Patricia M. Miron

Cytogenetic Abnormalities in a Series of 1029 Patients With Primary Myelodysplastic Syndromes A Report From the US With a Focus on Some Undefined Single Chromosomal Abnormalities

Conventional karyotype has an established role in myelodysplastic syndrome (MDS) and is included in the International Prognostic Scoring System (IPSS) for patient risk stratification and treatment selection. Although some chromosomal abnormalities have been well characterized, the significance of several miscellaneous, infrequent, single chromosomal abnormalities remains to be defined. In addition, the emerging therapeutic agents may change the natural course of disease in patients with MDS and the cytogenetic impact on risk stratification.

Systemic mastocytosis with associated clonal hematological non‐mast cell lineage disease: Clinical significance and comparison of chomosomal abnormalities in SM and AHNMD components

Some patients with systemic mastocytosis have concurrent hematological neoplasms, designated in the World Health Organization (WHO) classification as systemic mastocytosis with associated clonal hematological non‐mast cell lineage disease (SM‐AHNMD). In this study, we analyzed 29 patients with SM‐AHNMD and compared them to 40 patients with pure SM. The AHNMDs were classified as chronic myelomonocytic leukemia (CMML) (n = 10), myelodysplastic syndrome (MDS) (n = 7), myeloproliferative neoplasms (n = 4), B‐cell lymphoma/leukemia/plasma cell neoplasms (n = 7), and acute myeloid leukemia (n = 1). Patients with SM‐AHNMD were older, more frequently had constitutional symptoms and hematological abnormalities, less often had skin lesions, and had an inferior overall survival compared with pure SM patients (48 months vs. not‐reached, P < 0.001). Karyotypic abnormalities were detected in 9/28 (32%) patients with SM‐AHNMD but not in pure SM patients (P < 0.001). Combined imaging/ fluorescence‐in‐situ hybridization performed in four SM‐AHNMD cases revealed shared abnormal signals in mast cells and myeloid cells in two patients with SM‐CMML and one patient with SM‐MDS, but not in the mast cells of a case SM‐associated with chronic lymphocytic leukemia with ATM‐deletion. Quantitative mutation analysis showed higher levels of mutant KIT D816V in SM‐CMML and SM‐MDS than in pure SM (P < 0.001). Our data indicate that the SM‐AHNMD category in the WHO classification is heterogeneous, including clonally related and unrelated forms of AHNMD. The presentation, treatment, and outcome of patients with SM‐AHNMD is often dictated by the type of AHNMD. Am. J. Hematol. 88:219–224, 2013.

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells

A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL–induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.

Improved Detection Rate of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia and Other Mature B-Cell Neoplasms With Use of CpG-Oligonucleotide DSP30 and Interleukin 2 Stimulation

Detection of cytogenetic abnormalities requires successful culture of the clonal population to obtain metaphase chromosomes for study, and as such, has been hampered by low mitotic indices of mature B cells in culture. Our study presents data on the improved abnormality detection rate with the use of a CpG-oligonucleotide/interleukin 2 (OL/IL-2) culture protocol for mature B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and non-CLL specimens. The increased detection rate of abnormalities, compared with unstimulated culture and traditional pokeweed mitogen culture, was statistically significant for both CLL and non-CLL neoplasms. For CLL specimens, our data also showed that for cytogenetically visible aberrations, OL/IL-2 was as, if not more, sensitive than detection with interphase fluorescence in situ hybridization (iFISH). Use of OL/IL-2 allowed a number of abnormalities to be detected, which were not covered by specific iFISH panels, especially balanced translocations. Therefore, OL/IL-2 stimulation improves diagnostic sensitivity and increases discovery rate of novel prognostic findings.

Anaplastic lymphoma kinase–positive large B-cell lymphoma with complex karyotype and novel ALK gene rearrangements

Anaplastic lymphoma kinase-positive large B-cell lymphoma is a rare non-Hodgkin lymphoma that exhibits a characteristic immunoblastic/plasmablastic morphology and is frequently associated with t(2;17)(p23;q23) and expression of Clathrin-anaplastic lymphoma kinase fusion protein. Here, we report a refractory anaplastic lymphoma kinase-positive large B-cell lymphoma in a 49-year-old human immunodeficiency virus-positive man. The neoplastic cells expressed CD138, epithelial membrane antigen, CD45, and perforin, and showed a strong granular cytoplasmic anaplastic lymphoma kinase staining pattern. Conventional chromosome analysis revealed a clone with multiple anomalies and a chromosome count of 76 to 79. Fluorescence in situ hybridization studies showed 5 copies of the ALK gene with 2 intact signals and 3 signals resulting from 2 independent, previously unreported, rearrangements. One rearrangement, seen in 2 copies, involved translocation of ALK sequences to chromosome Xq21. The second rearrangement involved translocation of ALK sequences to chromosome 12q24.1. This case broadens the cytogenetic alterations in anaplastic lymphoma kinase-positive large B-cell lymphoma, and it also demonstrates the high genetic instability of this tumor.

Multicolor fluorescence in situ hybridization in clinical cytogenetic diagnostics

Multicolor fluorescence in situ hybridization is a technology that has vastly expanded the diagnostic repertoire of the clinical cytogenetics laboratory. The limitations of conventional chromosome banding analysis can often be overcome by the high sensitivity and specificity of multicolor fluorescence in situ hybridization tests. This article reviews the latest multicolor fluorescence in situ hybridization tests (including multiplex fluorescence in situ hybridization, spectral karyotyping, cross-species color banding, and comparative genomic hybridization) that are currently limited to a few select clinical cytogenetic laboratories, but may soon have more dominant roles in clinical cytogenetic practice.

Interphase FISH in plasma cell dyscrasia: increase in abnormality detection with plasma cell enrichment

Historically, cytogenetic studies of plasma cell neoplasms have been hampered by the fact that terminally differentiated plasma cells do not proliferate well in vitro. Although the use of interphase FISH (iFISH) has greatly improved the ability to detect cytogenetic abnormalities, cases with low numbers of neoplastic cells often do not demonstrate abnormalities. Using a four-assay, nine-probe iFISH panel, we compared the abnormality detection rate for overnight unstimulated bone marrow cultures (ONC) to that for plasma-cell enriched fractions obtained with use of CD138-coated immunomagnetic beads (PCE). In the ONC, an abnormality was detected in 11 of 29 cases (38%); in the PCE, an abnormality was detected in 30 of 33 cases (91%). For 28 cases in which iFISH results from ONC were compared directly with PCE samples, the overall abnormality rate was 36% for ONC and 89% for PCE (P < 0.01). The conventional GTG-banded chromosome analysis revealed only 2 of 34 cases with an abnormal karyotype (6%); both cases were hyperdiploid. We conclude that the plasma cell enrichment step for iFISH should be incorporated into the routine cytogenetic work-up for all patients with plasma cell neoplasms.

Chronic basophilic leukemia: a rare form of chronic myeloproliferative neoplasm

Chronic basophilic leukemia is a rare and poorly characterized entity. Only a limited number of cases have been described. Herein, we report a patient who presented with fatigue, weight loss, leukocytosis, persistent prominent basophilia, and mild eosinophilia. The bone marrow showed features characteristic of a myeloproliferative neoplasm with a marked increase in maturing basophils. The basophils exhibited nuclear hypersegmentation, abnormal granulation, and abnormally low CD38 expression. Conventional karyotyping revealed a t(5;12)(q31;p13). ETV6 but not PDGFRB rearrangement was detected by fluorescence in situ hybridization.

Mosaic tetraploidy and transient GFI1 mutation in a patient with severe chronic neutropenia

This report presents the case of a 15‐year‐old male with severe chronic neutropenia, leukopenia, and persistent tetraploid mosaicism in the bone marrow and peripheral blood. His father had mild neutropenia and bone marrow tetraploidy. Flow cytometric analysis of DNA content peripheral blood showed tetraploidy in 20% of granulocytes and 15% of monocytes. Sequence analysis of the ELA2 gene was normal, but the GFI1 gene exhibited transient appearance of single base changes the coding region and promoter. We speculate that an underlying genetic defect, inherited in an autosomal dominant pattern, leads to both disordered mitosis and neutropenia in this kindred. Pediatr Blood Cancer 2008;50:630–632.

Eosinophilia with FIP1L1-PDGFRA fusion in a patient with chronic myelomonocytic leukemia

A 67-year-old woman with a past medical history of alfathalassemia trait was referred to our medical center for evaluation of anemia, thrombocytopenia, and leukocytosis. CBC on admission revealed a WBC of 55.5 10/L, platelets 81 10/L, and hemoglobin 11.3 g/dL. The differential showed 86% neutrophils, 6% monocytes, 0.1% eosinophils, 0% basophils, 8% lymphocytes with 1% circulating blasts, and dysplastic granulocytes noted on the blood film. An enlarged spleen was discovered on physical examination, and confirmed by a computed tomography scan. The initial bone marrow (BM) examination revealed 95% cellularity with trilineage dysplasia. The BM aspirate smears showed 3% blasts, 4% monocytes, and less than 1% eosinophils. Study of 20 giemsa-trypsin-giemsa– banded metaphase cells showed a normal female karyotype. The patient was diagnosed with myelodysplastic/myeloproliferative disease, with clinicopathologic features of atypical chronic myeloid leukemia and chronic myelomonocytic leukemia 1 (CMML-1). She was started on an experimental trial with combined thalidomide, arsenic trioxide, dexamethasone, and ascorbic acid. Two months later, her peripheral monocyte proportion increased and exceeded 10%, with absolute monocytosis (up to 52.6 10/L). Her diagnosis met the criteria for CMML-1. Four months after the initial BM biopsy, she developed peripheral eosinophilia, with an absolute eosinophil count ranging from 1.5 to 4.2 10/L. Her peripheral eosinophilia persisted for a period of 2 to 3 months under a close follow-up. BM biopsy now revealed a 100% cellularity with large clusters/small sheets of eosinophils (Fig 1A). The BM aspirate showed 4% blasts, 5% monocytes, and 10% eosinophils, including many eosinophilic precursors (Fig 1B). Conventional G-banding again showed a normal female karyotype. Fluorescent in situ hybridization (FISH) with a probe set containing FIP1L1 (green), CHIC2 (red) and PDGFRA (green) loci (MP Biomedicals, Solon, OH) was performed on interphase nuclei, and a hybridization pattern consistent with deletion of CHIC2 and fusion of FIP1L1-PDGFRA was observed in 16 of 100 nuclei. Figure 2 shows three nuclei containing abnormal fusion signals (arrows), with deletion of CHIC2 and a resultant FIP1L-PDGFRA (green/green) fusion. The patient was started on imatinib 400 mg orally daily, with a rapid resolution of her peripheral eosinophilia. However, her other hematologic indices showed no improvement. Imatinib was discontinued after 6 weeks. A repeated BM biopsy showed a 100% cellular marrow with only occasional eosinophils (Fig 1C). The BM aspirate revealed