Publications by authors named "Frank C Kuo"

44 Publications

A Germinal Center-Associated Microenvironmental Signature Reflects Malignant Phenotype and Outcome of DLBCL.

Blood Adv 2021 Oct 12. Epub 2021 Oct 12.

Kyushu University Hospital, Japan.

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy with varying prognosis after the gold standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Several prognostic models have been established by focusing primarily on characteristics of lymphoma cells themselves, including cell-of-origin, genomic alterations, and gene/protein expressions. However, the prognostic impact of the lymphoma microenvironment and its association with characteristics of lymphoma cells are not fully understood. Using the nCounter-based gene expression profiling of untreated DLBCL tissues, we here assess the clinical impact of lymphoma microenvironment on the clinical outcomes and pathophysiological, molecular signatures in DLBCL. The presence of normal germinal center (GC)-microenvironmental cells, including follicular T cells, macrophage/dendritic cells, and stromal cells, in lymphoma tissue indicates a positive therapeutic response. Our prognostic model, based on quantitation of transcripts from distinct GC-microenvironmental cell markers, clearly identified patients with graded prognosis independently of existing prognostic models. We observed increased incidences of genomic alterations and aberrant gene expression associated with poor prognosis in DLBCL tissues lacking GC-microenvironmental cells relative to those containing these cells. These data suggest that the loss of GC-associated microenvironmental signature dictates clinical outcomes of DLBCL patients reflecting the accumulation of "unfavorable" molecular signatures.
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http://dx.doi.org/10.1182/bloodadvances.2021004618DOI Listing
October 2021

Clinical and molecular characterization of virus-positive and virus-negative Merkel cell carcinoma.

Genome Med 2020 03 18;12(1):30. Epub 2020 Mar 18.

Merkel Cell Carcinoma Center of Excellence, Dana-Farber/Brigham Cancer Center, Boston, MA, USA.

Background: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC.

Methods: In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure.

Results: Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival.

Conclusions: These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.
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http://dx.doi.org/10.1186/s13073-020-00727-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081548PMC
March 2020

Next-generation sequencing informs diagnosis and identifies unexpected therapeutic targets in lung squamous cell carcinomas.

Lung Cancer 2020 02 16;140:35-41. Epub 2019 Dec 16.

Department of Pathology, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115, USA.

Objectives: Potentially targetable genomic alterations have been identified in lung squamous cell carcinoma (LUSC), but none have yet translated into effective therapy. We examined potential benefits of next generation sequencing (NGS) in a cohort of consecutive LUSC patients with emphasis on distinctions between smokers and light/never smokers and implications for clinical trial enrollment.

Methods: We retrospectively evaluated results from an internally developed NGS assay (OncoPanel) targeting ∼300 genes with a mean overall target coverage of >200x for consecutive LUSC seen at our institution over 30 months.

Results: Tissue was obtained from 172 patients for targeted NGS. 42 (24 %) samples were insufficient for testing. Median age of tested patients was 66, including 87 % moderate/heavy versus 13 % light/never smokers; 66 % were stage IIIB or IV. Of 130 patients with evaluable NGS results, 49 (38 %) had at least 1 alteration qualifying for enrollment to a LungMAP treatment arm (PIK3CA, MET, FGFR family, cell cycle, or homologous recombination pathways) or for an approved therapy or other clinical trial (e.g. EGFR sensitizing mutations, MET exon 14 splice mutations, TSC1/2 mutation, or microsatellite instability). Therapeutic targets were enriched in light/never smokers (47 % vs 35 % moderate/heavy smokers). Unexpectedly, genomic features suggested an alternative diagnosis (metastatic cutaneous squamous carcinoma; mesothelioma) in 7 patients, including 35 % of never/light smokers.

Conclusion: NGS in a real-world LUSC cohort yields potentially targetable genomic alterations informing clinical trial enrollment and approved therapies and critical diagnostic insights. Our findings strongly support current guidelines recommending mutational profiling of LUSC arising in light/never smoking patients; the utility of sequencing in smokers with LUSC appears to be limited to identification of research targets.
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http://dx.doi.org/10.1016/j.lungcan.2019.12.005DOI Listing
February 2020

Alisertib plus induction chemotherapy in previously untreated patients with high-risk, acute myeloid leukaemia: a single-arm, phase 2 trial.

Lancet Haematol 2020 Feb 11;7(2):e122-e133. Epub 2019 Dec 11.

Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Background: Increased aurora A kinase (AAK) expression occurs in acute myeloid leukaemia; AAK inhibition is a promising therapeutic target in this disease. We therefore aimed to assess the activity of alisertib combined with 7 + 3 induction chemotherapy in previously untreated patients with high-risk acute myeloid leukaemia.

Methods: We did a single-arm, phase 2 trial of patients recruited from the Dana-Farber/Harvard Cancer Center in the USA. Eligible patients had previously untreated acute myeloid leukaemia, an Eastern Cooperative Oncology Group performance status of 0-2, and were at high risk of disease as defined by the presence of an adverse-risk karyotype, the presence of secondary acute myeloid leukaemia arising from previous myelodysplastic syndrome or myeloproliferative neoplasm, the presence of therapy-related acute myeloid leukaemia, or being 65 years or older. Enrolled patients received 7 + 3 induction chemotherapy of continuous infusion of cytarabine (100 mg/m per day on days 1-7) and intravenous bolus of idarubicin (12 mg/m per day on days 1-3). Oral alisertib (30 mg) was given twice per day on days 8-15. Patients could receive up to four consolidation cycles with cytarabine and alisertib, and alisertib maintenance for 12 months. The primary endpoint was a composite including the proportion of patients achieving complete remission and those with a complete remission with incomplete neutrophil or platelet count recovery. Analyses were per-protocol. This study is registered with Clinicaltrials.gov, number NCT02560025, and has completed enrolment.

Findings: Between Dec 31, 2015, and Aug 1, 2017, we enrolled a total of 39 eligible patients. 19 (49%) of 39 patients had secondary acute myeloid leukaemia and three (8%) had therapy-related acute myeloid leukaemia. At mid-induction, 33 (85%) of 39 patients showed marrow aplasia, six (15%) received re-induction. The median follow-up was 13·7 months (IQR 12·7-14·4). Composite remission was 64% (two-stage 95% CI 48-79), with 20 (51%) of 39 patients achieving complete remission and five (13%) achieving complete remission with incomplete neutrophil or platelet count recovery. The most common grade 3 or 4 adverse events included febrile neutropenia (16 [41%] of 39), neutropenia (12 [31%]), thrombocytopenia (13 [33%]), anaemia (11 [28%]), anorexia (nine [23%]), and oral mucositis (four [10%]). No treatment-related deaths were observed.

Interpretation: These results suggest that alisertib combined with induction chemotherapy is active and safe in previously untreated patients with high-risk acute myeloid leukaemia. This study met criteria to move forward to a future randomised trial.

Funding: Millennium Pharmaceuticals.
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http://dx.doi.org/10.1016/S2352-3026(19)30203-0DOI Listing
February 2020

Premalignant Clonal Hematopoietic Proliferations.

Am J Clin Pathol 2019 08;152(3):347-358

Department of Pathology, Massachusetts General Hospital, Boston.

Objectives: The 2017 Workshop of the Society for Hematopathology/European Association for Hematopathology aimed to review premalignant clonal hematopoietic proliferations.

Methods: The workshop panel reviewed 27 cases of clonal proliferations of indeterminate significance or potential (18 myeloid, nine lymphoid) and rendered consensus diagnoses.

Results: Immunophenotyping and genetic studies on peripheral blood, bone marrow, and lymph node samples have led to the incidental detection of small clonal populations in asymptomatic individuals. These premalignant clonal myeloid and lymphoid proliferations include monoclonal gammopathy of uncertain significance, monoclonal B-cell lymphocytosis, in situ follicular neoplasia, in situ mantle cell neoplasia, clonal hematopoiesis of indeterminate potential, and clonal cytopenia of undetermined significance.

Conclusions: Current diagnostic criteria for the diagnoses of premalignant clonal hematopoietic proliferations are reviewed and discussed in the context of the cases presented at the workshop.
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http://dx.doi.org/10.1093/ajcp/aqz079DOI Listing
August 2019

Next generation sequencing in hematolymphoid neoplasia.

Authors:
Frank C Kuo

Semin Hematol 2019 01 27;56(1):2-6. Epub 2018 May 27.

Director of Clinical Cancer Genomics and Molecular Pathology, Department of Pathology, UCLA Medical Center, Los Angeles, CA. Electronic address:

Large scale sequencing projects over the past 2 decades have led to the identification of many common genomic alterations in hematolymphoid neoplasms, some of which with diagnostic, therapeutic, and prognostic implications [1-3]. Although these alterations can be tested individually with high sensitivity and specificity using dedicated single gene tests, it is increasingly impractical and costly to test them separately as the number of alterations grows. Instead, multiplex testing platforms that can test multiple targets in a specimen have been developed. Among these platforms, massively parallel sequencing technologies (so-called next-generation sequencing [NGS] [4]) prove to be most versatile and is increasingly being used to build tests to meet the clinical testing need. In hematolymphoid neoplasms, the early incorporation of molecular findings into the diagnostic criteria by WHO [5] has further accelerated the adoption of NGS-based tests in routine clinical practice. This article focuses on what is used in the clinical diagnostic laboratories today and is not intended to be a review of the NGS technology or its future direction. Following discussion of the 2 families of sequencing instruments from Illumina and Thermo Fisher and 3 target enrichment methods, aspects of the analysis and report of NGS results that is clinically relevant are discussed.
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http://dx.doi.org/10.1053/j.seminhematol.2018.05.006DOI Listing
January 2019

High -mutant allele burden at diagnosis predicts unfavorable outcomes in de novo AML.

Blood 2018 06 3;131(25):2816-2825. Epub 2018 May 3.

Department of Pathology, Brigham and Women's Hospital, Boston, MA.

Acute myeloid leukemia (AML) with mutated is a newly recognized separate entity in the revised 2016 World Health Organization classification and is associated with a favorable prognosis. Although previous studies have evaluated in a binary fashion, little is known about the significance of its mutant allele burden at diagnosis, nor has the effect of comutations (other than ) been extensively evaluated. We retrospectively used targeted sequencing data from 109 patients with de novo AML with mutated to evaluate the potential significance of variant allele frequency (VAF), comutations, and clinical parameters with regard to patient outcomes. We observed that high VAF (uppermost quartile) correlated with shortened overall survival (median, 12.1 months vs not reached; < .0001) as well as event-free survival (median, 7.5 vs 65.44 months; < .0001) compared with the other -mutated cases. In both univariate and multivariable analyses, high VAF had a particularly adverse prognostic effect in the subset of patients treated with stem-cell transplantation in first remission ( = .0004) and in patients with mutated ( < .0001). Our findings indicate that the prognostic effect of mutation in de novo AML may be influenced by the relative abundance of the mutated allele.
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http://dx.doi.org/10.1182/blood-2018-01-828467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265642PMC
June 2018

Frameshift events predict anti-PD-1/L1 response in head and neck cancer.

JCI Insight 2018 02 22;3(4). Epub 2018 Feb 22.

Department of Medical Oncology, and.

Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti-PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti-PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti-PD-1/L1 response.
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http://dx.doi.org/10.1172/jci.insight.98811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916245PMC
February 2018

A distinct immunophenotype identifies a subset of NPM1-mutated AML with TET2 or IDH1/2 mutations and improved outcome.

Am J Hematol 2018 08 25;93(4):504-510. Epub 2018 Jan 25.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Recent work has identified distinct molecular subgroups of acute myeloid leukemia (AML) with implications for disease classification and prognosis. NPM1 is one of the most common recurrently mutated genes in AML. NPM1 mutations often co-occur with FLT3-ITDs and mutations in genes regulating DNA methylation, such as DNMT3A, TET2, and IDH1/2. It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis. We identified 133 cases of NPM1-mutated AML and correlated sequencing data with immunophenotypic and clinical findings. Of 84 cases (63%) that lacked monocytic differentiation ("myeloid AML"), 40 (48%) demonstrated an acute promyelocytic leukemia-like (APL-like) immunophenotype by flow cytometry, with absence of CD34 and HLA-DR and strong myeloperoxidase expression, in the absence of a PML-RARA translocation. Pathologic variants in TET2, IDH1, or IDH2 were identified in 39/40 APL-like cases. This subset of NPM1-mutated AML was associated with longer relapse-free and overall survival, when compared with cases that were positive for CD34 and/or HLA-DR. The combination of NPM1 and TET2 or IDH1/2 mutations along with an APL-like immunophenotype identifies a distinct subtype of AML. Further studies addressing its biology and clinical significance may be especially relevant in the era of IDH inhibitors and recent work showing efficacy of ATRA therapy in NPM1 and IDH1-mutated AML.
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http://dx.doi.org/10.1002/ajh.25018DOI Listing
August 2018

The relative utilities of genome-wide, gene panel, and individual gene sequencing in clinical practice.

Blood 2017 07 9;130(4):433-439. Epub 2017 Jun 9.

Department of Pathology, Brigham and Women's Hospital, Boston, MA.

Advances in technology that have transpired over the past 2 decades have enabled the analysis of cancer samples for genomic alterations to understand their biologic function and to translate that knowledge into clinical practice. With the power to analyze entire genomes in a clinically relevant time frame and with manageable costs comes the question of whether we ought to and when. This review focuses on the relative merits of 3 approaches to molecular diagnostics in hematologic malignancies: indication-specific single gene assays, gene panel assays that test for genes selected for their roles in cancer, and genome-wide assays that broadly analyze the tumor exomes or genomes. After addressing these in general terms, we review specific use cases in myeloid and lymphoid malignancies to highlight the utility of single gene testing and/or larger panels.
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http://dx.doi.org/10.1182/blood-2017-03-734533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813726PMC
July 2017

Validation of OncoPanel: A Targeted Next-Generation Sequencing Assay for the Detection of Somatic Variants in Cancer.

Arch Pathol Lab Med 2017 Jun 3;141(6):751-758. Epub 2017 Mar 3.

From the Center for Advanced Molecular Diagnostics, Brigham and Women's Hospital (Drs Garcia, Minkovsky, Jia, Ligon, Sholl, Kuo, MacConaill, Lindeman, and Dong, Messrs Ducar and Gong, and Ms. Shivdasani), and the Center for Cancer Genome Discovery, Dana Farber Cancer Institute (Mr Ducar and Dr MacConaill), Harvard Medical School, Boston, Massachusetts.

Context: - The analysis of somatic mutations across multiple genes in cancer specimens may be used to aid clinical decision making. The analytical validation of targeted next-generation sequencing panels is important to assess accuracy and limitations.

Objective: - To report the development and validation of OncoPanel, a custom targeted next-generation sequencing assay for cancer.

Design: - OncoPanel was designed for the detection of single-nucleotide variants, insertions and deletions, copy number alterations, and structural variants across 282 genes with evidence as drivers of cancer biology. We implemented a validation strategy using formalin-fixed, paraffin-embedded, fresh or frozen samples compared with results obtained by clinically validated orthogonal technologies.

Results: - OncoPanel achieved 98% sensitivity and 100% specificity for the detection of single-nucleotide variants, and 84% sensitivity and 100% specificity for the detection of insertions and deletions compared with single-gene assays and mass spectrometry-based genotyping. Copy number detection achieved 86% sensitivity and 98% specificity compared with array comparative genomic hybridization. The sensitivity of structural variant detection was 74% compared with karyotype, fluorescence in situ hybridization, and polymerase chain reaction. Sensitivity was affected by inconsistency in the detection of FLT3 and NPM1 alterations and IGH rearrangements due to design limitations. Limit of detection studies demonstrated 98.4% concordance across triplicate runs for variants with allele fraction greater than 0.1 and at least 50× coverage.

Conclusions: - The analytical validation of OncoPanel demonstrates the ability of targeted next-generation sequencing to detect multiple types of genetic alterations across a panel of genes implicated in cancer biology.
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http://dx.doi.org/10.5858/arpa.2016-0527-OADOI Listing
June 2017

Determination of VH Family Usage in B-Cell Malignancies via the BIOMED-2 IGH PCR Clonality Assay.

Am J Clin Pathol 2017 Jun;147(6):549-556

From the Department of Pathology, Center for Advanced Molecular Diagnostics, Brigham and Women's Hospital, Boston, MA.

Objectives: To determine whether V H family usage in B-cell lymphoproliferative disorders can be deduced from polymerase chain reaction (PCR) product-length information obtained through the BIOMED-2 (Invivoscribe, San Diego, CA) clonality assay.

Methods: We develop an algorithm that uses the sizing information of the BIOMED-2 immunoglobulin heavy chain (IGH) clonality assay to deduce V H family usage. PCR with family-specific primers on 51 clinical samples containing 54 rearranged alleles were used to validate the algorithm.

Results: The clonal PCR products in different framework reactions contain the same NDN segment (because they are from the same allele). Subtracting the size of the framework III product from the size of the framework I and II products yields the relative position of the framework primer binding sites for the V H segment used. The V H family can be assigned with these relative positions because they are V H family specific in the BIOMED-2 assay. The V H family assigned by the algorithm was concordant with family-specific PCR results for 49 (96%) of the 51 specimens.

Conclusions: We have developed an algorithm that can correctly assign V H family usage when all three BIOMED-2 framework reactions produced clonal products. Given the wide adoption of BIOMED-2 assay, the algorithm can facilitate collection of IGH V H usage data without additional cost to the laboratories.
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http://dx.doi.org/10.1093/ajcp/aqx007DOI Listing
June 2017

Molecular Analysis of Genetic Markers for Non-Hodgkin Lymphomas.

Curr Protoc Hum Genet 2017 04 6;93:10.14.1-10.14.29. Epub 2017 Apr 6.

Brigham and Women's Hospital, Boston, Massachusetts.

Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen-receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to be used in identifying concurrent disease at disparate sites or recurrence at future time points. Certain lymphoid malignancies harbor a characteristic chromosomal translocation, a finding that may have significant implications for an individual's prognosis or response to therapy. The polymerase chain reaction (PCR) is typically used to detect antigen-receptor gene rearrangements as well as specific translocations that can be supplemented by fluorescence in situ hybridization (FISH) and karyotype analysis. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cphg.37DOI Listing
April 2017

Clinical and Technical Aspects of Genomic Diagnostics for Precision Oncology.

J Clin Oncol 2017 Mar 13;35(9):929-933. Epub 2017 Feb 13.

Yuri Sheikine, Beth Israel Deaconess Medical Center, Harvard Medical School; and Frank C. Kuo and Neal I. Lindeman, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.

The emergence of precision medicine has been predicated on significant recent advances in diagnostic technology, particularly the advent of next-generation sequencing (NGS). Although the chemical technology underlying NGS is complex, and the computational biology expertise required to build systems to facilely interpret the results is highly specialized, the variables involved in designing and deploying a genomic testing program for cancer can be readily understood and applied by understanding several basic considerations. In this review, we present key strategic decisions required to optimize a genomic testing program and summarize the technical aspects of different technologies that render those methods more or less suitable for different types of programs.
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http://dx.doi.org/10.1200/JCO.2016.70.7539DOI Listing
March 2017

Molecular Analysis of Gene Rearrangements and Mutations in Acute Leukemias and Myeloid Neoplasms.

Curr Protoc Hum Genet 2017 01 11;92:10.4.1-10.4.49. Epub 2017 Jan 11.

Brigham and Women's Hospital, Boston, Massachusetts.

A subset of acute leukemias and other myeloid neoplasms contains specific genetic alterations, many of which are associated with unique clinical and pathologic features. These alterations include chromosomal rearrangements leading to oncogenic fusion proteins or alteration of gene expression by juxtaposing oncogenes to enhancer elements, as well as mutations leading to aberrant activation of a variety of proteins critical to hematopoietic progenitor cell proliferation and differentiation. Molecular analysis is central to diagnosis and clinical management of leukemias, permitting genetic confirmation of a clinical and histologic impression, providing prognostic and predictive information, and facilitating detection of minimal residual disease. This unit will outline approaches to the molecular diagnosis of the most frequent and clinically relevant genetic alterations in acute leukemias and myeloid neoplasms. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cphg.31DOI Listing
January 2017

Conventional cytogenetics for myeloid neoplasms in the era of next-generation-sequencing.

Am J Hematol 2017 03;92(3):227-229

Brigham and Women's Hospital, Boston, Massachusetts, USA.

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http://dx.doi.org/10.1002/ajh.24642DOI Listing
March 2017

Case 37-2016. An 86-Year-Old Woman with Leukocytosis and Splenomegaly.

N Engl J Med 2016 12;375(23):2273-2282

From the Departments of Medicine (A.T.F., T.A.G.), Radiology (N.M.K.), and Pathology (R.P.H.), Massachusetts General Hospital, the Department of Pathology, Brigham and Women's Hospital (F.C.K.), and the Departments of Medicine (A.T.F., T.A.G.), Radiology (N.M.K.), and Pathology (F.C.K., R.P.H.), Harvard Medical School - all in Boston.

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http://dx.doi.org/10.1056/NEJMcpc1509539DOI Listing
December 2016

Institutional implementation of clinical tumor profiling on an unselected cancer population.

JCI Insight 2016 11 17;1(19):e87062. Epub 2016 Nov 17.

Mirati Therapeutics, San Diego, California.

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. 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. 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 and , 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. 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. This work was supported by DFCI, BWH, and the National Cancer Institute (5R33CA155554 and 5K23CA157631).
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http://dx.doi.org/10.1172/jci.insight.87062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111542PMC
November 2016

Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing.

J Mol Diagn 2017 01 15;19(1):84-91. Epub 2016 Nov 15.

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

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome-associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.
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http://dx.doi.org/10.1016/j.jmoldx.2016.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225299PMC
January 2017

Molecular Pathology: Prognostic and Diagnostic Genomic Markers for Myeloid Neoplasms.

Authors:
Frank C Kuo

Surg Pathol Clin 2016 Sep;9(3):475-88

Center for Advanced Molecular Diagnostics, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA. Electronic address:

Application of next-generation sequencing (NGS) on myeloid neoplasms has expanded our knowledge of genomic alterations in this group of diseases. Genomic alterations in myeloid neoplasms are complex, heterogeneous, and not specific to a disease entity. NGS-based panel testing of myeloid neoplasms can complement existing diagnostic modalities and is gaining acceptance in the clinics and diagnostic laboratories. Prospective, randomized trials to evaluate the prognostic significance of genomic markers in myeloid neoplasms are under way in academic medical centers.
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http://dx.doi.org/10.1016/j.path.2016.04.010DOI Listing
September 2016

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies.

J Mol Diagn 2016 07;18(4):507-15

Center for Advanced Molecular Diagnostics, Brigham and Women's Hospital, Boston, Massachusetts. Electronic address:

Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total. Rapid heme panel's average coverage is 1500× with <5% of the amplicons with <50× coverage, and it reproducibly detects single nucleotide variants and small insertions/deletions at allele frequencies of ≥5%. Comparison with a capture-based next-generation sequencing assay showed that there is >95% concordance among a wide array of variants across a range of allele frequencies. Read count analyses that used rapid heme panel showed high concordance with karyotypic results when tumor content was >30%. The average turnaround time was 7 days over a 6-month span with an average volume of ≥40 specimens per week and a low sample fail rate (<1%), demonstrating its suitability for clinical application.
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http://dx.doi.org/10.1016/j.jmoldx.2016.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707186PMC
July 2016

Complete hematologic response of early T-cell progenitor acute lymphoblastic leukemia to the γ-secretase inhibitor BMS-906024: genetic and epigenetic findings in an outlier case.

Cold Spring Harb Mol Case Stud 2015 Oct;1(1):a000539

Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA;

Notch pathway antagonists such as γ-secretase inhibitors (GSIs) are being tested in diverse cancers, but exceptional responses have yet to be reported. We describe the case of a patient with relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) who achieved a complete hematologic response following treatment with the GSI BMS-906024. Whole-exome sequencing of leukemic blasts revealed heterozygous gain-of-function driver mutations in NOTCH1, CSF3R, and PTPN11, and a homozygous/hemizygous loss-of-function mutation in DNMT3A. The three gain-of-function mutations were absent from remission marrow cells, but the DNMT3A mutation persisted in heterozygous form in remission marrow, consistent with an origin for the patient's ETP-ALL from clonal hematopoiesis. Ex vivo culture of ETP-ALL blasts confirmed high levels of activated NOTCH1 that were repressed by GSI treatment, and RNA-seq documented that GSIs downregulated multiple known Notch target genes. Surprisingly, one potential target gene that was unaffected by GSIs was MYC, a key Notch target in GSI-sensitive T-ALL of cortical T-cell type. H3K27ac super-enhancer landscapes near MYC showed a pattern previously reported in acute myeloid leukemia (AML) that is sensitive to BRD4 inhibitors, and in line with this ETP-ALL blasts downregulated MYC in response to the BRD4 inhibitor JQ1. To our knowledge, this is the first example of complete response of a Notch-mutated ETP-ALL to a Notch antagonist and is also the first description of chromatin landscapes associated with ETP-ALL. Our experience suggests that additional attempts to target Notch in Notch-mutated ETP-ALL are merited.
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http://dx.doi.org/10.1101/mcs.a000539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850884PMC
October 2015

Myeloid neoplasm demonstrating a STAT5B-RARA rearrangement and genetic alterations associated with all-trans retinoic acid resistance identified by a custom next-generation sequencing assay.

Cold Spring Harb Mol Case Stud 2015 Oct;1(1):a000307

Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA;

We describe the case of a patient presenting with several weeks of symptoms related to pancytopenia associated with a maturation arrest at the late promyelocyte/early myelocyte stage of granulocyte differentiation. A diagnosis of acute promyelocytic leukemia was considered, but the morphologic features were atypical for this entity and conventional tests for the presence of a PML-RARA fusion gene were negative. Additional analysis using a custom next-generation sequencing assay revealed a rearrangement producing a STAT5B-RARA fusion gene, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and supplementary cytogenetic studies, allowing the diagnosis of a morphologically atypical form of acute promyelocytic leukemia to be made. Analysis of the sequencing data permitted characterization of both chromosomal breakpoints and revealed two additional alterations, a small deletion in RARA exon 9 and a RARA R276W substitution, that have been linked to resistance to all-trans retinoic acid. This case highlights how next-generation sequencing can augment currently standard testing to establish diagnoses in difficult cases, and in doing so help guide selection of therapy.
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http://dx.doi.org/10.1101/mcs.a000307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850893PMC
October 2015

KRAS and NKX2-1 Mutations in Invasive Mucinous Adenocarcinoma of the Lung.

J Thorac Oncol 2016 Apr 30;11(4):496-503. Epub 2016 Jan 30.

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

Introduction: Mucinous differentiation is observed in a subset of lung adenocarcinomas with unique clinical and pathological features, but the biology of these neoplasms is poorly understood.

Methods: We apply targeted next-generation sequencing to characterize the mutational profiles of 21 invasive mucinous adenocarcinomas, mixed mucinous/nonmucinous adenocarcinomas, and adenocarcinomas with mucinous features of the lung and validate key findings on 954 additional lung adenocarcinomas from our institution and 514 lung adenocarcinomas from The Cancer Genome Atlas.

Results: Sequencing identifies pathogenic mutations in the oncogenes Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), erb-b2 receptor tyrosine kinase 2 (ERBB2), and anaplastic lymphoma receptor tyrosine kinase (ALK) and recurrent mutations in tumor protein p53 (TP53), serine/threonine kinase 11 (STK11), NK2 homeobox 1 (NKX2-1), and SET domain containing 2 (SETD2). In the combined discovery and validation cohorts, we identify nine neoplasms with distinct molecular and pathological features. All are invasive mucinous adenocarcinomas or mixed mucinous/nonmucinous adenocarcinomas with mutations of KRAS and frameshift or nonsense mutations of NKX2-1. Immunohistochemical analysis shows that these neoplasms are associated with altered differentiation states, including loss of expression of the pulmonary marker thyroid transcription factor 1 (also called Nkx2.1) and expression of gastrointestinal markers.

Conclusions: These findings describe recurrent NKX2-1 mutations in invasive mucinous adenocarcinomas of the lung and support NKX2-1 as a lineage-specific tumor suppressor gene in lung carcinogenesis.
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http://dx.doi.org/10.1016/j.jtho.2016.01.010DOI Listing
April 2016

Next-generation sequencing-based panel testing for myeloid neoplasms.

Curr Hematol Malig Rep 2015 Jun;10(2):104-11

Center for Advanced Molecular Diagnostics, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA, 02115, USA,

Our ability to interrogate a broad array of genetic alterations in myeloid neoplasm has increased significantly with the advance in next-generation sequencing (NGS). In addition to morphologic examination, flow cytometry, and cytogenetics, NGS-based testing can add additional information to the diagnostic workup. More than a dozen myeloid-focused NGS-based panels are now available from commercial and academic laboratories. In this review, we examine the content of these panels in the context of our current understanding of driver alterations in myeloid neoplasms. With improved turnaround time, decreasing costs, and an expanding knowledge of the therapeutic and prognostic significance of the detected variants, NGS-based panel testing is likely to play a major role in the management of patients with myeloid neoplasm in the coming decade.
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http://dx.doi.org/10.1007/s11899-015-0256-3DOI Listing
June 2015

Age-related clonal hematopoiesis associated with adverse outcomes.

N Engl J Med 2014 Dec 26;371(26):2488-98. Epub 2014 Nov 26.

The authors' affiliations are listed in the Appendix.

Background: The incidence of hematologic cancers increases with age. These cancers are associated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders.

Methods: We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide variants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events.

Results: Detectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respectively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8).

Conclusions: Age-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the latter possibly due to an increased risk of cardiovascular disease. (Funded by the National Institutes of Health and others.).
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http://dx.doi.org/10.1056/NEJMoa1408617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306669PMC
December 2014

The ongoing evolution of the core curriculum of a clinical fellowship in pathology informatics.

J Pathol Inform 2014 30;5(1):22. Epub 2014 Jul 30.

Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA.

The Partners HealthCare system's Clinical Fellowship in Pathology Informatics (Boston, MA, USA) faces ongoing challenges to the delivery of its core curriculum in the forms of: (1) New classes of fellows annually with new and varying educational needs and increasingly fractured, enterprise-wide commitments; (2) taxing electronic health record (EHR) and laboratory information system (LIS) implementations; and (3) increasing interest in the subspecialty at the academic medical centers (AMCs) in what is a large health care network. In response to these challenges, the fellowship has modified its existing didactic sessions and piloted both a network-wide pathology informatics lecture series and regular "learning laboratories". Didactic sessions, which had previously included more formal discussions of the four divisions of the core curriculum: Information fundamentals, information systems, workflow and process, and governance and management, now focus on group discussions concerning the fellows' ongoing projects, updates on the enterprise-wide EHR and LIS implementations, and directed questions about weekly readings. Lectures are given by the informatics faculty, guest informatics faculty, current and former fellows, and information systems members in the network, and are open to all professional members of the pathology departments at the AMCs. Learning laboratories consist of small-group exercises geared toward a variety of learning styles, and are driven by both the fellows and a member of the informatics faculty. The learning laboratories have created a forum for discussing real-time and real-world pathology informatics matters, and for incorporating awareness of and timely discussions about the latest pathology informatics literature. These changes have diversified the delivery of the fellowship's core curriculum, increased exposure of faculty, fellows and trainees to one another, and more equitably distributed teaching responsibilities among the entirety of the pathology informatics asset in the network. Though the above approach has been in place less than a year, we are presenting it now as a technical note to allow for further discussion of evolving educational opportunities in pathology informatics and clinical informatics in general, and to highlight the importance of having a flexible fellowship with active participation from its fellows.
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http://dx.doi.org/10.4103/2153-3539.137717DOI Listing
September 2014
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