CD4+ CD56+ cutaneous neoplasm with hematological relapse is a rare malignant disease and has been described recently in the literature as blastic or agranular NK-cell leukemia/lymphoma. The origin of this neoplasm is uncertain. We describe a 75-year-old patient with a primary cutaneous neoplasm CD4+ CD56+ who evolved to leukemic phase despite standard lymphoma chemotherapy. Morphologically, the cells were undifferentiated without granules in the cytoplasm. The immunophenotype showed the expression of CD4, CD56, CD68, CD33, CD7, CD2, CD45RA, and CD38. Histological analysis revealed a cell infiltration mainly located in the dermis. T-cell receptor and immunoglobulin heavy chain genes were in germline configuration. Cytogenetic study showed complex structural abnormalities with a deletion of the chromosome 5 del(5q). The clinical course was aggressive with an early hematological relapse.

The previous studies assessing the relationship of cytogenetic abnormalities to JAK2 status have not shown any significant associations, although numbers have generally been too small to make a definitive comment. We therefore assessed the JAK2 V617F mutation status of 337 patients from 39 centers (175 males, 161 females; median age at time of study 71 years old, range 32–98). Patients were classified as follows: 100 with typical MDS, 10 with 5q- syndrome, 17 with AREB-2, 2 with RARS, 13 with RARS-T, 23 with MPD/MDS, 12 with CMML, 142 with typical MPDs, 5 with hypereosinophilic syndrome (HES), 7 with AML with multi-lineage dysplasia from Ph- MPD, 10 with aCML Ph- or MPD/MDS-u and 25 with typical CML Ph+. Bone marrow or blood derived genomic DNA was screened for the JAK2 mutation. The JAK2 V617F mutation was found in 109/337 pts (32%): typical MPD-Ph− (47%) cases, MDS (4.5%), 5q- syndrome (60%), AREB-2 (64%), CMML (42%), MDS/MPD (36%). A clonal cytogenetic abnormality was detected by R-banding in 159/337 cases (47%). The JAK2 mutation was associated with a chromosomal abnormality in 81/159 cases (51%). The most common chromosome abnormality associated with JAK2 mutation was the gain of chromosome 9 (n= 22, Associated with JAK2 mutation: 19/22 cases 86%). The second most common abnormality was partial deletion of 20q (n=30, Associated with JAK2 mutation: 19/30 cases 63%). The partial or complete loss of chromosome 7 (n=16, Associated with JAK2 mutation: 6/11), deletion of 5q- (n=10, Associated with JAK2 mutation: 6/10), gain of chromosome 8 (n=11, Associated with JAK2 mutation: 7/11), partial deletion of 11q (n=8, Associated with JAK2 mutation: 5/8), partial deletion of 12p (n=6 Associated with JAK2 mutation 4/6), partial deletion of 13q (n=5, Associated with JAK2 mutation: 4/5). Patients with trisomy 9 and JAK2 mutation were classified as follows : polycytemia vera (PV) n=4 (100% JAK2 mutated), essential thrombocytemia (ET) n=3 (100% JAK2 mutated), idiopathic myelofibrosis (IMF), n=2 (100% JAK2 mutated), MPD/MDS, n=3 (67% JAK2 mutated), secondary AML post MDS, n=1 (JAK2 mutated), atypical MPD, n=6 (100% JAK2 mutated), MMCL, n=1(JAK2 mutated). The V617 mutation was not found in the case of de novo AML, and 2 typical MDS cases. 9 males, 10 females, the median age: 68 years old, range 40–98 years old, median white cell count (WCC) 11 G/l, range 2.4–50.7 G/l (13/19 pts> 10G/l), median hemoglobin 13.5 g/dl, range 9–20.2 g/dl (5/19 pts> 17g/dl), median platelet count 667G/l range 9–1769 G/l (9/19 pts> 500 G/l). The BCR/ABL transcript (multiplex PCR) was not detected in all of these cases. In this study the gain of chromosome 9 associated with JAK2 V617F mutation was the most frequent chromosome abnormality (100%) observed in typical MPDs and atypical syndrome such as MDS/MPD. In summary, previous studies assessing the relationship of cytogenetic abnormalies to JAK2 status did not show any significant association (ref). The del(20q), del(13q), trisomy 8 and del(5q) are known to be recurring non-specific cytogenetic abnormalities, and some of them are also detectable in patients with JAK2 mutation positive or negative. We describe here a significant association the JAK2 V617F mutation and trisomy of chromosome 9 that was detected in a cohort of patients with gain of chromosome 9. Clearly, in addition to PV, IMF, and ET these associations were found in other disease entities, high frequency in the case of atypical MPDs particularly in the case of aCML. Previously, Campbell et al. reported 10 patients with a trisomy 9, in typical MPDs, all of them were V617F+. A longer follow-up and morphological diagnosis is however necessary to determine the prognostic signification of JAK2 mutation and +9 in the cases of classic myeloproliferative syndromes and atypical syndrome such as MDS/MPD overlap syndrome.

5064 Introduction, Multiple myeloma (MM) is characterized by a huge clinical heterogeneity despite the homogenous morphologic appearance of malignant plasma cells (PCs). The advent of interphase fluorescence in situ hybridization (FISH) or MicroArrays (MA) allows an increased rate of aberration detection and identification of some recurrent cryptic changes, which have been increasingly implemented as additional diagnostic and prognostic factors. To heighten sensitivity of Single Nucleotide Polymorphism (SNP) arrays, or FISH it is necessary to have a purified population of cells as starting material. Screening must be performed systematically on the purified CD138+ PCs. After testing different systems for cell purification, we encountered some challenges. We didn't obtain enough PCs for FISH and SNP array studies. This was due to excess M-protein accumulating in the blood stream, increasing hyper viscosity and also due to the morphology and size variations of PCs at various stages of differentiation. Additionally, downstream DNA extraction can be a challenge since EDTA found in most buffers is an inhibitor for chemical PCR reaction for some MA chips. Given the challenges, CERBA laboratory and Miltenyi Biotec GmbH have developed a fully automated process (FAP) for purification for CD138+ PCs. In a study of 100 BM patient samples, we compared the specificity, efficiency, performance, purity, ease of use, technologists' time and the quality of DNA after CD138+ PCs purification. Two methods were compared. In the first method, cells were directly purified from bone marrow samples by FAP using Automated Magnetic Cell Sorter (AMCS). In the second method, mononuclear cells from fresh whole bone marrow (WBM) were enriched by Ficoll, followed by cell selection procedure with anti-CD138+ MicroBeads using the AutoMACS®. Before separation and following the separation, the percentage of PCs was determined by Flow cytometry (FC) on WBM by multiparameter FC (MFC) for CD138/CD38 expression. Additionally, DNA quality on separated cells was assessed by Nanodrop. A fraction of the CD138+ PCs were used after hypotonic shock and Carnoy fixation, applied to glass slides for FISH application and another fraction for DNA extraction for MA (SNP.6 Affymetrix®) FISH was performed with the recommended unbalanced alterations & reciprocal rearrangements: del(13) (q14)(D13S25), del(17)(p13)(TP53),+3(D3Z), +9(D9Z1), +15(D15Z14), t(4;14)(p16;q32)/IGH-FGFR3. Results, the specificity and purity were the same for both process but the efficiency and performance were considerably better for FAP than mononuclear cells enriched by Ficoll (MCEFicoll) process. With FAP, in 95% of the MM cases we obtained enough PCs for performance of the recommended panel of FISH and for 50% of them we could extract DNA for SNP array. For the MCEFicoll, we observed inferior performance, with very few plasma cells after isolation. Having enough PSc for only 65% of the cases and we could only extract DNA for 28% of them. The quality of DNA was the same for both process and the technologists' time was longer by 30' /patient for MCEFicoll process than for FAP. Currently in CERBA lab, we realize more than 20 plasma cells isolation per week for patients with MM and from October 2007 to July 2011 we have separated more than 5.000 specimens using CD138 Whole Blood MicroBeads (CD 138 WBMB) from Miltenyi Biotec, in combination with the AMCS. This has allowed isolation directly from WBM without any sample preparation required, such as density gradient centrifugation (ficoll) or erythrocyte lysis. The detection rate of chromosomal abnormalities and the number of abnormalities per case in MM and PCs dyscrasia significantly improves when there are enough CD138+PCs for analysis. Conclusion, in this report we describe the benefits of fully automated isolations of CD138+ cells from WBM. We have developed an SOP for an automated reliable and standardized method which allows the processing of multiple samples in a single day, while maintaining sample integrity and increasing sensitivity of FISH analysis and WG arrays for a diagnosis lab. Disclosures: No relevant conflicts of interest to declare.