Sophia Yohe

Application of Immunohistochemistry to Soft Tissue Neoplasms

CONTEXT Soft tissue tumors are composed of numerous and complex diagnostic entities. Because of this complexity and the recognition of an intermediate malignancy category including some tumors with a deceptively bland histologic appearance, soft tissue tumors may represent a major diagnostic challenge to the general practicing pathologist. OBJECTIVE To correctly diagnose soft tissue tumors with the ancillary use of immunohistochemistry. DATA SOURCES Review of the current literature with emphasis on those tumors for which immunohistochemistry has proven to be particularly useful. CONCLUSIONS Immunohistochemistry plays an important role in the diagnosis of soft tissue tumors. One of its major utilities is to correctly identify a tumor as being of mesenchymal or nonmesenchymal origin. Once mesenchymal origin has been established, histologic subtyping according to specific cell lineage may be achieved with the use of lineage-specific markers. Tumors of uncertain cell lineage and tumors with primitive small round cell morphology are often characterized by a unique immunohistochemical phenotype. In this group of tumors, immunohistochemistry is most widely applied and is of greatest value. Despite the rapid development of molecular genetic techniques, immunohistochemistry still remains the most important diagnostic tool in the diagnosis of soft tissue tumors aside from recognition of morphologic features and clinical correlation.

EBV-positive mucocutaneous ulcer in organ transplant recipients: a localized indolent posttransplant lymphoproliferative disorder.

Epstein-Barr virus (EBV)-positive mucocutaneous ulcer (EBV MCU) is a B-cell lymphoproliferative disorder occurring in elderly or iatrogenic immunocompromised patients. It has not been reported in solid organ transplant recipients. We observed 7 patients with EBV MCU in a cohort of 70 transplant recipients with EBV+ posttransplant lymphoproliferative disorder (PTLD). Transplants included: 5 renal, 1 heart, and 1 lung. Median patient age was 61; 5 were male. EBV MCU was observed in oral mucosa in 4 and gastrointestinal tract in 3. Duration of immunosuppressive therapy before EBV MCU was 0.6 to 13 years. Ulcers were undermined by inflammatory cells and polymorphic or monomorphic large cell lymphoproliferation. Reed-Sternberg-like cells were present in 5/7. Large B cells were CD20, CD30, and EBV-encoded RNA positive in all cases. Diagnosis in 3 recent patients was EBV MCU; 4 patients diagnosed before familiarity with EBV MCU were classified as monomorphic large cell (n=3) and polymorphic (n=1) PTLD. None of the patients had EBV DNA in their blood (<1000 copies/mL) at diagnosis or follow-up versus 35/44 transplant patients with systemic PTLD (P<0.001). All lesions resolved with reduced immunosuppression (7/7), change in immunosuppression (2/7), and rituximab (3/7). Five patients are living: 4 healthy, 1 awaiting second renal transplant. Two patients died 3 and 5 years after resolution of EBV MCU. No patient recurred with EBV MCU or other PTLDs. EBV MCU mimics more aggressive categories of PTLD but lacks EBV DNA in blood, which may be a useful distinguishing feature. Lesions are likely to resolve with conservative management. Awareness of EBV MCU in the posttransplant setting is necessary for appropriate diagnosis and treatment.

Molecular Genetic Markers in Acute Myeloid Leukemia

Genetics play an increasingly important role in the risk stratification and management of acute myeloid leukemia (AML) patients. Traditionally, AML classification and risk stratification relied on cytogenetic studies; however, molecular detection of gene mutations is playing an increasingly important role in classification, risk stratification, and management of AML. Molecular testing does not take the place of cytogenetic testing results, but plays a complementary role to help refine prognosis, especially within specific AML subgroups. With the exception of acute promyelocytic leukemia, AML therapy is not targeted but the intensity of therapy is driven by the prognostic subgroup. Many prognostic scoring systems classify patients into favorable, poor, or intermediate prognostic subgroups based on clinical and genetic features. Current standard of care combines cytogenetic results with targeted testing for mutations in FLT3, NPM1, CEBPA, and KIT to determine the prognostic subgroup. Other gene mutations have also been demonstrated to predict prognosis and may play a role in future risk stratification, although some of these have not been confirmed in multiple studies or established as standard of care. This paper will review the contribution of cytogenetic results to prognosis in AML and then will focus on molecular mutations that have a prognostic or possible therapeutic impact.

Clinical Validation of Targeted Next-Generation Sequencing for Inherited Disorders

CONTEXT Although next-generation sequencing (NGS) can revolutionize molecular diagnostics, several hurdles remain in the implementation of this technology in clinical laboratories. OBJECTIVES To validate and implement an NGS panel for genetic diagnosis of more than 100 inherited diseases, such as neurologic conditions, congenital hearing loss and eye disorders, developmental disorders, nonmalignant diseases treated by hematopoietic cell transplantation, familial cancers, connective tissue disorders, metabolic disorders, disorders of sexual development, and cardiac disorders. The diagnostic gene panels ranged from 1 to 54 genes with most of panels containing 10 genes or fewer. DESIGN We used a liquid hybridization-based, target-enrichment strategy to enrich 10 067 exons in 568 genes, followed by NGS with a HiSeq 2000 sequencing system (Illumina, San Diego, California). RESULTS We successfully sequenced 97.6% (9825 of 10 067) of the targeted exons to obtain a minimum coverage of 20× at all bases. We demonstrated 100% concordance in detecting 19 pathogenic single-nucleotide variations and 11 pathogenic insertion-deletion mutations ranging in size from 1 to 18 base pairs across 18 samples that were previously characterized by Sanger sequencing. Using 4 pairs of blinded, duplicate samples, we demonstrated a high degree of concordance (>99%) among the blinded, duplicate pairs. CONCLUSIONS We have successfully demonstrated the feasibility of using the NGS platform to multiplex genetic tests for several rare diseases and the use of cloud computing for bioinformatics analysis as a relatively low-cost solution for implementing NGS in clinical laboratories.

Achieving stringent CR is essential before reduced-intensity conditioning allogeneic hematopoietic cell transplantation in AML

Reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (allo-HCT) can cure patients with AML in CR. However, relapse after RIC allo-HCT may indicate heterogeneity in the stringency of CR. Strict definition of CR requires no evidence of leukemia by both morphologic and flow cytometric criteria. We re-evaluated 85 AML patients receiving RIC allo-HCT in CR to test if a strict definition of CR had direct implications for the outcome. These patients had leukemia immunophenotype documented at diagnosis and analyzed at allo-HCT. Eight (9.4%) had persistent leukemia by flow cytometric criteria at allo-HCT. The patients with immunophenotypic persistent leukemia had a significantly increased relapse (hazard ratio (HR): 3.7; 95% confidence interval (CI): 1.3–10.3, P=0.01) and decreased survival (HR: 2.9; 95% CI: 1.3–6.4, P<0.01) versus 77 patients in CR by both morphology and flow cytometry. However, the pre-allo-HCT bone marrow (BM) blast count (that is, 0–4%) was not significantly associated with risks of relapse or survival. These data indicate the presence of leukemic cells, but not the BM blast count affects survival. A strict morphologic and clinical lab flow cytometric definition of CR predicts outcomes after RIC allo-HCT, and therefore is critical to achieve at transplantation.

Unrelated Cord Blood Transplantation in Adult and Pediatric Acute Lymphoblastic Leukemia: Effect of Minimal Residual Disease on Relapse and Survival

Data on pretransplantation minimal residual disease (MRD) and outcomes of umbilical cord blood transplantation (UCBT) are limited. Out of the 143 patients with acute lymphoblastic leukemia (ALL) who underwent UCBT at the University of Minnesota between 2004 and 2010, we evaluated 86 patients with available MRD assessment data by 4- and 8-color flow cytometry analysis immediately before transplantation. Ten patients (11.6%) were MRD-positive, and 76 were MRD-negative (88.4%). Most of the patients (82%) received myeloablative conditioning. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. In multivariate analysis, age, disease status (complete remission [CR] 1 versus CR2/CR3), disease group (precursor B cell ALL versus Philadelphia chromosome-positive ALL versus T cell ALL), and time to transplantation had no impact on relapse. Patients with MRD before UCBT had a greater incidence of relapse at 2 years (relapse rate, 30%; 95% confidence interval [CI], 4%-56%) and lower 3-year disease-free survival (30%; 95% CI, 7%-58%) compared with those without MRD (relapse rate, 16%; 95% CI, 8%-25%; P = .05; disease-free survival, 55%; 95% CI, 43%-66%; P = .02). Our data suggest that in patients with ALL, achieving an MRD-negative state before UCBT improves outcomes.

“Missed” Diagnoses of Phyllodes Tumor on Breast Biopsy: Pathologic Clues to Its Recognition

Fibroadenoma and phyllodes tumors of the breast exhibit a continuum of pathologic features. We examined phyllodes tumors initially called fibroadenoma for features that may accurately classify the tumor as phyllodes tumor on the first biopsy specimen. The phyllodes tumors initially called fibroadenoma for features that may accurately classify the tumor as phyllodes tumors on the first biopsy specimen are examined. Fifteen patients with phyllodes tumors were studied, initially called FA or another term short of PT. These tumors were compared with 16 true fibroadenomas, all with needle-core biopsy followed by excision. Resected phyllodes tumors were larger on average than fibroadenoma, 6.8 cm (range = 1.7-16.2 cm) versus 2.6 cm (range = 1.0-4.8 cm). In needle-core biopsy cases, sampling was limited, even in large breast masses. p53 and cleaved caspase-3 were noncontributory. Ki-67 showed higher proliferation indices in phyllodes tumors versus fibroadenoma (4.8% vs 0.6%). Features suggesting phyllodes tumors include tissue fragmentation, increased stromal cellularity especially around glands, stromal overgrowth, and increased mitoses. Increased sampling of a large tumor will likely yield more correct diagnoses.

Langerhans cell histiocytosis in acute leukemias of ambiguous or myeloid lineage in adult patients: support for a possible clonal relationship.

Four patients presented with acute leukemia of ambiguous or myeloid lineage in association with Langerhans cell histiocytosis and provide evidence suggesting a common origin of the two neoplasms. One patient had a non-constitutional trisomy 21 in both the leukemic blasts and the Langerhans cells indicative of a clonal relationship. A second case expressed CD2, CD13, and CD117 on both the Langerhans cells and the blasts suggesting a possible clonal relationship. All four cases exhibited geographic intermingling of the Langerhans cell histiocytosis and acute leukemia and shared unique features including extramedullary leukemia involving lymph nodes in all cases with Langerhans cell histiocytosis only present in sites involved by acute leukemia. T-cell antigen expression was present in all cases with one meeting criteria for mixed phenotype acute leukemia, T/myeloid, not otherwise specified. These findings support the concept that coexistent Langerhans cell histiocytosis and acute leukemia is clonally related in some cases. Furthermore, these cases of acute myeloid or acute leukemia of ambiguous lineage with Langerhans cell histiocytosis share some unique features suggesting a common underlying neoplastic hematopoietic stem cell.

Core-binding factor acute myeloid leukemia: Heterogeneity, monitoring, and therapy

Core binding factor acute myelogenous leukemia (CBF AML) constitutes 15% of adult AML and carries an overall good prognosis. CBF AML encodes two recurrent cytogentic abnormalities referred to as t(8;21) and inv (16). The two CBF AML entities are usually grouped together but there is a considerable clinical, pathologic and molecular heterogeneity within this group of diseases. Recent and ongoing studies are addressing the molecular heterogeneity, minimal residual disease and targeted therapies to improve the outcome of CBF AML. In this article, we present a comprehensive review about CBF AML with emphasis on molecular heterogeneity and new therapeutic options. Am. J. Hematol. 89:1121–1131, 2014.

Implementation of Cloud based Next Generation Sequencing data analysis in a clinical laboratory

BackgroundThe introduction of next generation sequencing (NGS) has revolutionized molecular diagnostics, though several challenges remain limiting the widespread adoption of NGS testing into clinical practice. One such difficulty includes the development of a robust bioinformatics pipeline that can handle the volume of data generated by high-throughput sequencing in a cost-effective manner. Analysis of sequencing data typically requires a substantial level of computing power that is often cost-prohibitive to most clinical diagnostics laboratories.FindingsTo address this challenge, our institution has developed a Galaxy-based data analysis pipeline which relies on a web-based, cloud-computing infrastructure to process NGS data and identify genetic variants. It provides additional flexibility, needed to control storage costs, resulting in a pipeline that is cost-effective on a per-sample basis. It does not require the usage of EBS disk to run a sample.ConclusionsWe demonstrate the validation and feasibility of implementing this bioinformatics pipeline in a molecular diagnostics laboratory. Four samples were analyzed in duplicate pairs and showed 100% concordance in mutations identified. This pipeline is currently being used in the clinic and all identified pathogenic variants confirmed using Sanger sequencing further validating the software.