Kaaren K. Reichard

Diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma is the most common lymphoma worldwide. Both morphologically and prognostically it represents a diverse spectrum of disease. Traditional morphologic subclassification often results in poor interobserver reproducibility and has not been particularly helpful in predicting outcome. Recent gene expression profiling studies have classified diffuse large B-cell lymphoma into 2 main subtypes, germinal center B-cell and activated B-cell, with the germinal center type showing an overall better survival. Validation of these subtypes has become possible for the practicing pathologist with the use of surrogate immunohistochemical markers. Importantly however, these prognostic studies were performed on material from the pre-rituximab treatment era. With the now well-accepted addition of rituximab (anti-CD20 antibody) to the typical large B-cell lymphoma chemotherapeutic regimen, a revalidation of any survival differences between the large B-cell lymphoma subgroups is necessary. This short review covers the current clinical, morphologic, immunophenotypic, genetic, gene expression profiling, and prognostic (studies before and after the addition of rituximab) features of de novo diffuse large B-cell lymphoma.

Acute Myeloid Leukemia With Myelodysplasia-Related Changes

OBJECTIVESnAcute myeloid leukemia with myelodysplasia-related changes (AML-MRC) is a heterogeneous disorder defined by morphologic, genetic, or clinical features. Genetic abnormalities associated with AML-MRC are often associated with adverse prognostic features, and many cases are preceded by a myelodysplastic syndrome (MDS) or a myelodysplastic/myeloproliferative neoplasm.nnnMETHODSnAlthough the criteria of 20% or more blasts in blood or bone marrow and multilineage dysplasia affecting 50% or more of cells in two or more of the myeloid lineages seem straightforward for AML-MRC, identification of morphologic dysplasia among observers is not always consistent, and there is morphologic overlap with other leukemic disorders such as acute erythroleukemia.nnnRESULTSnSession 3 of the workshop cases displayed heterogeneity as expected within AML-MRC, yet several cases suggested that recently recognized entities may exist within this category, such as familial MDS/AML predisposition syndromes and rare cases of high-risk AML associated with the cryptic t(5;11)(q35;p15);NUP98-NSD1 that may masquerade as a del(5q). However, most cases of AML-MRC were usually associated with adverse genetic abnormalities, particularly -5/del(5q), -7/del(7q), and/or complex karyotypes.nnnCONCLUSIONSnWhole-genome sequencing and array studies may identify genetic abnormalities, such as those affecting TP53, which may provide prognostic information.

Clonal mast cell disease not meeting WHO criteria for diagnosis of mastocytosis: clinicopathologic features and comparison with indolent mastocytosis

Clonal mast cell disease not meeting WHO criteria for diagnosis of mastocytosis: clinicopathologic features and comparison with indolent mastocytosis

Aberrant expression of CD123 (interleukin-3 receptor-α) on neoplastic mast cells

The high-affinity receptor for interleukin-3 (IL-3) comprises the ligand-binding α-subunit (CD123) plus the primary signaling common β (βc) subunit (CD131), the latter also being shared with receptors for IL-5 and granulocyte-macrophage colony stimulating factor (GM-CSF). IL-3 is a polyfunctional CSF that acts primarily on committed granulocyte-macrophage progenitor cells and their immediate progeny; in vivo administration of IL-3 in normal adult mice leads to increased levels of circulating eosinophils, granulocytes and monocytes as well as increased splenic mast cells. CD123 is expressed in a variety of hematological neoplasms including blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), Hodgkin’s lymphoma and hairy cell leukemia (reviewed by Testa et al.). In normal bone marrow (BM), o1% of CD34+/CD38− stem/progenitor cells express CD123; in contrast, 98% of corresponding cells from AML patients show strong expression of this marker. Similarly, CD123 expression has also been demonstrated in CD34+/CD38− cells in the majority of patients with myelodysplastic syndromes (MDS) and CML. In most AML patients, bulk CD34+ leukemic blasts virtually uniformly express CD123, and the putative leukemic stem cell (CD34+/CD38− / CD123+) has been shown to successfully engraft immunedeficient NOD/SCID mice. Early-phase therapeutic studies targeting CD123 in AML patients are currently underway. There is also ubiquitous, strong expression of functionally competent CD123 on CD4+/CD56+ blasts from BPDCN patients, an observation that has been successfully exploited in preliminary studies using an immunotoxin to target CD123 in these patients. Whereas CD123 is not expressed in normal or reactive mast cells, CD34+/CD38− stem/progenitor cells from systemic mastocytosis (SM) patients reportedly express this marker. However, there are conflicting data regarding CD123 expression on neoplastic mast cells from SM patients as detected by flow cytometry (FC) versus immunohistochemistry (IHC). In the current study, we analyzed, using both FC and IHC techniques, as to whether neoplastic mast cells in peripheral blood (PB) and BM from SM patients express CD123 in order to build a rationale for use of CD123-targeting therapies in this patient population. The current study was approved by our institutional review board. All patients provided written informed consent for PB and/or BM sample collection. Consecutive patients with confirmed SM were recruited from the Mastocytosis Clinic in the Division of Hematology; no other selection criteria were applied. The diagnosis of SM and its subclassification was according to WHO (World Health Organization) criteria. For FC, PB and BM samples were subjected to ammonium chloride lysis. Cells were then washed twice in staining buffer (phosphate-buffered saline containing 1mM EDTA and 0.5% bovine serum albumin), resuspended in 100 μl and appropriate dilution of corresponding fluorophore-conjugated surface antibody was added. Primary mouse anti-human antibodies used were as follows: PE-CD123 (cat. no. 340545), FITC-CD25 (cat. no. 555431), PerCP-CD45 (cat. no. 340665), FITC-CD34 (cat. no.555821), APC-CD117 (cat. no. 341096) (BD Biosciences, San Jose, CA, USA) and PE mouse anti-human FcεRI (cat. no. SM2257R) (Acris Antibody, San Diego, CA, USA). Samples were incubated at room temperature for 20min in the dark and then washed again. Surface antigen expression was interrogated using the 4-color multiparametric flow cytometer, FACSCalibur (BD Biosciences). Data analysis was performed using CellQuest Pro Software (BD Biosciences). Formalin-fixed and decalcified paraffinembedded BM biopsies were sectioned into 4 μm slices and affixed on glass slides. Samples were heated for half an hour at 56 °C, and then deparaffinized in xylene, rehydrated in a graded alcohol series and washed in water. Heat-induced epitope retrieval was used for both CD123 and CD117/KIT antigens. Enzyme pretreatment epitope retrieval was used for Tryptase antigen. Endogenous peroxidase activity was quenched in a bath of methanol and hydrogen peroxide. We stained samples for Tryptase (Dako, Carpinteria, CA, USA) at 1:1500, CD123 (clone 7G3, BD Pharmingen, Franklin Lakes, NJ, USA) at 1:2000 and CD117/KIT (Dako) at 1:500. Samples were incubated overnight with antibodies and peroxidase activity was localized for all samples with 3,3′-diaminobenzidine using Ventana staining and detection platforms (Ventana Medical Systems, Inc. Tucson, AZ, USA). Hematoxylin and eosin counterstaining was done per standard methods. Mast cell immunophenotyping by FC was performed on 14 SM patients; data were informative for 6 patients (8 samples) for whom an adequate number of mast cells were identified for immunophenotyping: (1) indolent SM (ISM, n= 2, PB = 1, paired PB/BM=1); (2) aggressive SM (ASM, n= 1, paired PB/BM=1) and (3) SM with an associated hematological neoplasm (SM-AHD, n= 3, PB (myelodysplastic syndrome/myeloproliferative neoplasmnot otherwise specified, (MDS/MPN-NOS) = 2, BM (polycythemia vera) = 1). Clinical and laboratory characteristics of these patients are described in Supplementary Table 1. A median total 653 625 cells per sample were interrogated (range 1 72 050–2 513 850). Neoplastic mast cells were defined as CD117++/SSC++/CD45+ +/CD34− /CD25+/FcεRI+ (Figure 1). The majority of mast cells for five patients expressed CD123 (no. 1–5, CD123 percentage positive, PB/BM): no. 1, CD123 = 91% (PB)/ BM not done; no. 2, CD123= 76% (PB)/ 82% (BM); no. 3, CD123 = 85% (PB)/ BM not done; no. 4, CD123 = 88% (PB)/ BM not done; and no. 5, CD123= 84% (BM)/PB not done. In contrast, one patient (no. 6) showed a minority of mast cells to be CD123 positive: (33% PB and 28% BM). For both SM patients who had paired PB and BM samples analyzed, CD123 expression was concordant in the two samples. FcεRI expression was brighter on mast cells from BM as compared with PB (data not shown). CD123 immunophenotyping by IHC was performed on 3 control samples (patients with clinically suspected SM, but with a negative workup) and 5 additional SM patients: (1) ISM, n= 3; (2) ASM, n= 1; and (3) SM-AHD (MDS/MPN-NOS), n= 1 (Supplementary Table 1). In control BM sections, rare scattered interstitially distributed cells showed strong CD123 staining, likely representing plasmacytoid dendritic cells. Weak staining of vascular endothelial cells in some venules/capillaries was observed; there was no readily perceptible staining of other hematopoietic cells. For the SM cases, CD123 staining was assessed on recognizable tight aggregates of mast cells, evident by their tryptase and CD117 staining characteristics. In the two cases with ASM and SM-AHD, morphologically atypical and tryptase-positive mast cells showed a strong, uniform CD123 staining pattern (Figures 2e and f). For the three ISM cases, mast cell CD123 staining appeared more variable relative to the former (Figures 2a–d). In one ISM case, a focal increase of

Next‐generation sequencing in systemic mastocytosis: Derivation of a mutation‐augmented clinical prognostic model for survival

In routine practice, the World Health Organization classification of systemic mastocytosis (SM) is also the de facto prognostic system; a core value is distinguishing indolent (ISM) from advanced SM (includes aggressive SM [ASM], SM with associated hematological neoplasm [SM‐AHN] and mast cell leukemia [MCL]). We sequenced 27 genes in 150 SM patients to identify mutations that could be integrated into a clinical‐molecular prognostic model for survival. Forty four patients (29%) had ISM, 25 (17%) ASM, 80 (53%) SM‐AHN and 1 (0.7%) MCL; overall KITD816V prevalence was 75%. In 87 patients, 148 non‐KIT mutations were detected; the most frequently mutated genes were TET2 (29%), ASXL1 (17%), and CBL (11%), with significantly higher mutation frequency in SM‐AHNu2009>u2009ASMu2009>u2009ISM (Pu2009<u20090.0001). In advanced SM, ASXL1 and RUNX1 mutations were associated with inferior survival. In multivariate analysis, ageu2009>u200960 years (HRu2009=u20092.4), hemoglobinu2009<u200910 g/dL or transfusion‐dependence (HRu2009=u20091.7), platelet countu2009<u2009150 × 109/L (HRu2009=u20093.2), serum albuminu2009<u20093.5 g/dL (HRu2009=u20092.6), and ASXL1 mutation (HRu2009=u20092.3) were associated with inferior survival. A mutation‐augmented prognostic scoring system (MAPSS) based on these parameters stratified advanced SM patients into high‐, intermediate‐, and low‐risk groups with median survival of 5, 21 and 86 months, respectively (Pu2009<u20090.0001). These data should optimize risk‐stratification and treatment selection for advanced SM patients. Am. J. Hematol. 91:888–893, 2016.

Conventional karyotyping and fluorescence in situ hybridization: an effective utilization strategy in diagnostic adult acute myeloid leukemia.

OBJECTIVESnCytogenetics defines disease entities and predicts prognosis in acute myeloid leukemia (AML). Conventional karyotyping provides a comprehensive view of the genome, while fluorescence in situ hybridization (FISH) detects targeted abnormalities. The aim of this study was to compare the utility of karyotyping and FISH in adult AML.nnnMETHODSnWe studied 250 adult AML cases with concurrent karyotyping and FISH testing. Karyotyping was considered adequate when 20 or more metaphases were analyzed.nnnRESULTSnIn total, 220 cases had adequate karyotyping and were classified as normal karyotype/normal FISH (n = 92), normal karyotype/abnormal FISH (n = 4), abnormal karyotype/normal FISH (n = 8), and abnormal karyotype/abnormal FISH (n = 116). The overall karyotype/FISH concordance rate was 97.7% with five discordant cases identified, four from the normal karyotype/abnormal FISH group and one from the abnormal karyotype/abnormal FISH group. No karyotype/FISH discordance was seen in the abnormal karyotype/normal FISH group for the FISH probes evaluated. FISH lent prognostic information in one (0.5%) of 220 cases with normal karyotype/abnormal FISH: CBFB-MYH11 fusion, indicating favorable prognosis.nnnCONCLUSIONSnIn adult AML, FISH rarely provides additional information when karyotyping is adequate. We therefore propose an evidence-based, cost-effective algorithmic approach for routine conventional karyotype and FISH testing in adult AML workup.

Targeted next generation sequencing and identification of risk factors in World Health Organization defined atypical chronic myeloid leukemia

Atypical chronic myeloid leukemia (aCML) is an aggressive myeloid neoplasm with overlapping features of myelodysplastic syndromes (prominent granulocytic dysplasia) and myeloproliferative neoplasms (neutrophilic leukocytosis). We studied 25 molecularly‐annotated and World Health Organization defined aCML patients; median age 70 years, 84% males. Cytogenetic abnormalities were seen in 36% and gene mutations in 100%. Mutational frequencies were, ASXL1 28%, TET2 16%, NRAS 16%, SETBP1 12%, RUNX1 12%, ETNK1 8%, and PTPN11 4%. Fifteen patients (60%) had >1 mutation, while 9 (36%) had ≥3. The median overall survival (OS) was 10.8 months and at last follow up (median 11 months), 17 (68%) deaths and 2 (8%) leukemic transformations were documented. On univariate analysis, survival was adversely impacted by advanced age (Pu2009=u2009.02), low hemoglobin (Pu2009=u2009.01), red blood cell transfusion dependence (Pu2009=u2009.03), high white blood cell count (Pu2009=u2009.02), TET2 (Pu2009=u2009.03), NRAS (Pu2009=u2009.04), PTPN11 (Pu2009=u2009.02) mutations and the presence of ≥3 gene mutations (Pu2009=u2009.006); ASXL1, SETBP1, and ETNK1 mutations did not impact OS. In multivariable analysis, advanced age (Pu2009=u2009.003) [age >67: HR 10.1, 95% CI 1.3‐119], low hemoglobin (Pu2009=u2009.008) [HB< 10 gm/dL: HR 8.2, 95% CI 1.6‐23.2] and TET2 mutations (Pu2009=u2009.01) [HR 8.8, 95% CI 1.6‐47.7] retained prognostic significance. We then used age >67 years, hemoglobin <10 gm/dL and the presence of TET2 mutations (each counted as one risk factor) to create a hazard ratio weighted prognostic model; effectively stratifying patients into two risk categories, low (0‐1 risk factor) and high (≥2 risk factors), with median OS of 18 and 7 months, respectively.

CD123 immunostaining patterns in systemic mastocytosis: differential expression in disease subgroups and potential prognostic value

CD123 is the α-subunit of the interleukin-3 receptor; it represents a potential therapeutic target in systemic mastocytosis (SM) given its absent expression on normal/reactive mast cells (MCs) and aberrant expression on neoplastic MCs. We studied 58 SM patients to define CD123 expression patterns by immunohistochemistry and its clinical significance. Two hematopathologists independently scored bone marrow slides using predefined histologic parameters. In all, 23 patients had indolent SM (ISM), 10 aggressive SM (ASM), 23 SM with associated hematological neoplasm (SM-AHN) and 2 had mast cell leukemia (MCL). MC_CD123 expression was demonstrable in 37 (64%) cases; expression rates were 100%, 61%, 57% and 0% in ASM, ISM, SM-AHN and MCL, respectively (P=0.02). Focal proliferation of plasmacytoid dendritic cells (PDCs) around MC aggregates, suggesting a tumor-promoting role for PDCs, was noted in 44 (76%) cases, and was significantly higher in CD123-positive versus -negative cases (87% versus 50%, P=0.005). CD123 expression and its staining intensity had prognostic value in SM-chronic myelomonocytic leukemia and nonindolent SM patients, respectively. These observations suggest that targeting CD123 in SM may have direct (via MCs) and indirect (via PDCs) antitumor effects and clinical trials to that effect require laboratory correlative studies to address the observed target expression heterogeneity.

Transient monosomy 7 in a chronic myelogenous leukemia patient during nilotinib therapy: a case report

Tyrosine kinase inhibitor treated chronic myelogenous leukemia patients with monosomy 7 arising in Philadelphia chromosome negative (Ph−) cells tend to evolve into MDS/AML. However, monosomy 7 in Ph− cells can be a transient finding, and it is not an absolute indication of the emergence of a new myeloid malignancy.

BCR-JAK2 fusion in a myeloproliferative neoplasm with associated eosinophilia.

Janus kinase 2 (JAK2) is located on chromosome 9 at band p24 and JAK2V617F is the most common mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPN). However, rearrangement of JAK2 is a rare event. We report a case of myeloproliferative neoplasm, unclassifiable (MPN-U) with BCR-JAK2 fusion confirmed by molecular studies. Conventional chromosome analysis (CC) revealed t(9;22)(p24;q11.2) and fluorescence in situ hybridization (FISH) showed a JAK2 gene rearrangement in 88% of interphase nuclei. The BCR-JAK2 fusion was confirmed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and demonstrated two in-frame 5BCR/3JAK2 transcripts with BCR exon 1 juxtaposed to JAK2 exon 15 and exon 17, respectively. Our results, together with literature review, reveal BCR-JAK2 fusions as oncogenic genetic alterations that are associated with myeloid or lymphoid neoplasms and are frequently characterized by eosinophilia. Further, patients with BCR-JAK2 are candidates for JAK2 inhibitor therapy. Given the distinct clinical and pathological characteristics, we believe that hematological neoplasms harboring BCR-JAK2 should be included as an additional distinct entity to the current WHO category of myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR, and testing for a JAK2 fusion should be pursued in neoplasms with a karyotypic 9p24 abnormality.