A decreased percentage of CD177pos neutrophils is frequently present in MDS and AML and is a useful flow cytometry (FCM) marker for the identification of MDS. The underlying mechanism leading to the low percentage of CD177pos neutrophils in MDS has not been explained. The aim of this study was to identify whether specific somatic mutations in myeloid neoplasms are associated with the low percentage of CD177pos neutrophils. 507 myeloid neoplasms with one or more pathogenic molecular abnormality identified by NGS and in which CD177 expression was assessed were evaluated. Correlation with CD177 expression was determined for 39 variables (including genes mutated, diagnostic groups and gender) using a 40 % cutoff level for low CD177 expression. In multivariate analysis mutations involving NPM1 (OD 0.26), RUNX1 (OD 0.39), TET2 (OD 0.58), and U2AF1 S34F (OD 0.25) were associated with low percentage of CD177pos neutrophils when all cases were evaluated. JAK2 (OD 2.5) alteration was associated with increased percentage of CD177pos neutrophils. Differences were noted between diagnostic subgroups with no single mutation associated with decreased CD177pos neutrophils in MDS and CCUS. The findings demonstrate an association between the percentage of CD177pos neutrophils and somatically acquired mutations involving several genes.

Objectives: Previously we demonstrated that a decreased percentage of CD177-positive granulocytes detected by flow cytometry (FCM) was associated with myelodysplastic syndrome (MDS). Here we expand on those findings to more rigorously evaluate the utility of CD177 for the detection of MDS. Methods: Two hundred patient samples (100 MDS and 100 controls) were evaluated for granulocyte expression of CD177 and 11 other flow cytometric parameters known to be associated with MDS. Results: We show that CD177, as a single analyte, is highly correlated with MDS with a receiver operating characteristic area under curve value of 0.8. CD177 expression below 30% demonstrated a sensitivity of 51% and a specificity of 94% for detecting MDS with a positive predictive value of 89.5%. In multivariate analysis of 12 MDS-associated FCM metrics, CD177 and the Ogata parameters were significant indicators of MDS, and CD177 increased sensitivity of the Ogata score by 16% (63%-79%) for predicting MDS. Finally, diagnostic criteria incorporating these parameters with a 1% blast cutoff level and CD177 resulted in a sensitivity of 90% and specificity of 91% for detecting MDS. Conclusions: The findings indicate CD177 is a useful FCM marker for MDS.

AIM:Develop an objective assay to detect c-MYC protein expression using multiparametric flow cytometry (FCM) as an alternative to immunohistochemistry (IHC). METHODS:57 patient samples and 11 cell line samples were evaluated. Cell suspensions were obtained and c-MYC staining was performed in combination with CD45 and CD19 and, in some samples, CD10. The percentage of c-MYC+ cells by FCM was correlated with the percentage determined by IHC. The relationship between c-MYC protein expression and the presence of a c-MYC gene rearrangement in aggressive and high-grade lymphomas was also assessed. RESULTS:c-MYC expression by FCM and IHC demonstrated a high degree of correlation in a training set of 33 patient cases, r=0.92, 11 cell line samples, r=0.81 and in a validation set of 24 aggressive and high-grade B-cell lymphomas, r=0.85. c-MYC gene was rearranged by fluorescence in situ hybridisation in 6/9 samples with high c-MYC expression (>40%) by FCM and 6/14 by IHC. CONCLUSIONS:We have developed a reliable multicolour FCM assay to detect c-MYC expression suitable for clinical laboratories that should be helpful to accurately quantify c-MYC expression in B-cell lymphomas.

Langerhans cell histiocytosis is characterized by a localized or systemic proliferation of Langerhans cells. BRAF mutations have been reported in 40–70% of cases and MAP2K1 mutations have been found in BRAF -negative cases, supporting that Langerhans cell histiocytosis is a true neoplasm, at least in mutated cases. In a small subset of patients, Langerhans cell histiocytosis is detected incidentally in a biopsy involved by lymphoma. These lesions are usually minute and rarely have been assessed for mutations. We assessed for BRAF and MAP2K1 mutations in seven cases of Langerhans cell histiocytosis detected incidentally in biopsies involved by lymphoma. We performed immunohistochemical analysis for phosphorylated (p)-ERK. There were four men and three women (median age, 54 years; range, 28–84). The biopsies included lymph nodes ( n =6) and chest wall ( n =1). The lymphomas included five classical Hodgkin lymphoma, one mantle cell lymphoma, and one angioimmunoblastic T-cell lymphoma. All cases were negative for BRAF V600E and MAP2K1 mutations. Nevertheless, three of seven cases showed ERK activation as shown by expression of p-ERK. We performed mutation analysis using a panel of 134 commonly mutated genes (including BRAF and MAP2K1) by next-generation sequencing on three cases, including two cases positive for p-ERK by immunohistochemistry. No mutations were detected in any of the three cases assessed. Six patients received therapy appropriate for their lymphoma. With a median follow-up of 21 months (range, 6–89), no patients developed disseminated or recurrent Langerhans cell histiocytosis. We conclude that lymphoma-associated Langerhans cell histiocytosis is a clinically benign process that is not associated with BRAF V600E or MAP2K1 mutations and, as suggested by others, the designation Langerhans cell hyperplasia may be more appropriate. Nevertheless, the expression of p-ERK in three cases suggests that the RAS–RAF–MAP2K–ERK pathway is activated, perhaps by non-mutational mechanisms induced by the presence of lymphoma or lymphoma–microenvironment interactions.

Langerhans cell histiocytosis (LCH) is a proliferation of Langerhans cells, often associated with lymphocytes, eosinophils, macrophages, and giant cells. BRAF mutations, usually V600E, have been reported in 40%-70% of cases, and recently, MAP2K1 mutations have been reported in BRAF-negative cases. We assessed 50 cases of LCH for BRAF mutations and assessed a subset of cases for MAP2K1 mutations. The study group included 28 men and 22 women (median age, 36.5 years; range, 1-78 years). BRAF V600E mutation was detected in 8 (16%) cases including 3 (30%) skin, 2 (11%) bone, 1 (50%) colon, 1 (20%) lung, and 1 (33%) extradural, intracranial mass. MAP2K1 mutations were detected in 6 of 13 (46%) BRAF-negative cases including 2 (100%) lymph node, 2 (50%) bone, 1 (25%) skin, and 1 (100%) orbit. Patients with BRAF mutation were younger than patients with wild-type BRAF (median age, 28 versus 38 years; P = .026). The median age of MAP2K1-mutated patients was 34.5 years, similar to patients without MAP2K1 mutation (41 years; P = .368). In agreement with 2 recent studies, we showed a high frequency of MAP2K1 mutations in BRAF-negative LCH cases. Unlike other studies, the overall frequency of BRAF mutation in this cohort is substantially lower than what has been reported in pediatric patients, perhaps because most patients in this study were adults. Moreover, we showed a high concordance between mutational and immunohistochemical analysis for BRAF mutation. There was no statistically significant association between BRAF or MAP2K1 mutation and anatomic site, unifocal versus multifocal presentation, or clinical outcome.

333Acarcinoma in situ (CIS) with nonkeratinizing (NK) histology distinct from the 'bowenoid' pattern that extensively involved the UAT.Here, we characterize the clinicopathologic features of HPV+ CIS/SD of the UAT.Design: All cases of UAT CIS/SD from July 2012 to March 2015 were categorized into histologic types: keratinizing (K), NK, mixed or 'bowenoid'.Exclusion criteria were: A) superficial biopsies where the base was not seen and B) CIS/SD within 1 mm of an invasive carcinoma, in continuity with a tonsil or base of tongue invasive carcinoma, or arising in another characterized lesion (Schneiderian papilloma, verrucous or papillary proliferation).P16 immunohistochemistry (positive if > 70%) and high-risk HPV RNA in situ hybridization (ISH) were performed on all NK/mixed and p16 on a subset of K cases.Results: Of 99 patients with CIS/SD, 84 were K, 3 NK, 12 mixed, and 0 'bowenoid'.All 3 (100%) NK and 6 (50%) mixed cases were p16 and HPV RNA ISH+ (9% of all CIS/SD HPV+) with 100% concordance between p16 and HPV status.All representative (14) K cases were p16-and thus were not HPV tested.Among the HPV+ cases, the floor of mouth (FOM) was the most commonly involved site and large (≥ 4 cm) or extensive/ unresectable disease was common [see Table of HPV+ cases].Case CIS/SD Type Age Sex Site Clinically Extensive 1 NK 63 M Oral/larynx Y 2 NK 60 M Subglottis/trachea Y 3 NK 69 M Buccal/FOM/oral tongue/palate

Plasmablastic lymphoma (PBL) is a rare aggressive neoplasm with lymphoid and plasmacytic differentiation that is commonly associated with immunodeficiency and an unfavorable prognosis. Clinicopathologic features have been largely derived from cases reports and small series with limited outcome analyses.

Acute promyelocytic leukemia (APL) is characterized by promyelocytic leukemia (PML)-retinoic acid receptor, alpha (RARA) fusion gene as a result of t(15;17)(q24.1;q21.2).1 Current advances in APL therapy have dramatically improved patient outcome.2,3 However, patients with APL are at risk of potentially lethal coagulopathy if appropriate therapy is delayed.4

OBJECTIVES:To assess CD105 (endoglin) expression in 119 acute myeloid leukemia (AML) and 13 control cases using immunohistochemistry.METHODS:CD105 expression was assessed retrospectively by using immunohistochemistry in bone marrow specimens.RESULTS:CD105 was strongly and diffusely positive in all 9 (100%) AMLs with t(15;17)(q24.1;q21.2), 2 (100%) AMLs with t(8;21)(q22;q22), 1 (100%) AML with t(6;9)(p23;q34), 7 (28%) of 25 AMLs with myelodysplasia-related changes, 1 (33%) of 3 therapy-related AMLs, 3 (16%) of 19 AMLs unclassifiable, 1 (14%) of 7 AMLs with inv(16)(p13.1q22), and 5 (11%) of 45 AMLs not otherwise specified. Uninvolved bone marrow in these cases showed no CD105 expression by erythroid precursors, megakaryocytes, or endothelial or stromal cells. Two of 13 control bone marrow specimens showed partial CD105 positivity in myeloid cells. In 21 strongly CD105+ AML cases tested for the IDH2 mutation, 9 (42%) were mutated (P = .004).CONCLUSIONS:These data suggest that CD105 could be a therapeutic target in a subset of patients with AML.

Distinguishing blastic plasmacytoid dendritic cell neoplasm (BPDCN) from acute myeloid leukemia (AML) is gaining increased importance because of emerging differences in therapeutic approaches, and this distinction can be problematic in bone marrow specimens. We identified retrospectively 16 patients with bone marrow involvement by BPDCN: 11 men and 5 women with a median age of 62.5 years (range, 19–86 years). Myelodysplastic changes were observed in five patients. Immunophenotypic analysis showed that the neoplastic cells were positive for CD4, CD123, TCL‐1, and HLA‐DR and were negative for CD3, CD8, CD13, CD19, CD34, and myeloperoxidase. Other antigens expressed by subsets of BPDCN cases included the following: CD56 (13/15; 81%), CD33 (7/10; 70%), CD7 (11/14; 69%), TdT (5/15; 33%), CD2 (5/11; 31%), CD117 (2/9; 22%), and CD5 (2/13; 15%). Conventional cytogenetic analysis showed chromosomal abnormalities in 6 of 13 (46%) cases analyzed, of which 3 cases had −13/13q−. Targeted next‐generation sequencing performed on five BPDCN cases identified TET2 (ten eleven translocation 2) mutations and no other AML‐associated mutations. In conclusion, BPDCN in the bone marrow has a characteristic immunoprofile (CD4+, CD56+, CD123+, and TCL‐1+) and appears to be commonly associated with myelodysplastic features and a high frequency of TET2 mutations in the absence of other mutations commonly observed in AML. Am. J. Hematol. 88:1055–1061, 2013. © 2013 Wiley Periodicals, Inc.

CONTEXT Anti-promyelocytic leukemia (PML) immunofluorescence staining is a known diagnostic tool for rapid diagnosis of acute promyelocytic leukemia (APL). OBJECTIVE We describe our methods using the recently developed, commercially available, tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 anti-PML antibody for APL testing. DESIGN Immunofluorescence staining with the tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 antibody was used to detect PML-RARA in bone marrow aspirate and/or peripheral blood smears from 30 patients with acute leukemia. The results were compared with those of concurrent testing with our in-house polyclonal anti-PML antibody and with established tests. RESULTS All APL cases showed a positive (fine/microgranular) immunofluorescence staining pattern, whereas non-APL cases showed a negative (chunky/macrogranular) pattern. These results, which were available within 2 hours, were validated by testing with the polyclonal anti-PML antibody and with established cytogenetic and molecular testing methods. CONCLUSIONS We validated the utility of the tetramethylrhodamine-5-isothiocyanate-labeled anti-PML antibody PG-M3 for the diagnosis of APL. Our results indicate that immunofluorescence staining with this antibody is a rapid and reliable method for the diagnosis of APL.

We read with interest the article by Popplewell and colleagues entitled “Primary anaplastic large-cell lymphoma associated with breast implants” in this journal [1]. The authors summarized 24 previ...