas (n = 9). Gene rearrangements were identified in 17/25 (68%) lipoblastomas, including PLAG1 (15/25, 60%) and HMGA2 (2/25, 8%). Five novel fusion partners (DDX6, KLF10, and KANSL1L with PLAG1 and EP400 and FGD6 with HMGA2) were found. PLAG1 immunohistochemistry was positive (nuclear, moderate/strong) in myxoid and classic subtypes lipoblastomas with preferential expression in mesenchymal cells within myxoid stroma and fibrous septa and negative in all controls. When comparing PLAG1 immunohistochemistry with molecular testing (FISH and/or RNA sequencing and/or karyotype), concordant results were noted in 13/25 (52%) cases, increasing to 15/25 (60%) after slight adjustment of the PLAG1 FISH positive threshold. In myxoid and classic lipoblastomas, PLAG1 immunohistochemistry seems to be a better surrogate marker for PLAG1 rearrangement, as compared with lipoma-like subtypes. In lipoma-like subtypes, targeted RNA sequencing appears to detect PLAG1 fusions better than FISH and immunohistochemistry. The preferential expression of PLAG1 in the mesenchymal and fibroblast-like cells deserves further investigation as the putative cell of origin in lipoblastoma.

Key Points We report a patient with hepatosplenic T-cell lymphoma (HSTL) and compare the disease to the derived xenograft model. Enhancer of zeste homolog 2 (EZH2) inhibitor treatment of an EZH2-mutant HSTL patient-derived xenograft model led to prolonged survival.

Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care. One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences. At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach. The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies. Genet Med advance online publication 26 January 2017

Background: Vascular anomalies are diverse entities and can range in severity from self-limiting to life-threatening. Diagnosis and care of these patients is challenging due to overlapping clinical and histologic features. Recently, it has been established that many vascular anomalies arise from somatic mutations in cancer genes (PIK3CA, AKT, NRAS). Use of cancer genomics in patients with vascular anomalies may establish a genetic diagnosis and expand use of targeted medical therapies. We evaluated the utility of targeted next generation sequencing for vascular anomalies patients at a single pediatric center. Methods: Using OncoPanel, a hybrid-capture and massively parallel sequencing assay that surveys DNA sequences of 447 genes implicated in cancer, we analyzed genetic variants in lesional tissue from vascular anomalies patients evaluated at Boston Children's Hospital between 5/2/2017 and 3/23/2020. Results: A total of 276 patients were consented and sequenced under the Dana Farber Cancer Institute Profile protocols DFCI 11-104 (n= 68) and DFCI 17-000 (n= 208). Clinical diagnoses prior to testing were varied and 11 patients (7%) had an unknown diagnosis. Tissue was analyzed for 138 patients. Targeted sequencing resulted in diagnostically significant alterations in 80 of 138 (57%) of patients and therapeutically significant alterations in 58 of 138 (42%) patients. To date, 18 patients in our cohort have been treated with medical therapy informed by their genetic diagnosis. Several more await enrollment on clinical trials. For patients with diagnoses previously categorized as unknown (n=11), sequencing led to identification of a genetic variant in 6 patients (54%). Additionally, 8/138 patients had variants requiring further evaluation for potential germline involvement. Discussion: Next generation sequencing in vascular anomalies patients identified actionable variants in a large proportion of the patients in our cohort. The mTOR inhibitor sirolimus has been used to treat a variety of vascular anomalies, but not all patients respond to this treatment. Targeted therapies based on specific genotypes hold promise as clinical trials in vascular anomalies are emerging. Additionally, sequencing in this cohort identified several variants suggesting a germline cancer predisposition requiring follow-up. Use of next generation sequencing has clinical utility and increased use of this testing may improve diagnosis, prognosis, and treatment for patients with vascular anomalies. Disclosures Adams: Novartis: Consultancy; Venthura: Consultancy. OffLabel Disclosure: Sirolimus is used off-label for the treatment of vascular anomalies.

There is a growing interest in immunotherapy in childhood cancers. Osteosarcoma is a compelling potential target as there are few targeted options available for this aggressive cancer. We provide a description of the landscape of programmed cell death-1 (PD-1), programmed cell death-ligand 1 (PD-L1) and relevant immune markers in serial samples from 15 osteosarcoma patients. PD-1 and PD-L1 expression was present in biopsy samples (47% and 53%, respectively), absent in resections, and present in metastases (40% and 47%). Both decalcified and nondecalcified specimens demonstrated expression of PD-1 and PD-L1. The results suggest that biopsy or metastatic specimens maybe most valuable in assessing expression of PD-1 and PD-L1.

Purpose:Several aggressive pediatric cancers harbor alterations in SMARCB1, including rhabdoid tumors, epithelioid sarcoma, and chordoma. As tumor profiling has become more routine in clinical care, we investigated the relationship between SMARCB1 genetic variants identified by next-generation sequencing (NGS) and INI1 protein expression. Therapeutic approaches for INI1-deficient tumors are limited. Early reports suggest a potential role for immune checkpoint inhibition in these patients. Thus, we also investigated PD-L1 and CD8 expression in INI1-negative pediatric brain and solid tumors.Experimental Design:We performed immunohistochemistry (IHC) for INI1 and immune markers (PD-L1, CD8, and CD163) and NGS on tumor samples from 43 pediatric patients who had tumors with INI1 loss on previous IHC or SMARCB1 genomic alterations on prior somatic sequencing.Results:SMARCB1 two-copy deletions and inactivating mutations on NGS were associated with loss of INI1 protein expression. Single-copy deletion of SMARCB1 was not predictive of INI1 loss in tumor histologies not known to be INI1-deficient. In the 27 cases with INI1 loss and successful tumor sequencing, 24 (89%) had a SMARCB1 alteration detected. In addition, 47% (14/30) of the patients with INI1-negative tumors had a tumor specimen that was PD-L1 positive and 60% (18/30) had positive or rare CD8 staining. We report on 3 patients with INI1-negative tumors with evidence of disease control on immune checkpoint inhibitors.Conclusions:A significant proportion of the INI1-negative tumors express PD-L1, and PD-L1 positivity was associated with extracranial tumor site. These results suggest that clinical trials of immune checkpoint inhibitors are warranted in INI1-negative pediatric cancers.

10005 Background: Next generation sequencing (NGS) assays are now a standard part of clinical care for many adult solid cancers. The significance of molecular tumor profiling for the care of children with cancer is not well understood.We aimed to determine the clinical impact of identifying genomic alterations by NGS for young patients with relapsed, refractory, or high-risk extracranial solid tumors. Methods: We report on the first 389 participants in a prospective cohort study enrolling patients at 12 institutions with extracranial solid tumors diagnosed at age 30 years or less. Targeted DNA NGS was performed on one or more tumor samples from each patient. Selected patients also had tumors subjected to RNA sequencing. Test results were returned to the treating oncologist and follow-up treatment and response data were collected.Identified genomic alterations were classified according to evidence of impact on diagnosis, prognosis or response to targeted therapy matched to an identified alteration (matched targeted therapy, MTT) using established guidelines. Response to MTT was determined and reported as a response if either there was radiographic response according to RECIST or the duration of therapy was > 4 months. Results: Molecular tumor profiling (MTP) was successful in 345 (89%) patients (mean age 11 years at diagnosis; 65% with sarcoma). Two hundred and ninety-nine patients with MTP results (87%) had one or more alterations of clinical significance. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 208 (60%), 51 (15%) and 240 (70%) patients, respectively. Of the 240 patients with tumors harboring genomic alterations designated as having therapeutic impact, 23 (11%) had Tier 1 molecular findings. 205 patients were eligible to receive MTT based on having a molecular alteration with therapeutic significance and sufficient follow-up; 31 of these patients (15%) received MTT. Seven patients (23%) receiving MTT responded, 6 of these were kinase fusions. All of the responders received targeted therapy matched to a fusion and 78% of diagnostically significant alterations were fusions. Conclusions: Molecular tumor profiling has a significant impact on diagnosis and treatment recommendations for young patients with extracranial solid tumors. These results emphasize the importance of fusion detection for patients with sarcomas and rare tumors. Clinical trial information: NCT02520713.

Introduction: Tumor profiling is becoming a more routine part of clinical care. Many academic centers and commercial entities offer tumor sequencing of cancer-related genes without matched germline profiling. We hypothesize that tumor-only sequencing may limit full clinical interpretation and have decreased sensitivity to identify significant germline variants. Methods: The Genomic Assessment Improves Novel Therapy (GAIN) Consortium is a clinical cancer genomics study for patients with high-risk solid malignancies. Patients in this study were selected for subanalysis if panel sequencing of 447 genes was performed on a tumor and interpreted by an expert panel prior to the availability of matched germline sequencing. Interpretation of tumor sequencing included both therapeutic recommendations and a curation of cancer-related variants of potential clinical significance if present in the germline. Germline sequencing was separately performed targeting 147 genes (a subset of the somatic panel) and analyzed with a germline-specific pipeline to identify and filter variants. We examined clinical recommendations in the somatic reports that were based on single-nucleotide variants identified from the 147 overlapping genes. We compared these interpretations with results from the matched germline data. Results: We identified 159 participants with somatic and germline sequencing reports meeting the eligibility criteria. Germline sequencing identified 38 pathogenic or likely pathogenic (P/LP) germline variants in 35 of 159 patients (22%). Of those 35 patients, 17 (49%) had a P/LP variant in an autosomal dominant cancer predisposition gene, 19 (54%) in an autosomal recessive gene, and 1 (2.9%) in a noncancer gene. Of the 38 total variants, 21 (55%) were identified by the analytic pipeline used for somatic sequencing and noted as potential germline variants in the somatic reports. Forty treatment recommendations were made from the somatic data within the overlapping genes. Ten (25%) treatment recommendations were based on variants that were later determined to be germline. These included variants in TP53, SDHA, SMARCA4, TSC2, FAM175A, CHEK2, and AKT1, many of which were noted in the somatic reports to be variants of uncertain significance or possibly germline. Conclusions: In this study, we found that clinically actionable germline variants were under-reported when relying on analytical pipelines and clinical interpretations developed for the analysis of tumor samples. In the absence of germline sequencing, we also found that cancer treatment recommendations can be made based on mutations identified from tumor sequencing that are germline variants. In many cases, these recommendations remain appropriate (e.g., PARP inhibitors for BRCA1/2) while in other cases germline data facilitated a more nuanced interpretation of actionability. These findings support the use of germline genetic testing and paired tumor-germline analysis in precision cancer medicine studies. Citation Format: Jaclyn Schienda, Catherine M. Clinton, Laura B. Corson, Alma Imamovic-Tuco, Navin Pinto, Luke Maese, Theodore W. Laetsch, AeRang Kim, Susan I. Vear, Margaret E. Macy, Mark A. Applebaum, Rochelle Bagatell, Amit J. Sabnis, Daniel A. Weiser, Julia L. Glade-Bender, Samuel L. Volchenboum, Wenjun Kang, Danielle Manning, Jonathan Nowak, Joshua Schiffman, Neal I. Lindeman, Alanna J. Church, Katherine A. Janeway, Brian D. Crompton, Junne Kamihara. The added value of examining germline variants in a precision cancer therapy study [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A06.

Introduction: In pediatric cancer, there is an urgent need for research that can identify and validate new therapeutic modalities for pediatric cancers. Diagnostic biopsy samples remain the ideal tissue samples for research and must be collected from surplus biopsy material, which is often extremely limited. Here we describe our efforts to optimize tissue allocation for clinical care and research as a joint effort between Department of Pathology at Boston Children’s Hospital and the Pediatric Solid Tumor Program at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center. Methods: Oncologists, pathologists, surgeons, interventional radiologists, pathology technical staff, and clinical research coordinators participated in the workflow design. The group agreed to develop a formalized procedure to address these five steps: 1) patient identification and consent, 2) prioritization of research objectives, 3) advance communication of tissue requests to the pathology staff, 4) tissue preparation, and 5) tissue distribution. It was unanimously agreed that all tissue must flow through the pathology department. On or before the day of surgery, clinical research teams sent the pathologist a patient-specific electronic communication indicating research consent and detailing the prioritized disease-specific research requests, including the procedure planned, all tissue types and tissue volumes requested, and details of any special preparation needed, such as avoiding decalcification in bone tumors. The communication was optimized to be clear but brief, with the goal of minimizing the impact to the pathologist’s work load. Pathologists were surveyed about the change in process. Results: Over a five-year period (2013-2018), 662 pediatric DFCI/BCH solid tumor patients have consented to one or more trials that request FFPE, frozen, or fresh tissue. Tumor types represent a spectrum of cases, with many rare and singular diagnoses. Of 1,768 research tissue requests, 1,121 (63%) were fulfilled. Clinical study requests from resection specimens were the most likely to be fulfilled (95% of 390 requests fulfilled), while basic research requests from core biopsies were the least likely to be fulfilled (26% of 255 requests fulfilled). In an anonymous survey, 7 of 7 pathologists report that the process had improved since the introduction of the electronic communication. Conclusions: A collaborative and informed model for tissue allocation is successful in distributing tissue for clinical studies and basic research projects. Our workflows and policies have gained pathologist approval and streamlined our processes. As clinical and research programs evolve, a thoughtful tissue allocation process will facilitate ongoing research. Citation Format: R. Seth Pinches, Catherine Clinton, Abigail Ward, Stephanie C Meyer, Alyaa Al-Ibraheemi, Suzanne Forrest, Gianna R. Strand, Hillary Detert, Anne Piche-Schulman, Kristen Gil, Tamara Restrepo, Rosemarie Tavares-Proulx, Jeffrey Goldsmith, Raja Shaikh, Christopher Weldon, Sanda Alexandrescu, Allison F. O’Neill, Monica Hollowell, Marian H. Harris, Katherine A. Janeway, Brian D. Crompton, Alanna Church. Improving tissue allocation for research in pediatric solid tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A67.

Introduction: The Clinical Genome Resource (ClinGen) Somatic Working Group (sWG) is a multi-institution team engaged in developing processes, resources, and standards to support accurate classification of somatic variants in cancer. Existing decision support resources in cancer knowledgebases are heavily skewed towards genes and variants relevant in adult cancers; however, information to support variant interpretation in childhood cancers is limited. Here we report on the goals and progress of the Pediatric Cancer Taskforce, created within the ClinGen sWG, to lead curation efforts of actionable alterations in childhood cancers. Methods: The ClinGen sWG Pediatric Cancer Taskforce (PCT) consists of a core group of twelve members comprising geneticists, pathologists, and oncologists with expertise in different pediatric cancers and with representation from 9 leading pediatric institutions. The taskforce has a total of 35 members including volunteer-curators who work under guidance of the expert members. Curation of childhood cancer variants is conducted in collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) team at Washington University in Saint Louis, using the CIViC knowledgebase (civicdb.org) and the ClinVar database as open-access curation and data-sharing platforms. Diagnostic, prognostic, and therapeutic evidence is tiered according to the AMP/ASCO/CAP guidelines for the clinical interpretation of somatic variants. PCT members are assigned specific genetic variant-tumor type associations for curation, which are then reviewed in monthly conferences to finalize assertions in CIViC. Results: The PCT has prioritized 40 genetic alterations relevant to pediatric cancer for curation based on their clinical relevance and the lack of sufficient existing curated evidence in clinical knowledgebases. To date, 4 assertions have been created and added to the database: HEY1-NCOA2 fusion in mesenchymal chondrosarcoma, KIAA1549-BRAF fusion and ACVR1 p.G328V variant in pediatric glioma, and EBF1-PDGFRB fusion in pediatric B-cell precursor acute lymphoblastic leukemia. Active curation has been initiated for NTRK fusions agnostic of tissue histology, targetable kinase fusions in Ph-like B-lymphoblastic leukemia, and common variants in selected pediatric sarcomas and brain tumors, focusing heavily on driver gene fusions in childhood cancers. 119 evidence items have been created in CIViC by the members. The PCT also works to implement more standardized and accurate classification of pediatric cancers in CIViC and other cancer resources, and to enhance search for pediatric-specific data through appropriate tagging of evidence using ontology terms. Conclusions: As molecular alterations are increasingly relevant to the care of children with cancer, the ClinGen PCT will work to develop standards, processes, and resources for efficient and accurate determination of clinical relevance of pediatric cancer variants. Citation Format: Alanna J. Church, Shruti Rao, Deborah Ritter, Arpad Danos, Kilann Krysiak, Laura B. Corson, Kevin E. Fisher, Matthew Hiemenz, Katherine A. Janeway, Jianling Ji, Chimene A. Kesserwan, Theodore W. Laetsch, Donald W. Parsons, Ryan J. Schmidt, Kristen L. Sund, Wan-Hsin Lin, Malachi Griffith, Obi L. Griffith, Shashikant Kulkarni, Subha Madhavan, Angshumoy Roy, Gordana Raca. Curation of pediatric cancer variants within the Clinical Genome Resource (ClinGen) [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A58.

Background: Gene variants with potential therapeutic significance have been reported in 30-60% of childhood malignancies. The 12-institution Genomic Assessment Informs Novel therapy (GAIN) consortium is conducting the individualized cancer therapy 2 (iCat2) study (NCT02520713) with the objective of evaluating the impact of tumor profiling on outcome. We provide an interim report on patients enrolled on the ongoing GAIN/iCat2 study. Methods and Objectives: Patients are eligible if they have a high-risk, recurrent/refractory (RR), or difficult-to-diagnose extracranial solid tumor diagnosed at ≤30 years and adequate sample available for sequencing. A next-generation targeted panel assay is performed. Results are returned with a GAIN report containing clinical interpretation, including an individualized cancer therapy (iCat) recommendation if there is evidence supporting a link between an identified variant and response to molecularly targeted therapy. iCat recommendations are tiered from 1 to 5 based on the level of clinical and preclinical support, with tier 1 being the highest and tier 5 the lowest. Potential extraordinary responders are selected for further review based on having treatment duration of ≥1 year for chemotherapy or ≥4 months or a partial response for targeted therapy. Results: 388 eligible patients were enrolled by 1/1/2019 with the most common diagnoses being osteosarcoma, Ewing sarcoma, and rhabdomyosarcoma. 366 patients (94%) have had at least one successful sequencing result, with 349 having molecular and GAIN reports suitable for inclusion in this analysis. 68% of patients (237/349) have received iCat recommendations, with 41% (143/349) having the highest tier of 1-2 and 27% (94/349) having a highest tier of 3-5. Common genes for which tier 1-2 iCat recommendations were made include TP53 (15%), SMARCB1 (4%), PIK3CA (3%), CDK4 (2%), and KRAS (2%). Common alterations for which tier 3-5 recommendations were made include EWSR1 fusions (12%), MYC/MYCN amplifications (8%), and CDKN2A deletions (7%). Of 170 RR patients with treatment follow-up data entered as of June 2019, 15% (25/170) have received matched targeted therapy. Six of these (24%) are considered extraordinary responders. Of note, extraordinary responses were also seen with some second-line chemotherapy and multitargeted kinase inhibitors. Conclusions: The proportion of patients with clinically significant gene variants is higher in this study than in some previous reports. Providing an iCat recommendation for alterations in genes such as TP53 where evidence is mixed, increased availability of molecularly targeted therapy trials, and more evidence may all be responsible for this increased rate. Reassessment of iCat recommendation tiers based on current evidence is ongoing. Extraordinary responses occur in a subset of children with extracranial solid malignancies who receive matched targeted therapy. Study enrollment is ongoing with further assessments of the impact of tumor profiling on outcome planned. Citation Format: Laura B. Corson, Alanna J. Church, Deirdre Reidy, Pei-Chi Kao, Wenjun Kang, Navin Pinto, Luke Maese, Theodore W. Laetsch, AeRang Kim, Susan I. Vear, Margaret E. Macy, Mark A. Applebaum, Lobin A. Lee, Duong Doan, R. Seth Pinches, Seong Choi, Suzanne J. Forrest, Catherine M. Clinton, Brian D. Crompton, Laura E. MacConaill, Samuel L. Volchenboum, Neal I. Lindeman, Steven G. DuBois, Wendy B. London, Katherine A. Janeway. Targeted sequencing in 388 patients with high-risk or recurrent/refractory pediatric extracranial solid malignancies: An interim report from the GAIN Consortium/iCat2 Study [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A28.

Introduction: Molecular techniques have been incorporated into the diagnostic algorithms for many specific tumors, but the diagnostic role of next-generation sequencing has not been described at a population level. We report diagnostically relevant alterations identified by large-scale sequencing in a prospective cohort of pediatric solid tumors. Methods and Objectives: Patients are eligible for the GAIN / iCat2 study if they have a high-risk, recurrent, or refractory extracranial solid tumor diagnosed at age 30 or less and have an adequate sample for sequencing available. After informed consent, tumor was sequenced using a next-generation sequencing assay that evaluates 447 genes and includes data about sequence variants, copy number alterations, and, in selected genes, translocations. Some cases received additional sequencing via RNASeq or targeted RNA sequencing for further evaluation of fusions. Diagnostic relevance was determined according to AMP/ASCO/CAP standards and guidelines for the reporting of sequence variants in cancer. Results: 349 patients were enrolled as of December 31, 2018, and had tumor tissue successfully sequenced. These patients represent 60 unique diagnoses according to the WHO ICD-O classification. The most common single diagnoses were osteosarcoma (n=64), Ewing sarcoma (n=44), and alveolar rhabdomyosarcoma (n=32). For 349 patients, 184 (53%) had one or more genetic alterations that were diagnostically relevant, of which 159 (86%) were structural variants, 16 (8%) were sequence variants, and 9 (5%) were copy number variations. Alterations of high diagnostic relevance include CIC-DUX4 fusions in sarcoma (n=8), TP53 intron 1 rearrangements in osteosarcoma (n=26), DICER1 sequence variants in various tumors (n=7), and BCOR internal tandem duplications in clear-cell sarcoma of kidney and primitive myxoid mesenchymal tumor of infancy (n=3). Conclusions: Diagnostically relevant alterations were identified in over half of pediatric solid tumor patients evaluated. Gene fusions are particularly prevalent. These results support a role for sequencing that includes robust fusion assessment to inform diagnosis in patients with pediatric solid tumors. Citation Format: Alanna J. Church, Laura B. Corson, Alma Imamovic-Tuco, Gianna R. Strand, Dierdre Reidy, Duong Doan, Robert S. Pinches, Mark A. Applebaum, Rochelle Bagatell, Brian D. Crompton, Steven G. DuBois, Julia L. Glade Bender, Theodore W. Laetsch, Lobin A. Lee, Neal I. Lindeman, Marian H. Harris, Margaret E. Macy, Luke Maese, Navin Pinto, Amit J. Sabnis, Eliezer M. Van Allen, Susan I. Vear, Daniel A. Weiser, Catherine M. Clinton, Katherine A. Janeway. Sequencing identifies diagnostically relevant alterations in pediatric solid tumor patients [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A59.

Introduction: Gene fusions are important oncogenic drivers with significant clinical impact in some cancer types. This is particularly true in pediatric cancers that often have low mutational burden and lack other diagnostic markers and therapeutic targets. Many gene fusions are rare or private to the individual patient and can be difficult to detect with methods optimized for common fusions. Unbiased sequencing methods and expansive computational resources are needed for expanding our ability to characterize fusions. Building a comprehensive catalog of oncogenic gene fusions will improve our understanding of their diversity and fully harness their potential for clinical impact. Methods: Patients are eligible for the GAIN/iCat2 study if they have been diagnosed with high-risk or recurrent/refractory extracranial solid tumor at age 30 or less and have a sample available for sequencing. Enrolled patients with an unclear diagnosis after standard clinical testing are nominated for transcriptome sequencing by the study investigators. We developed a computational pipeline in Google Cloud for gene fusion discovery utilizing paired end Illumina RNA-Seq data, multiple fusion callers, and a custom algorithm for integrative data analysis. The multicaller fusion detection approach enables us to address the high false-positive rate typical for gene fusion calling in transcriptomic data while improving the sensitivity to detect the more challenging fusions. After filtering, the fusions are annotated using the databases of known fusions and cancer genes. The predicted fusion transcripts are inspected visually, and the fusions are selected based on relevance to diagnostic classification or therapy to be validated by an orthogonal method. Results: 41 tumor samples were sequenced and analyzed for gene fusions. A total of 203 candidate fusions were detected by two or more fusion callers. Based on functional annotations and potential impact on diagnosis or therapeutic approaches, 12 fusion transcripts of interest were identified, 10 of which were validated by either pre-enrollment testing or an orthogonal method. Of 16 mesenchymal cases, 6 validated fusions had diagnostic relevance and 3 validated fusions had therapeutic implications (ERC1-BRAF, RBPMS-NTRK2, and VCAN-IL23R). Two patients responded to matched targeted therapy. In one case, diagnostic classification was revised. Conclusions: Whole-transcriptome sequencing in this selected patient population identified some fusion transcripts with clinical relevance. Determining the biologic significance of previously unreported fusions will require orthogonal sequencing such as whole genome, functional studies, and analysis of larger patient populations. Improved accuracy and scalability of methods for large-scale gene fusion analysis in the growing public datasets are likely to expand the landscape of gene fusions in cancer. Citation Format: Alma Imamovic, Alanna J. Church, Laura B. Corson, Deirdre Reidy, Navin Pinto, Luke Maese, Theodore W. Laetsch, AeRang Kim, Susan I. Vear, Margaret E. Macy, Mark A. Applebaum, Rochelle Bagatell, Amit J. Sabnis, Daniel A. Weiser, Julia L. Glade-Bender, Gianna R. Strand, Lobin A. Lee, R. Seth Pinches, Catherine M. Clinton, Brian D. Crompton, Neal I. Lindeman, Steven G. DuBois, Katherine A. Janeway, Eliezer M. Van Allen. Leveraging cloud-based computational resources for gene fusion discovery with potential clinical implications for pediatric solid tumor patients [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B13.

Several aggressive cancers impacting children are characterized by alterations in the SWI/SNF complex, including rhabdoid tumors, epithelioid sarcoma, and chordoma. With recent early-phase trials showing responses to EZH2 inhibitors, it is important to understand the optimal approach to identifying INI1-deficient cancers. As tumor profiling is becoming a more routine part of clinical care, this study was designed to determine the relationship between SMARCB1 genetic variants identified by a sequencing panel test and INI1 protein expression. Beyond EZH2 inhibitors, therapeutic approaches for INI1-deficient tumors are limited. Thus, we also sought to investigate PD-L1 expression in a cohort of INI1-deficient pediatric brain and solid malignancies. Patients were identified by two methods: 1) search of our institutional pathology database from 2000-2015 for INI1-deficient tumors and 2) presence of SMARCB1 genomic alteration in a database of 280 cases with somatic panel sequencing results. Patients were included in the study if sufficient archival tumor tissue was available for repeat, confirmatory sequencing and immunohistochemistry (IHC). Somatic next-generation sequencing (NGS) was performed via a panel assay, OncoPanel, which surveys exonic DNA sequences of 447 cancer genes and 191 regions across 60 genes for rearrangement detection. IHC stains for INI1 and PD-L1 were performed according to standardized procedure on Leica Bond automated platforms and expression was assessed by two investigators (AA, JP) who were blinded to sequencing results. The study included 43 patients. IHC was performed on at least one specimen for all patients and repeat, confirmatory NGS was successful in 91% (39/43). Single-copy deletion of SMARCB1 on NGS panel was not predictive of loss of INI1 expression by IHC with only 1/10 (10%) SMARCB1 single-copy deletion cases having INI1 loss. In the 26 cases with INI1 loss by IHC and successful tumor sequencing, 23 (89%) had a genomic alteration in SMARCB1 detected. Five cases (22%) had 1-copy deletion, 12 (52%) had 2-copy deletion, 2 (9%) had nonsense mutations, and 4 (17%) had two inactivating alterations. 40% (12/30) of the patients with INI1-deficient tumors had at least one tumor specimen that was PD-L1 positive (≥1%). PD-L1 status was not associated with timing of tumor sampling or prior treatment. TMB ranged from 0.76 to 9.13 mut/Mb of DNA. We have observed 2 patients with INI1-deficient cancers with evidence of efficacy of immune checkpoint inhibitors. SMARCB1 2-copy deletions and inactivating mutations are associated with loss of INI1 protein expression, but 1-copy deletion of INI1 in histologies other than those already known to be INI1-deficient is not predictive of loss of protein expression. These results, along with two case reports of successful disease control with immune checkpoint inhibitors, suggest that clinical trials of PD-1 or PD-L1 inhibitors, either as single agents or in combination with an EZH2 inhibitor, are warranted in malignancies with INI1 loss. Citation Format: Suzanne J. Forrest, Alyaa Al-Ibraheemi, Abigail Ward, Duong Doan, Catherine Clinton, Juan Putra, R. Seth Pinches, Cigall Kadoch, Susan Chi, Steve G. Dubois, Patrick Leavy, Natalie Collins, Alanna Church, Katherine A. Janeway. Genomic and immunologic characterization of a cohort of INI1-deficient pediatric cancers [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A16.

TPS10560 Background: In children, fusions of the NTRK1/2/3 genes (TRK fusions) occur in soft tissue sarcomas, including infantile fibrosarcoma (IFS), congenital mesoblastic nephroma, high- and low-grade gliomas, secretory breast carcinoma, and papillary thyroid cancer. Rarely, TRK fusions also occur in Ph-like acute lymphoblastic leukemia and acute myeloid leukemia. Larotrectinib is a selective TRK inhibitor FDA-approved for the treatment of TRK fusion solid tumors in patients with no satisfactory alternative treatments or whose cancer has progressed following initial treatment. In children, larotrectinib demonstrated a 94% overall response rate (ORR) with a 12-month progression free survival rate of 75% (1). Methods: Patients 2/dose BID (max of 100 mg/dose) continuously in 28-day cycles. Patients with solid tumors who achieve CR will discontinue larotrectinib at the completion of at least 12 total cycles of therapy and 6 cycles after achieving CR. Those whose tumors become surgically resectable may undergo on study resection and discontinue therapy if an R0/R1 (IFS) or R0 (other tumors) resection is obtained. All other patients will receive 26 cycles in the absence of unacceptable toxicity or progressive disease. The primary endpoint is the ORR to larotrectinib according to RECIST 1.1 in children with IFS. The study uses a Simon 2-stage minimax design, and the regimen will be considered of sufficient interest if 16 of 21 (76%) patients with IFS demonstrate response. Patients with other solid tumors and leukemias will be analyzed in separate cohorts as secondary objectives. Correlative studies include serial sampling of circulating tumor DNA and neurocognitive assessments. This is the first Children’s Oncology Group study to assign frontline therapy based on the presence of a molecular marker independent of histology, and the first clinical trial to evaluate larotrectinib for the treatment of leukemia. Enrollment began in October 2019 (NCT03834961). 1. Tilburg CMv, DuBois SG, Albert CM, et al: Larotrectinib efficacy and safety in pediatric TRK fusion cancer patients. Journal of Clinical Oncology 37:10010-10010, 2019 Clinical trial information: NCT03834961.

Purpose: Novel targeted therapeutics have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, with simple tumor genomes and infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The cyclin-dependent kinase (CDK)4/6 pathway recently emerged as a dependency in Ewing sarcoma. Given the heightened efficacy of this class with targeted drug combinations in other cancers, as well as the propensity of resistance to emerge with single agents, we aimed to identify genes mediating resistance to CDK4/6 inhibitors and biologically relevant combinations for use with CDK4/6 inhibitors in Ewing. Experimental Design: We performed a genome-scale open reading frame (ORF) screen in 2 Ewing cell lines sensitive to CDK4/6 inhibitors to identify genes conferring resistance. Concurrently, we established resistance to a CDK4/6 inhibitor in a Ewing cell line. Results: The ORF screen revealed IGF1R as a gene whose overexpression promoted drug escape. We also found elevated levels of phospho-IGF1R in our resistant Ewing cell line, supporting the relevance of IGF1R signaling to acquired resistance. In a small-molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6 inhibitor treatment. The combination of CDK4/6 inhibitors and IGF1R inhibitors was synergistic in vitro and active in mouse models. Mechanistically, this combination more profoundly repressed cell cycle and PI3K/mTOR signaling than either single drug perturbation. Conclusions: Taken together, these results suggest that IGF1R inhibitors activation is an escape mechanism to CDK4/6 inhibitors in Ewing sarcoma and that dual targeting of CDK4/6 inhibitors and IGF1R inhibitors provides a candidate synergistic combination for clinical application in this disease.

Infantile fibrosarcoma and congenital mesoblastic nephroma are tumors of infancy traditionally associated with the ETV6-NTRK3 gene fusion. However, a number of case reports have identified variant fusions in these tumors. In order to assess the frequency of variant NTRK3 fusions, and in particular whether the recently identified EML4-NTRK3 fusion is recurrent, 63 archival cases of infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma (tumor types that are known to carry recurrent ETV6-NTRK3 fusions) were tested with NTRK3 break-apart FISH, EML4-NTRK3 dual fusion FISH, and targeted RNA sequencing. The EML4-NTRK3 fusion was identified in two cases of infantile fibrosarcoma (one of which was previously described), and in one case of congenital mesoblastic nephroma, demonstrating that the EML4-NTRK3 fusion is a recurrent genetic event in these related tumors. The growing spectrum of gene fusions associated with infantile fibrosarcoma and congenital mesoblastic nephroma along with the recent availability of targeted therapies directed toward inhibition of NTRK signaling argue for alternate testing strategies beyond ETV6 break-apart FISH. The use of either NTRK3 FISH or next-generation sequencing will expand the number of cases in which an oncogenic fusion is identified and facilitate optimal diagnosis and treatment for patients.

Background Clinical genomics platforms are needed to identify targetable alterations, but implementation of these technologies and best practices in routine clinical pediatric oncology practice are not yet well established. Methods Profile is an institution-wide prospective clinical research initiative that uses targeted sequencing to identify targetable alterations in tumors. OncoPanel, a multiplexed targeted exome-sequencing platform that includes 300 cancer-causing genes, was used to assess single nucleotide variants and rearrangements/indels. Alterations were annotated (Tiers 1-4) based on clinical significance, with Tier 1 alterations having well-established clinical utility. OncoCopy, a clinical genome-wide array comparative genomic hybridization (aCGH) assay, was also performed to evaluate copy number alterations and better define rearrangement breakpoints. Results Cancer genomes of 203 pediatric brain tumors were profiled across histological subtypes, including 117 samples analyzed by OncoPanel, 146 by OncoCopy, and 60 tumors subjected to both methodologies. OncoPanel revealed clinically relevant alterations in 56% of patients (44 cancer mutations and 20 rearrangements), including BRAF alterations that directed the use of targeted inhibitors. Rearrangements in MYB-QKI, MYBL1, BRAF, and FGFR1 were also detected. Furthermore, while copy number profiles differed across histologies, the combined use of OncoPanel and OncoCopy identified subgroup-specific alterations in 89% (17/19) of medulloblastomas. Conclusion The combination of OncoPanel and OncoCopy multiplex genomic assays can identify critical diagnostic, prognostic, and treatment-relevant alterations and represents an effective precision medicine approach for clinical evaluation of pediatric brain tumors.

The GAIN iCat2 Project is a collaboration between Dana-Farber/Boston Children's Cancer and Blood Disorder Center and eleven pediatric oncology centers across the United States to sequence relapsed, metastatic, difficult-to-diagnose, and high-risk extracranial solid tumors from 825 patients. The goals are to gain a better understanding of the genomic events in pediatric cancers and determine the clinical impact of matched targeted therapy. Tumor samples are sequenced on one of four gene panels performed in CLIA certified, CAP accredited laboratories, most often utilizing OncoPanel at the Center for Advanced Molecular Diagnostics, Brigham Women’s Hospital. This panel assesses SNVs and CNVs in 447 cancer-associated genes and interrogates intronic regions of 60 genes frequently involved in oncogenic translocation. For undifferentiated sarcomas and tumors in which oncogenic drivers are not identified by the gene panel, whole exome sequencing or RNA sequencing for fusion detection may be done. Interpretation of genomic results, including potential implications for diagnosis and hereditary risks, as well as assessment of possible matched targeted therapies and suitable trials are summarized in a report to the primary oncology provider. An interim analysis of tumors from the first 275 patients enrolled who have OncoPanel results was performed to assess genomic alterations most prevalent in this group of pediatric cancers. 50% (137/275) have structural alterations in their tumors with over half of these (74/137) harboring an oncogenic fusion that is the main, or only identified, driver of the cancer. These include fusions pathognomonic for diseases such as Ewing sarcoma, alveolar rhabdomyosarcoma, synovial sarcoma, desmoplastic small round cell tumors, mesenchymal chondrosarcoma, low grade fibromyxoid sarcoma, and NUT midline carcinoma. Other cases showed recurrent disruption of key tumor suppressors, such as TP53 intron 1 translocations in osteosarcoma. Lastly, more generalized, key, cancer-driving fusions were seen with rearrangements involving BRAF, NOTCH, and NTRK. In addition to aiding in diagnosis, identification of fusions has led to targeted therapy recommendations for many patients. SNVs and CNVs also helped clarify diagnoses, especially in the case of DICER1 and SMARCB1 alterations, and identified potential targeted therapies to consider for relapsed patients. Although patient recruitment is ongoing, this study shows promise for advancing our understanding and treatment of pediatric cancers and highlights the critical importance of incorporating techniques for fusion detection in tumor profiling. Citation Format: Laura B. Corson, Alma Imamovic-Tuco, Gianna R. Strand, Deirdre Reidy, Duong Doan, Mark A. Applebaum, Rochelle Bagatell, Brian D. Crompton, Steven G. DuBois, Julia L. Glade Bender, AeRang Kim, Theodore W. Laetsch, Lobin A. Lee, Neal I. Lindeman, Laura E. MacConaill, Margaret E. Macy, Luke Maese, Seth Pinches, Navin Pinto, Amit J. Sabnis, Eliezer M. Van Allen, Susan I. Vear, Daniel A. Weiser, Catherine M. Clinton, Katherine A. Janeway, Alanna J. Church. A high prevalence of chromosomal translocations as drivers in high-risk pediatric solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3104.

BACKGROUND: TP53 mutations are rare across pediatric cancers. Recent work using CRISPR-Cas9 screens has demonstrated that MDM2 and MDMX are strong dependencies in a range of TP53-wildtype pediatric malignancies. Increased expression of MDM2 and MDMX is a common mechanism for suppressing p53 in pediatric malignancies and can occur by copy number gain or amplification, as has been reported in retinoblastoma and hepatoblastoma. In pediatric high-grade gliomas, activating PPM1D mutations drive p53 suppression, likely through MDM2 stabilization. ALRN-6924 is a novel, first-in-class, cell-permeating stapled peptide that disrupts the inhibitory interactions between MDM2/MDMX(MDM4) and p53. ALRN-6924 has been evaluated in two adult Phase I trials, with good tolerability and evidence of clinical activity across a range of cancer subtypes. Given the oncogenic roles of MDM2 and MDMX in pediatric malignancies, we developed a Phase I clinical trial designed to evaluate the tolerability, pharmacokinetics, and pharmacodynamic and antitumor activity of ALRN-6924. METHODS: This is a Phase I, open-label, investigator-initiated multicenter study of ALRN-6924 in children 1-21 years of age with relapsed/refractory cancer (NCT03654716). The primary objectives are to determine the recommended phase 2 dose, and to describe toxicities and pharmacokinetic parameters of ALRN-6924 in this population. The monotherapy arm consists of two cohorts: Cohort A for patients with TP53-wildtype solid tumors and lymphomas; and Cohort B for patients with retinoblastoma, or TP53-wildtype tumors that meet any of the following criteria: hepatoblastoma, malignant rhabdoid tumor, MDM2 or MDMX amplification, TET2 loss, or PPM1D activating mutations. Patients with CNS primary tumors are only eligible for Cohort B. In Cohorts A and B, patients receive ALRN-6924 intravenously on Days 1, 4, 8 and 11 of a 21-day cycle starting at a dose of 2.2 mg/kg. An expansion cohort for patients eligible for Cohort B will open following completion of monotherapy dose escalation. Patients with relapsed/refractory leukemias enroll to Cohort C and receive ALRN-6924 in combination with low-dose cytarabine on days 1, 8 and 15 of a 28-day cycle starting at a dose of 2.7 mg/kg. Pharmacokinetic sampling and pharmacodynamic testing (serum MIC-1 modulation) is required for all patients. Correlative biology studies will include evaluation of circulating tumor DNA for TP53 mutations in patients with solid tumors and serial assessment of leukemic blasts in patients with relapsed leukemia. Enrollment began in October 2018. Up to 69 patients will be enrolled. Citation Format: David S. Shulman, Kieuhoa T. Vo, Elizabeth Fox, Jodi A. Muscal, Loren D. Walensky, Yana Pikman, Kimberly Stegmaier, Alanna Church, Brian D. Crompton, Andrew E. Place, Susan N. Chi, Allison F. O'Neill, Junne Kamihara, Suzanne Ezrre, Cecilia Carlowicz, Dawn Pinchasik, Hasan Al-Sayegh, Clement Ma, Wendy B. London, Steven G. DuBois. A Phase I multicenter trial of the dual MDM2/MDMX inhibitor ALRN-6924 in children and young adults with relapsed/refractory pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT112.

Purpose Mantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis. Methods We assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL. Results Fourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores. Conclusion Eleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.

BACKGROUND. Comprehensive genomic profiling of a patient's cancer can be used to diagnose, monitor, and recommend treatment. Clinical implementation of tumor profiling in an enterprise-wide, unselected cancer patient population has yet to be reported. METHODS. We deployed a hybrid-capture and massively parallel sequencing assay (OncoPanel) for all adult and pediatric patients at our combined cancer centers. Results were categorized by pathologists based on actionability. We report the results for the first 3,727 patients tested. RESULTS. Our cohort consists of cancer patients unrestricted by disease site or stage. Across all consented patients, half had sufficient and available (>20% tumor) material for profiling; once specimens were received in the laboratory for pathology review, 73% were scored as adequate for genomic testing. When sufficient DNA was obtained, OncoPanel yielded a result in 96% of cases. 73% of patients harbored an actionable or informative alteration; only 19% of these represented a current standard of care for therapeutic stratification. The findings recapitulate those of previous studies of common cancers but also identify alterations, including in AXL and EGFR, associated with response to targeted therapies. In rare cancers, potentially actionable alterations suggest the utility of a "cancer-agnostic" approach in genomic profiling. Retrospective analyses uncovered contextual genomic features that may inform therapeutic response and examples where diagnoses revised by genomic profiling markedly changed clinical management. CONCLUSIONS. Broad sequencing-based testing deployed across an unselected cancer cohort is feasible. Genomic results may alter management in diverse scenarios; however, additional barriers must be overcome to enable precision cancer medicine on a large scale. FUNDING. This work was supported by DFCI, BWH, and the National Cancer Institute (5R33CA155554 and 5K23CA157631).

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers., Identifying therapeutic targets in rare cancers is challenging due to the lack of relevant pre-clinical models. Here, the authors generate a cancer cell line from a paediatric patient with a rare undifferentiated sarcoma and through functional genomics and chemical screens identified CDK4 and XPO1 as potential therapeutic targets in this cancer.

Renal medullary carcinomas (RMCs) are thought to be driven by the loss of tumor suppressor, SMARCB1. These rare kidney cancers carry a very poor prognosis and primarily affect African American adolescents and young adults with sickle cell trait. From two patients with RMC, we have identified by whole-genome sequencing mechanisms of SMARCB1 loss (e.g., inactivating fusion events involving SMARCB1). We developed in vitro models of primary and relapsed metastatic disease. We performed biochemical and functional studies to conclusively show that RMC is dependent on loss of SMARCB1, similar to rhabdoid tumors and atypical teratoid/rhabdoid tumors. Furthermore, we performed small-molecule screens, pooled CRISPR-Cas9 knockout, and RNAi suppression screens focused on druggable cancer targets. Integration of these orthogonal methods identifies a core set of targets that may provide a rational approach to therapeutic targeting for this rare kidney cancer and other SMARCB1-deficient cancers. Citation Format: Andrew L. Hong, Yuen-Yi Tseng, Bryan D. Kynnap, Mihir B. Doshi, Jeremiah Wala, Gabriel Sandoval, Alanna J. Church, Elizabeth Mullen, Cigall Kadoch, Charles W.M. Roberts, Rameen Beroukhim, Jesse S. Boehm, William C. Hahn. Modeling renal medullary carcinomas identifies druggable vulnerabilities in SMARCB1-deficient cancers [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B18.

Infantile myofibromas (IM) are rare benign neoplasms thought to derive from perivascular myoid cells. The prognosis is generally good, although about one-third of cases with multicentric involvement will also have visceral involvement, and the prognosis for these patients is poor. The use of combination cytotoxic chemotherapy (vincristine/dactinomycin and vinblastine/methotrexate) has proven effective in cases of multicentric disease with visceral involvement and in cases in which the disease has progressed and has threatened the life of the patient. The pathologic diagnosis of IM can be challenging, particularly with small biopsies, since IM exist on a morphologic spectrum with myopericytomas, glomangiopericytomas, and glomus tumors, and other spindle cell neoplasms are also on the histologic differential. Most families with autosomal dominant infantile myofibromatosis have germline variants in PDGFRB. In one case of autosomal dominant IM a NOTCH3 variant was identified in the absence of a PDGFRB variant. Most sporadic infantile myofibromas also have identifiable variants in PDGFRB. To date, however, there have been no genetic variants or immunohistochemical stains that are 100% sensitive or specific in diagnosing IM. Nine cases of infantile myofibroma/myofibromatosis were identified from the case archives of Boston Children’s Hospital. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections and sequenced using a clinically validated targeted panel of 447 genes for 9 cases. Immunohistochemical stain for activated NOTCH3 was performed. The histopathology of each case was reviewed by two pediatric pathologists. Of the nine IM cases, 7 had one or more sequence variants in PDGFRB. One case had a NOTCH3 sequence variant and one had a NOTCH2 sequence variant. All nine cases (100%) had a distinctive pattern of copy alterations including gains of PDGFRB and NOTCH3. One case failed immunohistochemical staining; of the remaining 8 IM cases, all 8 showed nuclear staining for activated NOTCH3. We describe a distinctive pattern of genomic alterations in infantile myofibromatosis, including a high prevalence (7/9 cases) of alterations in PDGFRB, several of which are known to be activating, and copy number alterations including copy number gains of PDGFRB and NOTCH3 in all 9 cases. The p.N666K, p.D850V (both seen in this study), and p.R561C variants have been shown to result in constitutive activation of PDGFRB, transform cells and induce tumor formation, and render tumor sensitivity to tyrosine kinase inhibitors. In vascular smooth muscle cells, PDGFRB has been shown to be an immediate downstream target of NOTCH3 signaling. The consistent presence of activated NOTCH3, detected by immunohistochemistry in all IM cases, confirms that the genomic changes have functional effect. The NOTCH signaling pathway is the target of inhibitors including gamma-secretase inhibitors; some NOTCH-mutated leukemias have been shown to be sensitive to inhibition, suggesting that these inhibitors may also be useful in NOTCH-mutated myofibromas. Our findings suggest a common pathway of PDGFRB/NOTCH3 activation in infantile myofibromas. Identification of the characteristic genomic alterations or immunohistochemical staining pattern may facilitate a difficult pathologic diagnosis. Furthermore, targeted inhibitors are available for both PDGRFB and NOTCH, and may be of use in patients with visceral or multicentric disease. Citation Format: Selene C. Koo, Calicchio Monica, Benjamin Ferland, Marian H. Harris, Jon C. Aster, Katherine A. Janeway, Alyaa Al-Ibraheemi, Alanna J. Church. A distinctive genomic and immunohistochemical profile for NOTCH3 and PDGFRB in infantile myofibroma with diagnostic and therapeutic implications [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A31.

Novel targeted drug combinations have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, where tumor genomes are typically simple with infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The CDK4/6 pathway, which has been extensively validated as a target in estrogen receptor-positive breast cancer, recently emerged as a dependency in Ewing sarcoma, an aggressive pediatric bone tumor. Given the heightened efficacy of CDK4/6 inhibitors (CDK4/6i) in combination with other targeted drugs in breast cancer, as well as the known propensity of resistance to emerge with single agent targeted cancer therapy, we aimed to identify CDK4/6i resistance mechanisms and biologically relevant drug combinations in Ewing. To identify candidate resistance mechanisms, we performed a genome-wide lentiviral open-reading frame (ORF) rescue screen in two Ewing cell lines sensitive to the CDK4/6 inhibitors palbociclib and ribociclib and looked for genes that confer resistance to these drugs. This revealed IGF1R as a gene whose overexpression promoted drug escape. In parallel to the ORF screen, we established resistance to CDK4/6i in Ewing sarcoma cell lines by chronic ribociclib exposure. We found elevated levels of phospho-IGF1R in resistant cells by immunoblotting, also supporting that this pathway's activation is relevant to acquired resistance. We performed CRISPR knockout of IGF1R in these drug resistant cells and demonstrated rescue of sensitivity to CDK4/6i. Furthermore, resistant lines maintained high sensitivity to an IGF1R inhibitor. Concurrently, in a small molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6i in Ewing cells. We thus tested ribociclib and the IGF1R tool compound AEW541 in a panel of six Ewing cell lines in vitro. We observed strong synergy utilizing the Chou-Talalay method. This finding was next validated in an in vivo cell line xenograft mouse model, where we demonstrated prolonged survival and decreased tumor volumes with combination drug treatment. In order to investigate mechanism of CDK4/6-IGF1R inhibitor synergy, we used proteomic analysis by reverse phase protein array (RPPA) in single and combined drug treated Ewing cells. RPPA revealed enhanced suppression of the PI3K/mTOR pathway in the combination, a central cancer cell signaling axis. Taken together, these results suggest that IGF1R activation is an escape mechanism to CDK4/6i in Ewing and that dual targeting of CDK4/6 and IGF1R provides a candidate synergistic drug combination for this disease. Citation Format: Lillian M. Guenther, Neekesh V. Dharia, Linda Ross, Amy S. Conway, Amanda L. Robichaud, Alanna J. Church, Rajarshi Guha, Mindy I. Davis, Gabriela Alexe, Jaume Mora, Federica Piccioni, Kimberly Stegmaier. Targeting resistance mechanisms to CDK4/6 inhibitors in Ewing sarcoma with an IGF1R inhibitor drug combination strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1629.

Although the histologic appearance of pure testicular teratomas (PTTs) is similar in children and adults, the prognosis is dramatically different. Prepubertal PTTs are rare, with a benign clinical course, whereas the adult cases typically have malignant outcomes. Chromosome 12p abnormalities are seen in most adult testicular germ cell tumors but have not been found in prepubertal PTTs. IMP3 is an oncofetal protein that is highly expressed in many malignancies. Recently, we demonstrated IMP3 is expressed in adult mature testicular teratomas but not in mature ovarian teratomas. The aim of this study was to evaluate prepubertal PTTs for chromosome 12p abnormalities and expression of IMP3. A total of 11 cases (excision, n=1; orchiectomy, n=10) were obtained from the surgical pathology archives of 2 large medical centers (1957-2013). All 11 cases were investigated for isochromosome 12p and 12p copy number gain using interphase fluorescence in situ hybridization analysis and were examined by immunohistochemistry for IMP3 expression. Patients ranged in age from 0.9 to 7.0 (mean, 2.4) years. A positive immunohistochemical stain for IMP3 (cytoplasmic staining) was identified in 5 (46%) of 11 cases. Isochromosome 12p was detected in 2 cases (18%) that also expressed IMP3. Somatic copy number alterations of 12p were not observed (0%). We are the first to describe 12p abnormalities and IMP3 expression in prepubertal PTTs. Our data demonstrate a small subset of PTTs harbor typical molecular alterations observed in adult testicular germ cell tumors. Although prepubertal PTTs are considered to be benign neoplasms, it may be a heterogeneous group.

Traumatic brain injury (TBI) is particularly common in young people, generating healthcare costs that can span decades. The cellular processes activated in the first minutes following injury are poorly understood, and the 3-4 h following trauma are crucial for reducing subsequent injury. Spreading depression (SD) is a profound inactivation of neurons and glia lasting 1-2 min that arises focally and migrates outward across gray matter. In the hours following focal stroke, the metabolic stress of energy reduction and recurring SD-like events (peri-infarct depolarizations, PIDs) interact to promote neuronal injury. Similar recurring depolarizations might evolve immediately following TBI and exacerbate neuronal damage peripheral to the impact site. To test this possibility and examine if certain drugs might limit damage by inhibiting what we term traumatic spreading depression (tSD), we developed a technique whereby a small weight was dropped onto a live slice of rat neocortex while imaging changes in light transmittance (LT). Imaging revealed a propagating front of increased LT arising at the border of the impact site. Traumatic SD significantly expanded the region of ensuing damage. Both tSD and subsequent damage were blocked by the NMDA receptor antagonist MK-801 (100 microM) or the sigma-1 receptor (sigma1R) ligands dextromethorphan (30 microM) or BD-1063 (100 microM). Co-application of the sigma1R antagonist (+)3-PPP with DM reversed the block as did lowering temperature from 35 degrees C to 32 degrees C. This study provides evidence that an event similar to peri-infarct depolarization can arise from an injury site in neocortex within seconds following impact and act to expand the region of acute neuronal damage.

Renal medullary carcinoma is a rare kidney cancer that is primarily seen in adolescent and young adult African American patients with sickle cell trait. Prognosis is poor and treatment options are limited. We have developed several cell line models that recapitulate the primary and relapsed metastatic samples from a patient who succumbed to this disease. We have confirmed by whole exome sequencing that our models have sickle cell trait and loss of heterozygosity of the SMARCB1 loci, both hallmarks of this disease. By RNA-sequencing, we see a lack of SMARCB1 transcription. We have further shown dependency of our models to SMARCB1 re-expression thus suggesting that this cancer is indeed driven by loss of SMARCB1 at a functional level. We performed pooled CRISPR-Cas9 and RNAi loss of function screens and a small molecule screen focused on druggable cancer targets based on our previous work in parallel to a genome-wide pooled CRISPR-Cas9 loss of function screen. Integrating these complementary and orthogonal methods, we identified a number of targets for further validation. These targets, when combined may provide a rational approach to therapeutic targeting for this rare kidney cancer. Citation Format: Andrew L. Hong, Yuen-Yi Tseng, Bryan D. Kynnap, Mihir B. Doshi, Gabriel Sandoval, Coyin Oh, Abeer Sayeed, Gill Shubhroz, Alanna J. Church, Paula Keskula, Anson Peng, Paul A. Clemons, Aviad Tsherniak, Francisca Vazquez, Carlos Rodriguez-Galindo, Katherine A. Janeway, Levi A. Garraway, Stuart L. Schreiber, David E. Root, Elizabeth Mullen, Kimberly Stegmaier, Cigall Kadoch, Charles W.M. Roberts, Jesse S. Boehm, William C. Hahn. Identification of Druggable Targets through Functional Multi-Omics in Renal Medullary Carcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B17.

INTRODUCTION: Clinical genomics platforms identify targetable alterations for precision medicine in pediatric neuro-oncology, allowing both the genomic profiling of tumors to inform clinical care and facilitating the potential discovery of novel driver alterations. METHODS: We applied two clinical genomic platforms (OncoPanel and OncoCopy) to profile pediatric brain tumors in a clinical setting. OncoPanel, a multiplexed targeted exome-sequencing platform that includes 300 cancer causing genes, was used to assess single nucleotide variants and rearrangements/indels. OncoCopy, a high-resolution genome-wide array comparative genomic hybridization (aCGH) assay, was used to evaluate copy number alterations and refine rearrangement breakpoints. RESULTS: Cancer genomes of 203 pediatric brain tumors were profiled across histological subtypes including 117 analyzed by OncoPanel, 146 by OncoCopy, and 60 evaluated using both methodologies. OncoPanel revealed clinically relevant alterations in 56% of patients (44 cancer mutations and 20 rearrangements), including BRAF alterations that directed the use of targeted inhibitors. EWSRa rearrangements were observed unexpectedly in two patients; one with a Atypical Teratoid Rhabdoid Tumor with loss of INI-1 (SMARCB1), and the second in an ependymoma. Whole Genome Sequencing of the rhabdoid tumor confirmed a rearrangement involving EWSR1 and PLAGL1.CONCLUSIONS: The combined deployment of OncoPanel and OncoCopy multiplex genomic assays can identify clinically relevant genomic alterations in pediatric brain tumors and facilitate the discovery of novel driver alterations. *equal contribution

Of pediatric solid tumors, as many as 10% of tumors are categorized as rare. Many of these rare tumors lack standard effective known therapy. The ability to identify vulnerabilities for many rare tumors has been significantly limited by the lack of in vitro and in vivo models. Furthermore, current approaches to study such vulnerabilities are usually limited to a specific compound or target. Our objectives were 1) to develop a platform to collect tumor samples and generate in vitro models and 2) to develop systematic and orthogonal approaches focused on currently known druggable cancer targets to identify vulnerabilities in these difficult to treat cancers. We have developed a proof of concept cell line from a patient who succumbed to progressive undifferentiated sarcoma treated on an aggressive multi-therapy regimen. This cell line, in its early passages, has novel gene fusions that match that of the primary tumor. Furthermore, even at early passages, this cell line was amenable to high throughput functional screens. Using a targeted pooled shRNA screen (employing matched seed controls) and an analogous CRISPR screen we identified dependencies to XPO1 and CDK4. In parallel, compounds against these targets were identified in a small molecule compound screen. These targetable dependencies were further validated in vivo with a micro-dosing device. These observations identify new targets in this rare malignancy. Furthermore, this suggests that the interrogation of patient derived cell lines facilitates the identification of testable therapeutic approaches. Citation Format: Andrew L. Hong, Glenn S. Cowley, Yuen-Yi Tseng, Jaime H. Cheah, Oliver Jonas, Mihir B. Doshi, Bryan D. Kynnap, Coyin Oh, Stephanie Meyer, Paul Clemons, Michael Burger, Francisca Vazquez, Barbara Weir, Gregory V. Kryukov, Alanna Church, Alma Imamovic, Aviad Tsherniak, Craig Bielski, Brian Crompton, Elizabeth Mullen, Charles Roberts, Carlos Rodriguez-Galindo, Katherine A. Janeway, Kimberly Stegmaier, Paul van Hummelen, Robert Langer, Levi A. Garraway, Stuart L. Schreiber, David E. Root, Jesse S. Boehm, William C. Hahn. Developing a functional genomics platform to interrogate rare pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B38.

We describe the clinical course of a recurrent case of congenital fibrosarcoma diagnosed in a 9-mo-old boy with a history of hemimelia. Following complete surgical resection of the primary tumor, the patient subsequently presented with bulky bilateral pulmonary metastases 6 mo following surgery. Molecular characterization of the tumor revealed the absence of the prototypical ETV6-NTRK3 translocation. However, tumor characterization incorporating cytogenetic, array comparative genomic hybridization, and RNA sequencing analyses, revealed a somatic t(2;15)(2p21;15q25) translocation resulting in the novel fusion of EML4 with NTRK3. Cloning and expression of EML4-NTRK3 in murine fibroblast NIH 3T3 cells revealed a potent tumorigenic phenotype as assessed in vitro and in vivo. These results demonstrate that multiple fusion partners targeting NTRK3 can contribute to the development of congenital fibrosarcoma.