Publications by authors named "Alanna J Church"

28 Publications

  • Page 1 of 1

Germline Sequencing Improves Tumor-Only Sequencing Interpretation in a Precision Genomic Study of Patients With Pediatric Solid Tumor.

JCO Precis Oncol 2021 22;5. Epub 2021 Dec 22.

Department of Pediatric Oncology, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA.

Purpose: Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks.

Materials And Methods: Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation.

Results: One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events.

Conclusion: A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.
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http://dx.doi.org/10.1200/PO.21.00281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8710335PMC
December 2021

Molecular Characterization of Inflammatory Tumors Facilitates Initiation of Effective Therapy.

Pediatrics 2021 12;148(6)

Department of Pediatrics, Dana-Farber/Boston Children's Cancer and Blood Disorders Center and Harvard Medical School, Harvard University, Boston, Massachusetts.

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.
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http://dx.doi.org/10.1542/peds.2021-050990DOI Listing
December 2021

Molecular Alterations in Pediatric Solid Tumors.

Surg Pathol Clin 2021 Sep 8;14(3):473-492. Epub 2021 Jul 8.

Department of Pathology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.path.2021.05.010DOI Listing
September 2021

Assessment of BCOR Internal Tandem Duplications in Pediatric Cancers by Targeted RNA Sequencing.

J Mol Diagn 2021 10 27;23(10):1269-1278. Epub 2021 Jul 27.

Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmoldx.2021.07.006DOI Listing
October 2021

Fibrolamellar carcinoma: An entity all its own.

Curr Probl Cancer 2021 08 1;45(4):100770. Epub 2021 Jul 1.

Department of Surgery, Boston Children's Hospital, Boston MA.

Fibrolamellar carcinoma (FLC) is a rare malignant entity arising from the liver and primarily affecting patients in late adolescence and young adulthood. FLC tumors are characterized by their unique histologic features and an only recently discovered genomic alteration: a chimeric fusion protein found in nearly all tumors. The rarity of these tumors coupled with the only recent acknowledgement of this genomic abnormality has likely led to disease under-recognition and de-prioritization of collaborative efforts aimed at establishing an evidence-guided standard of care. Surgical resection undoubtedly remains a mainstay of therapy and a necessity for cure but given the incidence of metastatic disease at diagnosis and high rates of distant relapse, systemic therapies remain a key component of disease control. There are few systemic therapies that have demonstrated proven benefit. Recent efforts have galvanized around single-institute or small consortia-based studies specifically focused on the enrollment of patients with FLC or use of agents with biologic rationale. This review will outline the current state of FLC epidemiology, histology, biology and trialed therapies derived from available published literature.
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http://dx.doi.org/10.1016/j.currproblcancer.2021.100770DOI Listing
August 2021

A Curriculum for Genomic Education of Molecular Genetic Pathology Fellows: A Report of the Association for Molecular Pathology Training and Education Committee.

J Mol Diagn 2021 10 7;23(10):1218-1240. Epub 2021 Jul 7.

Molecular Genetic Pathology Fellow Training in Genomics Task Force of the Training and Education Committee, Association for Molecular Pathology, Rockville, Maryland; Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmoldx.2021.07.001DOI Listing
October 2021

Undifferentiated Embryonal Sarcoma of the Liver With Rhabdoid Morphology Mimicking Carcinoma: Expanding the Morphologic Spectrum or a Distinct Variant?

Pediatr Dev Pathol 2021 Nov-Dec;24(6):564-569. Epub 2021 Jun 14.

Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

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 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.
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http://dx.doi.org/10.1177/10935266211018930DOI Listing
January 2022

Natural History of Thyroid Disease in Children with PTEN Hamartoma Tumor Syndrome.

J Clin Endocrinol Metab 2021 03;106(3):e1121-e1130

Thyroid Center, Boston Children's Hospital, Boston, MA, USA.

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.
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http://dx.doi.org/10.1210/clinem/dgaa944DOI Listing
March 2021

A case of metastatic adenocarcinoma of unknown primary in a pediatric patient: Opportunities for precision medicine.

Pediatr Blood Cancer 2021 04 12;68(4):e28780. Epub 2020 Dec 12.

Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Dana-Farber Cancer Institute, Boston, Massachusetts.

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http://dx.doi.org/10.1002/pbc.28780DOI Listing
April 2021

Clinical Pan-Cancer Assessment of Mismatch Repair Deficiency Using Tumor-Only, Targeted Next-Generation Sequencing.

JCO Precis Oncol 2020 Nov;4:1084-1097

Informatics and Analytics Department, Dana-Farber Cancer Institute, Boston, MA.

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.
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http://dx.doi.org/10.1200/PO.20.00185DOI Listing
November 2020

New molecular insights into the pathogenesis of lipoblastomas: clinicopathologic, immunohistochemical, and molecular analysis in pediatric cases.

Hum Pathol 2020 10 18;104:30-41. Epub 2020 Jul 18.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA; Division of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.humpath.2020.07.016DOI Listing
October 2020

Making the most of small samples: Optimization of tissue allocation of pediatric solid tumors for clinical and research use.

Pediatr Blood Cancer 2020 09 15;67(9):e28326. Epub 2020 Jul 15.

Department of Pathology, Boston Children's Hospital, Boston, Massachusetts.

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.
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http://dx.doi.org/10.1002/pbc.28326DOI Listing
September 2020

DICER1-associated central nervous system sarcoma in children: comprehensive clinicopathologic and genetic analysis of a newly described rare tumor.

Mod Pathol 2020 10 14;33(10):1910-1921. Epub 2020 Apr 14.

Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

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.
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http://dx.doi.org/10.1038/s41379-020-0516-1DOI Listing
October 2020

Genomic and Immunologic Characterization of INI1-Deficient Pediatric Cancers.

Clin Cancer Res 2020 06 2;26(12):2882-2890. Epub 2020 Mar 2.

Department of Pediatric Hematology/Oncology, Dana-Farber/Boston Children's Cancer and Blood Disorders Center and Harvard Medical School, Boston, Massachusetts.

Purpose: Several aggressive pediatric cancers harbor alterations in , including rhabdoid tumors, epithelioid sarcoma, and chordoma. As tumor profiling has become more routine in clinical care, we investigated the relationship between 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 genomic alterations on prior somatic sequencing.

Results: two-copy deletions and inactivating mutations on NGS were associated with loss of INI1 protein expression. Single-copy deletion of 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 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.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3089DOI Listing
June 2020

PD-1 and PD-L1 Expression in Osteosarcoma: Which Specimen to Evaluate?

J Pediatr Hematol Oncol 2020 11;42(8):482-487

Department of Pathology, Boston Children's Hospital.

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.
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http://dx.doi.org/10.1097/MPH.0000000000001685DOI Listing
November 2020

Recurrent and novel USP6 fusions in cranial fasciitis identified by targeted RNA sequencing.

Mod Pathol 2020 05 11;33(5):775-780. Epub 2019 Dec 11.

Department of Pathology, Boston Children's Hospital, Boston, MA, USA.

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.
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http://dx.doi.org/10.1038/s41379-019-0422-6DOI Listing
May 2020

A Distinctive Genomic and Immunohistochemical Profile for NOTCH3 and PDGFRB in Myofibroma With Diagnostic and Therapeutic Implications.

Int J Surg Pathol 2020 Apr 29;28(2):128-137. Epub 2019 Sep 29.

Boston Children's Hospital, Boston, MA, USA.

. 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 . The relationship of myofibroma and PDGFRB to the NOTCH pathway has not yet been described. . 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). . Alterations identified by next-generation sequencing included sequence variants in 8/10 cases (80%), a variant in 1/10 cases (10%), and a 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 and . 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. . Our findings suggest a common pathway of PDGFRB/NOTCH3 activation in myofibromas, even in cases that lack 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.
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http://dx.doi.org/10.1177/1066896919876703DOI Listing
April 2020

Renal medullary carcinomas depend upon loss and are sensitive to proteasome inhibition.

Elife 2019 03 12;8. Epub 2019 Mar 12.

Broad Institute of Harvard and MIT, Cambridge, United States.

Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor loss, which also require expression of the E2 ubiquitin-conjugating enzyme, . Our studies identify a synthetic lethal relationship between -deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.
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http://dx.doi.org/10.7554/eLife.44161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436895PMC
March 2019

A somatic activating NRAS variant associated with kaposiform lymphangiomatosis.

Genet Med 2019 07 13;21(7):1517-1524. Epub 2018 Dec 13.

Departments of Pathology & Laboratory Medicine and Medical Genetics, Alberta Children's Hospital Research Institute and Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.

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 polymerase chain reaction (dPCR) to validate the exome findings and to screen KLA samples from six other individuals.

Results: We identified a somatic activating NRAS variant (c.182 A>G, p.Q61R) in lesional tissue from 10/11 individuals, at levels ranging from 1% to 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 signaling pathways. This discovery will expand treatment options for these high-risk patients as there is potential for use of targeted RAS pathway inhibitors.
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http://dx.doi.org/10.1038/s41436-018-0390-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565516PMC
July 2019

A Combination CDK4/6 and IGF1R Inhibitor Strategy for Ewing Sarcoma.

Clin Cancer Res 2019 02 5;25(4):1343-1357. Epub 2018 Nov 5.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children's Hospital, Boston, Massachusetts.

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 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 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.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498855PMC
February 2019

Recurrent EML4-NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy.

Mod Pathol 2018 03 3;31(3):463-473. Epub 2017 Nov 3.

Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA.

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.
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http://dx.doi.org/10.1038/modpathol.2017.127DOI Listing
March 2018

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study.

Genet Med 2017 07 26;19(7):787-795. Epub 2017 Jan 26.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

Purpose: 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.

Methods: 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.

Results: 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.

Conclusion: 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.
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http://dx.doi.org/10.1038/gim.2016.191DOI Listing
July 2017

Clinical targeted exome-based sequencing in combination with genome-wide copy number profiling: precision medicine analysis of 203 pediatric brain tumors.

Neuro Oncol 2017 Jul;19(7):986-996

Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Department of Pathology, Department of Radiology, Department of Neurosurgery, Boston Children's Hospital, Boston, Massachusetts; Department of Medical Oncology, Oncologic Pathology, Department of Pediatric Oncology, Department of Cancer Biology, Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, Massachusetts; Department of Pathology, Department of Neurosurgery, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts; Pratiti Bandopadhayay, Broad Institute of MIT and Harvard, Boston, Massachusetts; Harvard Medical School, Boston, Massachusetts.

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.
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http://dx.doi.org/10.1093/neuonc/now294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570190PMC
July 2017

Integrated genetic and pharmacologic interrogation of rare cancers.

Nat Commun 2016 06 22;7:11987. Epub 2016 Jun 22.

Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, Massachusetts 02142, USA.

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.
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http://dx.doi.org/10.1038/ncomms11987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917959PMC
June 2016

Characterization of a novel fusion gene EML4-NTRK3 in a case of recurrent congenital fibrosarcoma.

Cold Spring Harb Mol Case Stud 2015 Oct;1(1):a000471

Columbia University Medical Center, Department of Pediatric Oncology/Hematology/Stem Cell Transplantation, Department of Pediatrics, New York, New York 10032, USA;

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.
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http://dx.doi.org/10.1101/mcs.a000471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850889PMC
October 2015

MicroRNA signature obtained from the comparison of aggressive with indolent non-Hodgkin lymphomas: potential prognostic value in mantle-cell lymphoma.

J Clin Oncol 2013 Aug 8;31(23):2903-11. Epub 2013 Jul 8.

Ontario Cancer Institute, University Health Network.

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.
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http://dx.doi.org/10.1200/JCO.2012.45.3050DOI Listing
August 2013

Spreading depression expands traumatic injury in neocortical brain slices.

J Neurotrauma 2005 Feb;22(2):277-90

Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada.

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.
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http://dx.doi.org/10.1089/neu.2005.22.277DOI Listing
February 2005
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