TuDung T. Nguyen

Validation of a Next-Generation Sequencing Assay for Clinical Molecular Oncology

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Clinical next-generation sequencing in patients with non-small cell lung cancer.

A clinical assay was implemented to perform next‐generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non–small cell lung cancer (NSCLC).

State of the art in myeloid sarcoma

Introduction:  Myeloid sarcomas are extramedullary lesions composed of myeloid lineage blasts that typically form tumorous masses and may precede, follow, or occur in the absence of systemic acute myeloid leukemia. They most commonly involve the skin and soft tissues, lymph nodes, and gastrointestinal tract and are particularly challenging to diagnose in patients without an antecedent history of acute myeloid leukemia.

CD163 Immunohistochemistry Is Superior to CD68 in Predicting Outcome in Classical Hodgkin Lymphoma

OBJECTIVES In recent years, research has increasingly focused on the microenvironment of classical Hodgkin lymphoma (CHL) as a predictor of treatment outcome. The focus of this study was to assess the interobserver reproducibility in interpreting macrophage-associated immunohistochemistry (IHC) for CD68 and CD163 in a retrospective cohort of 88 patients with CHL. METHODS Staining results were correlated with clinical outcome in all patients and those with a high international prognostic score (IPS). RESULTS The intraclass correlation (ICC) for the five hematopathologists interpreting the IHC was stronger for CD163 (0.70) than for CD68 (0.50). Using a cutoff of 25% mean macrophage reactivity and including all patients, a statistically significant difference in overall survival (OS) was seen only for CD163 (P = .0006) and not for CD68 (P = .414). Patients with a mean CD163 reactivity of 25% or more had a median OS of 71 months vs 101 months for patients with less than 25% reactivity. CD163 retained statistical significance in multivariate analysis. In patients with advanced-stage CHL with high IPS, OS was also significantly worse for those with a mean CD163 reactivity of 25% or higher. CONCLUSIONS Our study confirms previous reports of a prognostic role of tumor-infiltrating macrophages in CHL, but only for CD163. Although most of the literature supports an increasing role of macrophage IHC as a predictor of clinical outcome, successful clinical translation will require a standardized method and reporting system.

Use of classic and novel immunohistochemical markers in the diagnosis of cutaneous myeloid sarcoma

Cutaneous myeloid sarcoma is often challenging to diagnose based solely upon histopathological features. Although immunohistochemistry can aid in its diagnosis, specific markers have not been clearly identified. We evaluated the utility of immunohistochemical markers in 57 cutaneous myeloid sarcoma cases. In addition to classical markers (CD117, CD163, CD34, myeloperoxidase and lysozyme), we used CD33 and CD14, recently described markers in paraffin‐embedded tissue samples, and Kruppel‐like factor 4 (KLF‐4), a novel monocytic marker. Our results show that lysozyme was expressed in 91%, CD33 in 60%, myeloperoxidase in 54%, CD34 in 39% and CD117 in 36% of cases. An antibody panel that included lysozyme, CD117 and CD33 identified all cases. The monocytic markers CD14, KLF‐4 and CD163 were expressed in 60, 58 and 40% of all cases, respectively. CD14 and KLF‐4 expression was significantly more common in cases with monocytic differentiation. CD14 is the single most sensitive and specific marker for monocytic differentiation (79 and 80%). Although KLF‐4 in isolation is relatively insensitive (50 and 87%), it enhances sensitivity in detecting monocytic cutaneous myeloid sarcoma when combined with CD14. Our results indicate that in addition to classical immunohistochemical markers, targeted use of newer antibodies, including CD33, CD14 and KLF‐4 is useful in the diagnosis of cutaneous myeloid sarcoma and in the detection of monocytic differentiation.

Gastrointestinal lymphomas in a North American population: clinicopathologic features from one major Central-Midwestern United States tertiary care medical center.

BackgroundGastrointestinal (GI) lymphomas are very common types of extranodal lymphomas, and we hypothesize there are regional differences in subtype, distribution in the GI tract, and epidemiological features among the different populations.MethodsWe retrospectively evaluated the clinical, molecular and histologic features of North American primary and secondary GI lymphomas diagnosed from 2000–2009 seen at our institution. We utilized immunohistochemistry and fluorescence in situ hybridization to further evaluate a subset of the gastric lymphomas.ResultsExtranodal marginal zone lymphomas of mucosal associated lymphoid tissue (MALTs) and diffuse large B cell lymphomas (DLBCLs) were the most common subtypes of GI lymphomas. Select gastric DLBCLs (N = 6) and MALTs (N = 13) were further examined for API2-MALT1 and IGH translocations, and P16 and P53 protein expression. Gastric MALTs showed frequent API2-MALT1 (38%) but not IGH translocations (0%), and the DLBCLs showed neither translocation. Expression of P16 and P53 proteins and the proliferative index were compared between high grade gastric lymphomas (gastric DLBCLs) and low grade gastric lymphomas (gastric MALTs). P53 overexpression (P = 0.008) and a high proliferation index [Ki-67] (P = 0.00042) were significantly associated with gastric DLBCL, but no statistically significant difference was observed in P16 expression (p = 0.108) between gastric DLBCL and gastric MALT.ConclusionOur study revealed that GI lymphomas from a Central-Midwestern North American population showed differences and similarities to non-North American cohorts. In addition, API2-MALT1, P16 and P53 abnormalities occurred frequently in gastric lymphomas from this North American population.Virtual slidesThe virtual slides for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1415505838687793

Immunohistochemical Analysis of Monocytic Leukemias Usefulness of CD14 and Krüppel-like Factor 4, a Novel Monocyte Marker

Detection of monocytic differentiation in myeloid neoplasms by immunohistochemical analysis is challenging owing to a lack of sensitive and/or specific antibodies. We tested the usefulness of immunohistochemical analysis for CD14, an antigen commonly detected by flow cytometry, and Krüppel-like factor 4 (KLF4), a potentially novel marker of monocytic differentiation, in a series of myeloid leukemias, including 53 acute myeloid leukemias with monocytic differentiation. These findings were compared with immunohistochemical findings for CD68 (KP-1), CD34, and CD163 and were also correlated with flow cytometric and enzyme cytochemical results. CD163 and CD14 are the most specific markers of monocytic differentiation, followed by KLF4. CD68, in contrast, is the most sensitive monocytic marker, and KLF4 is also significantly more sensitive than CD14 and CD163. These studies show that KLF4 is another marker of monocytic differentiation and that the combination of CD14 and CD163 can increase the diagnostic sensitivity for monocytic neoplasms.

Identification of major factors associated with failed clinical molecular oncology testing performed by next generation sequencing (NGS).

DNA analysis by NGS has become important to direct the clinical care of cancer patients. However, NGS is not successful in all cases, and the factors responsible for test failures have not been systematically evaluated.

T-cell prolymphocytic leukemia frequently shows cutaneous involvement and is associated with gains of MYC, loss of ATM, and TCL1A rearrangement.

T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive mature T-cell leukemia with frequent cutaneous presentation, which has not been well characterized. Among the 25 T-PLLs diagnosed between 1990 and 2013 at our institution, 32% (8/25) showed cutaneous manifestations, presenting as rash, purpura, papules, and ulcers. The skin biopsies showed leukemia cutis with perivascular and periadnexal irregular, small to medium-sized lymphoid infiltrates without epidermotropism. The lymphoid infiltrates were composed of mature CD4+ T cells expressing other T-cell antigens, and a subset (48%) showed dual CD4+/CD8+ coexpression. Higher median absolute peripheral blood lymphocyte count (43.0 vs. 13.0k/mm3; P=0.031) and elevated lactate dehydrogenase levels (P=0.00018) at the time of diagnosis were significantly associated with T-PLLs with skin involvement compared with those without. The extent of bone marrow involvement (P=0.849) and overall survival (P=0.144) was similar in the 2 groups. Fluorescence in situ hybridization or karyotype revealed frequent gains of MYC (67%; n=9), loss of ATM (64%; n=11), and TCL1A rearrangement or inversion 14q (75%; n=12). Gains of TCL1A was also seen (78%; n=9), including in some cases that had concurrent TCL1A rearrangement, whereas TP53 loss was less common (30%; n=10). No correlation was seen between the immunophenotype and morphology versus the presence or absence of skin involvement. These data suggest that cutaneous involvement by T-PLL is relatively common and often associated with significant peripheral blood involvement. The frequent MYC, ATM, and TCL1A alterations identified support that these genes are integral to the pathogenesis of T-PLL.

Anaplastic Large-Cell Lymphoma With Aberrant Expression of Multiple Cytokeratins Masquerading As Metastatic Carcinoma of Unknown Primary

Case Report A 52-year-old woman without a significant past medical history presented to her primary care physician in July 2011 for evaluation of a large, nontender, firm, fixed left inguinal mass. A computed tomography (CT) scan of the abdomen and pelvis confirmed bulky left inguinal adenopathy measuring up to 5.6 cm and numerous left iliac nodes measuring up to 4.7 cm, but was otherwise negative. An excisional left inguinal lymph node biopsy showed a neoplasm with anaplastic morphology. The tumor cells formed cohesive clusters that were centered in the subcapsular spaces and near sinuses with extension into the paracortex (Fig 1A). On high-power examination, the tumor cells were pleomorphic, with vesicular chromatin and prominent to inconspicuous nucleoli (Fig 1B). Immunohistochemistry showed that the tumor cells were positive for CD30 and the cytokeratins epithelial membrane antigen (EMA), intercellular adhesion molecule 5 (CAM5.2), and OSCAR (Figs 1C through 1E). Focal positive staining for cytokeratins AE1/AE3 was also noted. CD45, CD15, S100, CD2, CD3, CD5, CD7, CD10, CD19, CD20, anaplastic lymphoma kinase (ALK1), cytokeratin 7 (CK7), CK20, thyroid transcription factor 1, granzyme B, T-cell intracellular antigen, synaptophysin, and chromogranin were negative. Tand B-cell receptor gene rearrangement studies were negative. A pathologic diagnosis: “consistent with metastatic poorly differentiated carcinoma,” was rendered by the initial institution after review and concurrence by pathologists at a National Cancer Institute–designated cancer center. Evaluation for a primary site of carcinoma included a negative upper endoscopy, colonoscopy, mammogram, and gynecologic exam. CA-125 and carcinoembryonic antigen were normal. The patient developed low-grade fevers, abdominal discomfort, and a diffuse, dry, flaky maculopapular pruritic rash. She received one cycle of carboplatin and Taxol (Bristol-Myers Squibb, Princeton, NJ) on October 14, 2011, for presumed carcinoma of unknown primary (CUP). A repeat CT scan on November 2, 2011, showed progression of her abdominal and pelvic adenopathy when compared with a CT scan from October 13, 2011. She was empirically treated with cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy, completing six cycles in February 2012, with a marked decrease in the bulky adenopathy. In January 2012, a sample from her initial biopsy was sent for a CancerType ID gene test (bioTheranostics, San Diego, CA) for molecular classification; this test measures and integrates the expression of 92 genes to distinguish 28 tumor types and 50 subtypes. On the basis of the gene expression profiling, there was a 96% chance that the primary malignancy was lymphoma. The patient’s bulky pelvic adenopathy recurred less than a month after completion of cyclophosphamide, doxorubicin, vincristine, and prednisone treatment. Positron emission tomography CT scanning showed additional new lesions involving the mediastinum, midesophagus, and bones. She developed daily fevers, drenching sweats, and increasing weakness, and her weight decreased by 30 pounds. A new right inguinal lymph node biopsy in April 2012 showed similar morphologic and immunohistochemical findings as her previous biopsy, although more numerous tumor cells were seen in this subsequent biopsy. The differential diagnosis on the repeat biopsy included classical Hodgkin lymphoma (cHL), anaplastic large-cell lymphoma (ALCL), and metastatic, poorly differentiated carcinoma. Numerous atypical mitoses and apoptotic figures were seen along with necrosis. Additional immunostains showed that the tumor cells also expressed CD4, CD33, CAM5.2, and Wilms’ tumor 1, but were negative for a large panel of antibodies including ALK1, paired box gene-5, and other T-cell antigens (CD1, CD2, CD3, CD5, CD7, and CD8). T-cell receptor (TCR) gamma rearrangement studies showed a biclonal TCR rearrangement (Fig 1F). The molecular results, the morphology, and the immunophenotype were most consistent with an anaplastic lymphoma kinase(ALK) –negative ALCL with aberrant cytokeratin expression. The patient began treatment with the CD30-targeted antibody-drug conjugate brentuximab vedotin; however, she died less than 1 week after treatment initiation.