TuDung T. Nguyen

p16 and p15, 2 inhibitors of cyclin-dependent kinases, are frequently hypermethylated in hematologic neoplasias. Decitabine, or 5-Aza-2'-deoxycytidine, reverts hypermethylation of these genes in vitro, and low-dose decitabine treatment improves cytopenias and blast excess in ~50% of patients with high-risk myelodysplastic syndrome (MDS). We examined p15 and p16 methylation status in bone marrow mononuclear cells from patients with high-risk MDS during treatment with decitabine, using a methylation-sensitive primer extension assay (Ms-SNuPE) to quantitate methylation, and denaturing gradient gel electrophoresis (DGGE) and bisulfite-DNA sequencing to distinguish individually methylated alleles. p15 expression was serially examined in bone marrow biopsies by immunohistochemistry. Hypermethylation in the 5' p15 gene region was detected in 15 of 23 patients (65%), whereas the 5' p16 region was unmethylated in all patients. Among 12 patients with hypermethylation sequentially analyzed after at least one course of decitabine treatment, a decrease in p15 methylation occurred in 9 and was associated with clinical response. DGGE and sequence analyses were indicative of hypomethylation induction at individual alleles. Immunohistochemical staining for p15 protein in bone marrow biopsies from 8 patients with p15 hypermethylation revealed low or absent expression in 4 patients, which was induced to normal levels during decitabine treatment. In conclusion, frequent, selective p15 hypermethylation was reversed in responding MDS patients following treatment with a methylation inhibitor. The emergence of partially demethylated epigenotypes and re-establishment of normal p15 protein expression following the initial decitabine courses implicate pharmacologic demethylation as a possible mechanism resulting in hematologic response in MDS.

BACKGROUND: 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). METHODS: Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. RESULTS: Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. CONCLUSIONS: NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing.

CD163, a hemoglobin scavenger receptor, is expressed in monocytes and macrophages. We tested the expression of the CD163 protein in 1,105 human malignancies and normal tissues using tissue microarrays and conventional paraffin-embedded tissue sections. Besides staining nonneoplastic monocytes and histiocytes (tissue macrophages), membranous/cytoplasmic staining for CD163 was primarily limited to neoplasms with monocytic/histiocytic differentiation. CD163 reactivity was not observed in normal tissues, lymphomas, carcinomas, and in a majority of mesenchymal neoplasms, including follicular dendritic cell tumors (0 of 4), although it stained admixed histiocytes. Staining for CD163 was seen in Rosai-Dorfman disease (5 of 6), histiocytic sarcoma (3 of 4), littoral cell angioma (6 of 6), and Langerhans cell histiocytosis (3 of 5). A subset of atypical fibrous histiocytomas (9 of 16), benign fibrous histiocytomas (6 of 9), and atypical fibroxanthomas (1 of 3) also showed CD163 staining. Our studies also confirm earlier work showing that CD163 is expressed in acute myeloid leukemia with monocytic differentiation (AML, FAB subtype M5) (2 of 6), as well as a majority of giant cell tenosynovial tumors (7 of 8). Its limited range of expression and tissue specificity indicate that CD163 may have significant diagnostic utility in separating specific tumors with monocytic and histiocytic derivation from other entities in their differential diagnosis.

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. METHODS: We conducted a search of the English language medical literature for recent studies of interest to individuals involved in the diagnosis of myeloid sarcoma. RESULTS: The differential diagnosis includes non-Hodgkin lymphoma, blastic plasmacytoid dendritic cell neoplasm, histiocytic sarcoma, melanoma, carcinoma, and (in children) small round blue cell tumors. The sensitivity and specificity of immunohistochemical markers must be considered when evaluating a suspected case of myeloid sarcoma. A high percentage of tested cases have cytogenetic abnormalities. CONCLUSION: A minimal panel of immunohistochemical markers should include anti-CD43 or anti-lysozyme as a lack of immunoreactivity for either of these sensitive markers would be inconsistent with a diagnosis of myeloid sarcoma. Use of more specific markers of myeloid disease, such as CD33, myeloperoxidase, CD34 and CD117 is necessary to establish the diagnosis. Other antibodies may be added depending on the differential diagnosis. Identification of acute myeloid leukemia-associated genetic lesions may be helpful in arriving at the correct diagnosis.