Normal Metaphase Research Articles

The role of FLT3 in sole trisomy 8 acute myeloid leukemia.

6562 Background: FLT3 (fms-related tyrosine kinase 3) with internal tandem duplication (ITD) is a known adverse prognostic factor in normal karyotype acute myeloid leukemia (AML). We asked whether FLT3 ITD carries any prognostic significance in sole trisomy 8 AML. Methods: A total of 37 AML patients (25 males:12 females) with sole trisomy 8 were seen at Roswell Park Cancer Institute between 1991 and 2008. Treatment differed according to available protocols at the time. Results: Nucleophosmin (NPM1) was detected in only one case (FLT3 ITD/D835 negative). Interestingly, FLT3 D835 was detected in five (20%) of the 25 patients without FLT3 ITD but was not detected in any of the 7 FLT3 ITD- positive patients. A total of 22 (59%) patients achieved complete remission (CR). There was no difference in achievement of CR, overall or disease-free survival between trisomy 8 patients with or without FLT3 ITD. Of interest, patients with FLT3 ITD were significantly younger (mean: 50 year old; 95% confidence interval [CI]: 33.51, 66.49) compared to patients without FLT3 ITD (mean: 69.8 year old; 95% CI: 65.87, 73.73) (p = 0.006). In addition, patients with FLT3 ITD were less likely to have normal metaphases in their karyotype than patients without FLT3 ITD (86.7% [95% CI: 0.693, 0.963] vs. 42.8% [95% CI: 0.099, 0.816]; p = 0.027). Conclusions: The presence of FLT3 ITD in trisomy 8 AML was associated with younger age and presence of trisomy 8 in all analyzed metaphases when compared to those without FLT3 ITD. AML patients with trisomy 8 and FLT3 ITD had similar CR, overall and progression-free survival as those without FLT3 ITD. No significant financial relationships to disclose.

Abstract 3664: Bendamustine induces a dose-dependent cell cycle arrest in Hela cells that results in different mitotic outcomes

Abstract Bendamustine (BDM) was originally synthesized in the 1960s but has only recently been approved by the FDA for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. BDM consists of a bifunctional alkylating nitrogen mustard group and a benzimidazole ring and has been suggested to have both alkylator and purine analogue activities. Insights into the molecular mechanisms by which efficacy is achieved are scarce. Furthermore, the effects of BDM on solid tumors also remains under-investigated. Here we evaluate the effects of BDM on Hela cells and attempt to discern its effects on the cell cycle. Hela cells were treated with different concentrations of BDM, with the antimetabolite gemcitabine, or with the alkylator MMS. Interestingly, we found dose-dependent effects of BDM on cell cycle progression. Using synchronized cells, we observed that a low dose of BDM blocked cells in G2, similar to MMS, while a 4-fold higher dose of BDM arrested cells in S phase, as seen with gemcitabine. We next investigated the effect of abrogating the BDM-induced S and G2 cell cycle arrests with the Chk1 inhibitor UCN-01. To track the fates of cells time-lapse microscopy was conducted on cells expressing H2B: GFP. Cells were first arrested in G2 with low BDM or MMS before addition of UCN-01. Cells were observed to enter mitosis, align their chromosomes but then were delayed from entering anaphase. The cells eventually exited mitosis, with a small number of cells displaying lagging chromosomes. In contrast, cells arrested in S phase with high dose BDM or with gemcitabine, followed by UCN-01 showed highly aberrant mitotic figures. Although these cells were also delayed in mitosis, many of them died in mitosis rather than exit. We stained the mitotic cells generated by G2 or S phase inhibitors and found that cells arrested in G2 and forced into mitosis displayed normal looking metaphases, with aligned centromere/kinetochores on the spindle. However, the cells arrested in S phase followed by UCN-01 led to the generation of ‘mitosis with unreplicated genomes’ (MUGs). This phenomenon is characterized by fragmented mitotic chromatin and detached centromeres and kinetochores away from bulk DNA, as confirmed by electron microscopy (see accompanying poster Beeharry et al.). Furthermore, UCN-01 only induces formation of MUGs in cells arrested in with high BDM or gemcitabine. Although cells arrested in G2 by low BDM or MMS can also enter mitosis after UCN-01 treatment, they do not exhibit fragmented centromeres (MUGs). These observations are consistent with the notion that BDM can act as a nucleoside analogue or as an alkylator, depending on the dose. BDM must therefore inhibit different processes in a dose-dependent manner. Furthermore, tumor cell response may differ depending on the sensitivity of these two cellular processes to BDM. These findings maybe critical for predicting sensitization by inhibitors of Chk1 kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3664.

Telomere shortening and chromosomal instability in myelodysplastic syndromes

Telomere shortening and chromosomal instability are believed to play an important role in the development of myeloid neoplasia. So far, published data are only available on the average telomere length in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but not on the telomere length of individual chromosomes. We used a new technique, telomere/centromere-fluorescence in situ hybridization (T/C-FISH), which combines fluorescence R-banding and FISH using a probe against the telomere repeats to measure the telomere length of each chromosome arm in 78 patients with MDS. In line with the previous results, patients with MDS showed significantly shorter telomeres than those of healthy controls. Telomere lengths did not differ significantly between distinct morphological subtypes of MDS. However, there was a significant difference in telomere length between patients with an isolated monosomy 7 and patients with a normal karyotype (P < 0.05). Notably, patients with an isolated monosomy 7 showed significantly longer telomeres than patients with a normal karyotype in many chromosome arms, among them 7p and 7q. Neo-telomeres were found in two patients with a complex karyotype, in one case at the fusion site of a dic(14;20). Normal and aberrant metaphases of the same patient did not differ in telomere length, thus indicating to telomere shortening as a basic mechanism affecting all hematopoietic cells in patients with MDS. In some MDS subtypes, like MDS with isolated monosomy 7, telomeres may be stabilized and even increase in length because of the activation of telomerase or alternative mechanisms.

The Role of Cytogenetic Abnormalities in Acute Myeloid Leukemia with NPM1 mutations and No FLT3 Internal Tandem Duplication.

Abstract 1594Poster Board I-620 BackgroundNucleophosmin (NPM1) gene mutations are observed in almost one-third of adult acute myeloid leukemia (AML). If the favorable genotype defined by the presence of NPM1 mutations without fms-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) is known to be associated with favorable outcome in patients with cytogenetically normal (CN) acute myeloid leukemia (AML), impact of these molecular abnormalities are still debated in those with cytogenetically abnormal (CA) AML. Patients and MethodsWe analyzed the role of these factors in newly diagnosed AML patients homogeneously treated in the Acute Leukemia French Association (ALFA) trials between 1990 and 2006 (ALFA 9000, 9801, 9802). Among 1529 patients (15-70 years of age), 554 patients were screened for NPM1 mutation, FLT3-ITD and have informative karyotype. Mutations screening were centrally performed according to previously described procedures. Karyotype was considered as normal if at least 20 normal marrow metaphases were present without any abnormal marrow metaphases. Favorable core binding factor (CBF) AML (74 patients) were excluded from this analysis. ResultsAmong the 480 remaining patients, 137 (29%; median age 48 years, range 17-68) were NPM1 mutated, 22 of them (16%) having CA-AML. We found no difference between NPM1 mutated CN-AML patients (n=115) and NPM1 mutated non-CBF CA-AML patients (n=22) in term of complete remission rate (88% vs 83%; P= .49), overall survival (OS) (39% vs 38 % at 5 years, P= .85), event-free survival (EFS) (33% vs 25% at 5 years, P= .39). However, in the context of the favorable genotype, EFS was significantly longer in the 79 CN- than in the 16 CA-AML patients (41% vs 19% at 5 years ; P= .04) with a trend to better OS (51% vs 31% at 5 years ; P= .12). No significant differences were observed in patients with NPM1 mutated AML but FLT3-ITD (5-year EFS, 12% vs 40%; 5-year OS, 20% vs 60%; P= .47 and .27, respectively), even if the numbers of patients were here very low (35 CN- vs 5 CA-AML). More importantly, the favorable genotype was predictive of a longer EFS and OS in the 267 patients with CN-AML (5-year EFS, 41% vs 19%; 5-year OS, 51% vs 30%; P= .0001 and .001, respectively), while no differences were observed in the 213 patients with non-CBF CA-AML (5-year EFS, 19% vs 16%; 5-year OS, 31% vs 27%; P= .38 and .36, respectively). ConclusionAs shown, patients with the favorable genotype but CA-AML had an outcome relatively similar to those with a non favorable genotype and either CA- or CN-AML. The favorable outcome associated with NPM1 mutation in the absence of FLT3-ITD might thus depend on the presence of a normal karyotype. We thus think that larger studies or overviews are needed to definitely answer this question. DisclosuresDombret:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Near-Tetraploidy in Childhood Acute Lymphoblastic Leukemias Does Not Adversely Affect Prognosis: An EGIL Retrospective Study On 36 Cases Petr Lemez, Andishe Attarbaschi, Marie-Christine Bene, Yves Bertrand, Gian-Luigi Castoldi, Erik Forestier, Richard Garand, Oskar A. Haas, Emmanuelle Homolle, Sandrine Kagialis-Girard, Wolf-Dieter Ludwig, Estella Matutes, Marie-Pierre Pages, Winfried Pickl, Anna Porwit, Alberto Orfao, Jan Stary, Herbert Strobl, Pascaline Talmant, Mars B. Van't

Abstract 1585Poster Board I-611The first larger study on near-tetraploid (metaphases with 80∼104 chromosomes) acute lymphoblastic leukemias (NT-ALL) described 20 children: 9 of the T-lineage (T-ALL) and 11 of the B-cell precursors (BCP-ALL), and concluded that: “NT-ALL represented a unique subgroup of ALL, characterized by preponderance of T-cell immunophenotype, specific morphologic features, and possibly a poorer prognosis” (Pui et al, Blood 1990;76:590). The aim of our study was to enlarge the limited knowledge on these rarely occurring NT-ALL. The EGIL collected retrospectively laboratory and clinical data of 36 children (under the age of 18 years) with NT-ALL from 6 European countries diagnosed and treated in international studies between 1992 and 2008. Bone marrow blasts of all 36 children were negative for peroxidase. Immunophenotyping of leukemic blasts showed that 10 cases were diagnosed as T-ALL and 26 cases as BCP-ALL. In 4 cases aberrant near-diploid metaphases were simultaneously present. At least a single normal metaphase was found in 27 cases. Leukemic blasts of all patients were BCR/ABL1 negative. T-ALL was diagnosed in 7 boys and three girls of a median age 11.9 (3.6 – 17.5) years. CNS infiltration was found in one child. Heterogeneity of NT-T-ALL cases was apparent with immunophenotyping because they belonged to all 4 EGIL categories: TI or TII were each diagnosed in two cases; TIII or TIV each in three cases. Their blasts were negative for HLA-DR in all 10 cases and for CD34 in all 6 examined cases. Cytogenetic examinations revealed different structural abnormalities in 6 cases. All 10 cases achieved complete remission (CR) after induction chemotherapy and 8 are remaining in the 1st CR, exhibiting event-free survival (EFS) 80 % with a median follow-up of 37+ months. One case died of septic shock in pancytopenia after his 3rd consolidation therapy and survived only 7 months. Another case relapsed in the bone marrow and maxilla after 25 months. He reached the 2nd CR with chemotherapy and is alive after unrelated HLA-identical stem cell transplantation (SCT) for 14 months. Survival of NT-T-ALL cases has been favorable - 90 % with a median of 41 (7-120) months. NT-BCP-ALL was further divided according to the cytogenetic and molecular results into 4 categories. ETV6/RUNX1 positive BCP-ALL was diagnosed in 7 boys and 5 girls with a median age of 7.0 (3.3-15.5) years. Immunophenotyping of bone marrow blasts lead to the diagnosis of EGIL categories BII in 10 cases and BIII in two. Blasts from all cases were positive for HLA-DR and for CD34 in 8 of 9 cases. All these 12 cases have achieved CR after induction chemotherapy and are alive in the 1st CR for 18-147 (median 65) months. Twelve cases of ETV6/RUNX1 negative BCP-ALL fall to at least two categories: two cases exhibited simultaneously hypodiploid metaphases with NT ones, 10 cases had only NT metaphases. These 10 cases were 6 boys and 4 girls with a median age of 9.5 (4.4-14.5) years and WBC 0.9-13.0 × 109/l. Immunophenotyping of blasts established the diagnosis of BII in 9 cases and BIII in one. All tested cases were positive for HLA-DR and CD34, three expressed CD2 antigen. All these cases reached CR after induction chemotherapy and are remaining in the 1st CR for 12-155 (median 73) months. Hypodiploid ETV6/RUNX1 negative BII BCP-ALL was diagnosed in two girls. One with a karyotype of 44 chromosomes relapsed after 37 months and died of infection in the 2nd CR after unrelated allogeneic SCT. The other with 45 chromosomes reached CR and is remaining in CR for 7 months. Leukemic blasts of two boys, 9.5 year-old, with BII NT-BCP-ALL were not examined for ETV6/RUNX1. One is in the 1st CR for 82 months. Another relapsed twice after 6 and 11 years. He underwent cord blood SCT in the 3rd CR and died 2.5 months later of cerebral toxoplasmosis. In summary, our data show immunophenotypic, cytogenetic, and molecular heterogeneity of childhood NT-ALL. In contrast to previous reports, we observed favorable prognosis of NT-ALL patients after current more intensive induction and consolidation chemotherapy. Near-tetraploidy did not deteriorate prognosis of childhood NT-ALL cases across different immunophenotypic, cytogenetic and molecular ALL types.Supported by grants MSM0021620813, MZOVFN2005. DisclosuresNo relevant conflicts of interest to declare.

Use of array comparative genomic hybridization for prenatal diagnosis of fetuses with sonographic anomalies and normal metaphase karyotype

To prospectively study the addition of array comparative genomic hybridization (CGH) to the prenatal evaluation of fetal structural anomalies. Pregnant women carrying fetuses with a major structural abnormality were recruited at the time of invasive procedure for chromosome analysis. Only women whose fetuses had a normal karyotype (n = 50) were subsequently evaluated by array CGH using one of two arrays (1887 clones covering 622 loci or subsequently 4685 clones covering 1500 loci). The mean gestational age of the fetuses was 24.5 weeks (range 11-38 weeks). The most prevalent anomalies were cardiac, central nervous system, skeletal, and urogenital. The median turnaround time for culturing and array CGH diagnosis was 18 days (range 2-72). Four of 50 fetuses had abnormal array results. One (2%) was clinically significant and three (6%) were inherited or benign variants. Array CGH studies in fetuses with sonographic anomalies and normal metaphase karyotype detected clinically significant copy number alterations in 1 of 50 cases. This percentage (2%) is consistent with prior prenatal reports. Further studies are warranted to more precisely identify which fetal anomalies are associated with copy number alterations of clinical significance.

Meiosis in Solanum phureja

Meiosis and pollen mitosis were studied in a clone of Solanum phureja in order to elucidate the generally high frequency (about 90 %) of tetraploid progeny in tetraploid X diploid crosses involving S. phureja. The first meiotic division was normal with 12 bivalents in metaphase and apparently normal chromosome disjunction. The second meiotic division showed normal metaphases with 12 chromosomes in each of the two spindles, along with cells showing a bi- or tripolar spindle (cell wall formation was simultaneous). At the end of the second division those disturbances resulted in unreduced cells either with two diploid nuclei or with one diploid and two haploid nuclei along with the normal tetrads with four haploid nuclei. The frequency of unreduced gametes was about 50 %. First and second pollen mitoses were also represented by normal haploid as well as unreduced diploid cells. Hence, the high frequency of tetraploid offspring in Solanum tuberosum X S. phureja crosses could be caused by selective fertilization with unreduced diploid pollen grains.

The relationship of Sendai virus-induced chromosome pulverization to cell cyclus in HeLa cells.

Chromosome pulverization is a premature condensation of interphase chromatin (PCC) influenced by the cytoplasm of a metaphase cell after virus-induced cell fusion. In chromosome preparations of such syncytial cells, there is one set of normal metaphase chromosomes and the prematurely condensed chromosomes of one or several interphase nuclei. In this work the PCC in HeLa cells was investigated with special reference to the relationship between the morphology of PCC and the cellular phase of the affected interphase nucleus. The PCC was classified morphologically into three groups: (1) Pro- or prometaphase-like PCC where the chromatin condensed into continuous strands. (2) Intermediate PCC in which chromatin fragments of varying size were connected by thin strands. (3) Pulverized PCC where the condensed chromatin formed numerous small and irregular fragments. The chromatin of G2 cells condensed into pro- and prometaphase-like PCC. The PCC of G1 cells was mostly pro- and prometaphaselike as well, but could be distinguished morphologically from PCC of G2 cells. In addition, the chromatin of some G1 cells condensed into intermediate (16%) or pulverized PCC (13%). The prematurely condensed chromosomes of S-cells were mainly pulverized, but intermediate as well as pro- and prometaphase-like PCC of the same type as in G2 cells also occurred at low frequencies (15 and 8% respectively).

Chromosome Polymorphism and Fanconi-Like Instability in the Scarabaeid Beetle Macraspis tristis from Guadeloupe

The karyotype of Macraspis tristis Laporte is described. It is composed of 18 chromosomes. C-band positive heterochromatin is very abundant and is located at centromeric regions and, for some chromosomes, at telomeric regions. There is a high inter-individual chromosomal polymorphism for the presence and extension of telomeric heterochromatin. In one of the 8 specimens studied, 2 populations were observed in testicular cells. Besides groups of normal metaphases, other groups displayed multiple chromatid and chromosome alterations such as breaks, exchanges (radials), deletions and translocations, resembling those described in Fanconi anemia. The insect had a normal phenotype, but its gametogenesis did not reach the spermatocyte II stage, and was quite poor in spermatocytes I. The clonal character of the chromosome instability was obvious owing to the incomplete cytodiaeresis of germ cells which remain associated by cytoplasmic bridges. This may be the first example of chromosome instability observed in animals from nature.

A case of generalized MALT lymphoma with IgM paraproteinemia and peripheral blood involvement

Dear Editor,Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma) comprise awell-defined entity of non-Hodgkin lymphomas (NHL) butmay also share clinical and histopathologic findings withother low-grade lymphomas, thereby causing diagnosticproblems. A 35-year-old woman was diagnosed withHelicobacter pylori (HP)-positive gastritis and MALT lym-phoma in July 2007. Without further staging, HP eradicationtherapy was initiated; however, control endoscopy after6 months showed persisting lymphoma, and the patient wasreferred to our hospital for further evaluation and therapy. Shepresented with B symptoms, and computed tomographyscans revealed a diffuse gastric infiltration of antrum andcorpus, enlargement of epigastric lymph nodes, as well as an8×7 cm hypodense liver lesion and multiple opacities in bothlungs. All of these lesions showed increased FDG uptake bypositron emission tomography scan. Histologic examinationof the stomach, liver, and lung lesions confirmed thediagnosis of generalized MALT lymphoma exhibiting patchyinfiltrates of small centrocyte-like monoclonal B cells withinterspersed reactive and colonized follicles in the involvedorgans, forming typical lymphoepithelial lesions in the gastricmucosa with accompanying HP infection. Bone marrowbiopsy revealed minimal infiltration with lymphoma cells.Peripheral blood parameters showed a normoquantitativewhite blood cell count with 50% well-differentiated lym-phoctes and 5% lymphoplasmacytic cells and a hypochromicanemia with a hemoglobin level of 9.3 g/dl due to irondeficiency. Flow cytometry (FACS) of the peripheral bloodrevealed 18% CD19+/20+/slgM+/5-/10-leukemic lambda-clonal B cells. Serum immunoglobulin analysis showed amonoclonal paraprotein of immunoglobulin M (IgM) lambdatype (IgM 11.2 g/l; range 0.4 –2.3 g/l). Cytogenetic analysis ofthe peripheral blood cells revealed normal metaphases;however, interphase fluorescence in situ hybridization (I-FISH) revealed 20% cells showing three signals with probesfor the MALT and BCL-2 locus on chromosomal region18q21 and three signals with a probe for the EVI-1 locus onregion 3q26 consistent with duplications or (partial) trisomiesof these chromosomal regions. I-FISH with MALT/API2break apart probes showed no pathologic fusion signals, andconsistent with this finding, reverse transcriptase polymerasechain reaction performed on tumor specimens failed to detectMALT-API2 specific RNA fusions, thereby excluding theMALT lymphoma specific t(11;18)(q21;q21). Normal I-FISHsignals on the peripheral blood cells with an immunoglobulinheavy chain (IgH) break apart probe excluded the otherMALT specific IgH related translocations t(1;14)/BCL10-IgH, t(14;18)/IgH-MALT1 and t(3;14)/FOXP1-IgH. Due towidespread dissemination, chemotherapy according to the R-CHOP protocol was initiated, and after two cycles, thedifferential blood count and FACS analysis of the peripheralblood showed no pathologic findings; after a total eightcycles, a complete remission was achieved, indicated byhistologic examination of bone marrow and stomach biopsy

Radiation Protective Potentiality of Adhatoda vasica.

The present study forecasts the extensive damage caused by radiotherapy and chemotherapy treatment for cancer in the genetic setup of an individual. To counteract this damage, radioprotective potentiality of 50% Methanolic extract of Adhatoda vasica was evaluated through Cyto-Geno analysis.50% Methanolic extract of Adhatoda vasica leaves was used as a drug whose radioprotective potentiality was to be investigated. Human peripheral lymphocytes were cultured using RPMI-1640 media, harvested and observed under microscope for chromosomal aberration assay.Total 200 metaphases were counted for each group. All the metaphases were found to be normal in Control group. The frequency of normal metaphases greatly increased after pretreatment with both low and high drug dose (50mg/Kg body weight and 100mg/Kg body weight) concentration. The average normal metaphase in Radiation Control was 118 out of 200 metaphases but after pretreatment with 50% Methanolic extract of Adhatoda vasica the number of normal metaphase increased to 179 at 50mg/Kg body weight and 184 at 100mg/Kg body weight. It was concluded that 50% Methanolic Extract of Adhatoda vasica with both low and high drug doses have shown its potentiality as a radio protector against the therapeutically induced mutations which can prove to be a contributor in cancer management in future. Such indigenous Indian, herbal, cost effective, poor man friendly drug will definitely be a potential adjuvant to cancer treatments like radiotherapy and chemotherapy since Amifostine a well known radioprotector given to the patients at the time of cancer therapy is expensive and has its own side effects. Keywords: Radiation Protective, Adhatoda vasica, metaphases

The usefulness of interphase fluorescence in situ hybridization at diagnosis of myeloma in addition to metaphase cytogenetics

e19558 Background: Multiple myeloma is characterized by the accumulation of malignant plasma cells within the bone marrow and regarded as incurable, but remissions may be induced with steroids, chemotherapy, thalidomide and stem cell transplants. The clinical heterogeneity of myeloma is dictated by the cytogenetic aberrations present in the clonal plasma cells. Fluorescence in situ hybridization (FISH) overcomes the limitations of standard cytogenetics and allows for the detection of numerical and structural chromosomal abnormalities in both metaphase spreads and interphase nuclei. Methods: We evaluated the chromosome abnormalities in 34 MM patients using conventional cytogenetics and interphase FISH with 6 probes such as IGH/CCND1, IGH/FGFR3, IGH/MAF, DS13S319/LAMP1, IGH/BAP, and p53/CEP17. Results: Cytogenetic abnormalities were found in 24 (70.6%) of the 28 MM patients. 10 (35.7%) patients had abnormal metaphases by conventional cytogenetics. Interphase FISH results were abnormal in 21 (61.8%) patients and 11 (52.3%) patients had abnormal interphase FISH but normal metaphases. The evidence of the loss of D13S319 with or without loss of LAMP1 was found in 6 (21.4%) patients, and loss of p53±CEP17 for 2 patients, IGH-BAP for 9 (26.5%) patients, IGH/FGFR3 for 2 patients, and IGH/CCND1 for 7 (20.6%) patients, respectively. However, there were none positive for IGH/MAF. Chromosome 13 abnormalities and IGH rearrangement is correlated with poor clinical outcome. Conclusions: Interphase FISH can provide useful information to evaluate the presence of prognostic chromosome abnormalities in addition to metaphase cytogenetics. And it should be used in the routine evaluation of multiple myeloma. No significant financial relationships to disclose.

Long-term persistence of nonpathogenic clonal chromosome abnormalities in donor hematopoietic cells after allogeneic stem cell transplantation

We describe the cases of two unrelated patients who exhibited multiple chromosomal abnormalities in donor cells after allogeneic peripheral blood stem cell transplantation (PBSCT). The patients were diagnosed with chronic myeloid leukemia and chronic lymphocytic leukemia, respectively, and both underwent nonmyeloablative conditioning with cyclophosphamide and fludarabine followed by PBSCT from their HLA-matched opposite-sex siblings. Post-transplant bone marrow cytogenetics showed full engraftment, and the early post-transplant studies demonstrated only normal donor metaphases. Subsequent studies of both patients, however, revealed a population of metaphase cells with abnormal, but apparently balanced, donor karyotypes. Chromosome studies performed on peripheral blood cells collected from both donors after transplantation were normal. Both patients remained in clinical remission during follow-up of approximately 8 years in one case, and 6 years in the other case, despite the persistence of the abnormal clones. Chromosomal abnormalities in residual recipient cells after bone marrow or PBSCT are not unusual. In contrast, only rare reports of chromosome abnormalities in donor cells exist, all of which have been associated with post-bone marrow transplant myelodysplastic syndrome or acute leukemias. The present cases demonstrate the rare phenomenon of persistent clonal nonpathogenic chromosome aberrations in cells of donor origin.

Additional chromosomal abnormalities and their prognostic significance in adult Philadelphia-positive acute lymphoblastic leukemia: with or without imatinib in chemotherapy

The study analyzed the characteristics and prognostic significance of additional chromosomal abnormalities in 110 Chinese adults with Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukemia (ALL). Secondary aberrations were present in 60.9% of the cases. All chromosomes were involved in secondary aberrations, and chromosomes 9, 7, 21, 18, and 14 were most frequently abnormal. Fifty of 110 patients (45.5%) had at least one normal metaphase cell in their chromosome preparations at diagnosis. Patients with additional aberrations had shorter disease-free survival (DFS) and overall survival (OS) in chemotherapy combined with imatinib (ICT) group and only shorter DFS in conventional chemotherapy (CT) group. The existence of normal metaphase cells was associated with a superior survival in CT group, but not in ICT group. Patients with loss of chromosomes 7, 7p, 9, and 9p had inferior outcome compared to patients with other secondary aberrations and those without secondary aberrations, in both CT and ICT group.

The hierarchically organized splitting of chromosomal bands for all human chromosomes.

BackgroundChromosome banding is widely used in cytogenetics. However, the biological nature of hierarchically organized splitting of chromosomal bands of human chromosomes is an enigma and has not been, as yet, studied.ResultsHere we present for the first time the hierarchically organized splitting of chromosomal bands in their sub-bands for all human chromosomes. To do this, array-proved multicolor banding (aMCB) probe-sets for all human chromosomes were applied to normal metaphase spreads of three different G-band levels. We confirmed for all chromosomes to be a general principle that only Giemsa-dark bands split into dark and light sub-bands, as we demonstrated previously by chromosome stretching. Thus, the biological band splitting is in > 50% of the sub-bands different than implemented by the ISCN nomenclature suggesting also a splitting of G-light bands. Locus-specific probes exemplary confirmed the results of MCB.ConclusionOverall, the present study enables a better understanding of chromosome architecture. The observed difference of biological and ISCN band-splitting may be an explanation why mapping data from human genome project do not always fit the cytogenetic mapping.

A Case Report of An Unusual Case of Myelodysplatic Syndrome in a 26 year old Female

A 25 y/o African American female was referred to hematology clinic for abnormal lab values (Hemoglobin 8 g/dL, White cell count 2.7 k/uL, Platelets 86 K/uL, Mean corpuscular volume 106, and retic count 3.55) of several years for which she had not been thoroughly investigated. The patient reported she was always anemic since childhood. She had no new symptoms or abnormal bleeding except for fatigue and feeling weak. Peripheral blood review showed moderate anisopoikilocytosis. Few macrocytes, schistocytes, teardrop cells, ovalocytes and schistocytes, and polychromasia were seen. Leukocytes were markedly decreased. Small mature-appearing lymphocytes represent the predominant circulating cell type. Rare hyposegmented neutrophils were noted. No blasts were seen. Platelets were markedly decreased. Iron studies including Ferritin were all within normal, EPO level 282. Hemoglobin electrophoresis revealed a hereditary persistence of HgF (HgF=8.9%) with HbA0 of 89.5 L % (normal 97 – 99%) and Hb A2 1.6 % (normal 1 – 3%). Genetic test for thalassemia revealed: b- globulin negative and a-globulin deletion - consistent with Alfa thalassemia carrier state. Since the degree of anemia was disproportionate to alpha thalassemia carrier state, BM biopsy was obtained: morphological exam showed hypocellular bone marrow for age with an average similarity of 30–40%. There was a preponderance of erythroid precursors seen in clusters. No excessive blastic activity was present. Megakaryocytes appeared to be normal in number with a few showing mono/hypolobated nuclei. Bony trabeculae were unremarkable. Flow cytometry revealed no abnormalities. On cytogenetics, the following abnormal karyotype was found: 46,XX,add(5)(p15),add(6)(p21.3)[17]/46,XX,der(10)(1;10)(q21;p15)[3]. Thus, two abnormal and different clones with atypical aberrations were detected. One clone (85% of metaphases) exhibited additional material at 5p and 6p and the other clone (15%) showed a derivative chromosome composed of the two long arms of chromosome 1 and 10, with net gain of 1q and loss of 10p. No normal metaphases were detected. Peripheral blood flowcytometry for PNH was negative. The etiology of the patient's pancytopenia was not immediately apparent from the examination of the bone marrow specimen. Differential diagnoses that were considered included effects of drugs/toxins, or medications, or infectious process, but none was identified. However, the karyotypic abnormality strongly argues for the diagnosis of a clonal hematological malignancy, in particular a myelodysplastic syndrome. The cytogenetic abnormalities, although atypical, are indicative of a neoplasm involving the bone marrow. Gain of 1q has been observed mainly in myeloid malignancies and multiple myeloma. Presence of two different clones and absence of normal metaphases is usually indicative of unfavorable prognosis. BM evaluation was repeated about 2 months from the initial evaluation, it confirmed initial findings including the cytogenetic findings. The patient has been managed conservatively; she is being evaluated for Hematopoeitic Stem Cell Transplantation. She had required red cells transfusion only once for symptomatic treatment of fatigue and dyspnea.Conclusion: This case documents the rare diagnosis of myelodsyplastic syndrome (hypoplastic) in a young patient. MDS seems to be the likely diagnosis based on the clonal evidence that has been confirmed on two occasions in this case; of particular interest is the multiclonal process that exists in this patient.

Mechanisms of the Formation of Multinuclear Malignant Plasma Cells in the Novel AL/MM Human Cell Lines, ALMC-1 and ALMC-2: Implications for Tumor Cell Growth Control.

Malignant plasma cell (PC) disorders are clonal diseases based on their immunoglobulin variable region sequence signature. However, intratumor phenotypic, genetic, and morphologic heterogeneity is often observed, fueling the notion that a subpopulation of the malignant PC clone may have extensive self-renewal properties whereas others may not. Little information exists regarding the relationship between these putative tumor subpopulations that is instrumental to our understanding of the growth, progression and therapeutic targeting of PC disorders. We recently described establishment of two novel cell lines from one patient initially diagnosed with primary amyloidosis (ALMC-1), who eventually progressed to a combination of AL and symptomatic multiple myeloma (MM; ALMC-2). Although both cell lines are clonally related, they exhibit a number of intriguing differences, including a marked variability in their makeup of mononuclear (MoN) and multinuclear cells (2 − >10 nuclei; MuN). The ALMC-1 cell line consistently displays a much larger fraction of MuN cells (~50%) than the ALMC-2 cell line (~20%). By contrast, the growth rate of the ALMC-2 cells is significantly higher than ALMC-1 cells suggesting an inverse relationship between growth rate and fraction of MuN cells. It has been demonstrated that MoN plasmablastic morphology is a predictor of poor survival in MM, however the clinical significance of MuN PCs, which are frequently observed in bone marrow biopsies, has yet to be determined. We therefore used the ALMC-1 and ALMC-2 cell lines to begin to test the hypothesis that the MoN fraction in both cell lines contains cells with extensive self-renewal properties whereas the MuN cells are no longer capable of cell division. The central questions become by what mechanism are MuN PCs generated and how are MoN PCs maintained? Two hypotheses that can explain the dual persistence of both types of cells are:random cell fusion;regulated or sporadic cell division of MoN cells into 2 daughter cells, one of which divides normally and the other one fails to undergo cytokinesis.To address the first possibility, each cell line was transfected with GFP or YFP. Single positive (GFP or YFP) cells were then mixed and cocultured for assessing the frequency of dual positive cells by flow cytometry at various time points. However, studies to date suggest this mechanism does not occur. To begin to address the second possibility and determine whether extrinsic factors, e.g., cytokines, may regulate the rate of generation of MuN PCs, we cultured both cell lines +/− IL-6 and IGF-I and assessed numbers of MoN and MuN PCs. Initial analyses suggest that IL-6, but not IGF-I, increases the proportion of MoN PCs in both cell lines. To address the hypothesis that MuN PCs are no longer capable of growth, cells were labeled with bromodeoxyuridine (BrDU) to assess the capability of MuN cells to synthesize DNA. We observed that MuN PCs incorporating BrDU did so uniformly in each nucleus present in the cell. We then assessed the ability of cell division of MuN cells by quantitating normal vs abnormal metaphases in both cell lines and observed that >90% of metaphases were present in MoN PCs, suggesting that MoN PCs largely give rise to MuN PCs and the latter are likely compromised in cytokinesis. As centrosomes play a critical role in regulating cell division and centrosome abnormalities have been noted in MM by others, we next used gamma-tubulin immunofluorescence to detect centrosomes. MoN PCs exhibited uniformly normal centrosome numbers and positioning while MuN PCs showed clear centrosomal abnormalities including accumulation of centrosomes, centrosome clustering, and multiple directional centrosome polarization, suggesting a mechanism by which the MuN cells may be generated. In summary, our data suggest that MuN PCs lack growth potential and that intrinsic and/or extrinsic factors may drive accumulation of these cells. These observations support the possibility that patients with a larger proportion of MuN PCs may have differences in clinical outcome from those patients with largely MoN PCs. To test this, we have begun hematopathological analysis of a large series of patients diagnosed with various plasma cell disorders. Knowledge of the signals that regulate the balance between MoN and MuN cells could lend itself toward identification of therapeutic modalities that may maximize differentiation of tumor cells into end-stage cells.

STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation

We analysed the phenotypic outcome of a Stra8-null mutation on male meiosis. Because the mutant spermatocytes (1) underwent premeiotic DNA replication, (2) displayed cytological features attesting initiation of recombination and of axial-element assembly, and (3) expressed Spo11 and numerous other meiotic genes, it was concluded that STRA8 is dispensable for meiotic initiation. The few mutant spermatocytes that progressed beyond leptonema showed a prolonged bouquet-stage configuration, asynapsis and heterosynapsis, suggesting function(s) of STRA8 in chromosome pairing. Most importantly, a large number of mutant leptotene spermatocytes underwent premature chromosome condensation, within 24 hours following the meiotic S phase. This phenomenon yielded aberrant metaphase-like cells with 40 univalent chromosomes, similar to normal mitotic metaphases. From these latter observations and from the wild-type pattern of Stra8 expression, we propose that, in preleptotene spermatocytes, STRA8 is involved in the process that leads to stable commitment to the meiotic cell cycle.

Myelodysplastic syndromes associated with interstitial deletion of chromosome 5q: Clinicopathologic correlations and new insights from the prelenalidomide era

To better estimate prognosis for patients with myelodysplastic syndromes (MDS) associated with clonal interstitial deletions of the long arm of chromosome 5 (del(5q)), we reviewed the medical records of 130 adults with del(5q) MDS seen at our institution over a 15-year period. Overall median survival of this cohort was 9.5 months, shorter than reported in earlier series. The least favorable outcomes are associated with complex cytogenetics, lack of any normal metaphases, normocytic rather than macrocytic erythrocyte indices, and low baseline lymphocyte counts. Lymphopenia but not neutropenia at the time of diagnosis appears to be a new adverse prognostic indicator. Cytogenetic breakpoints defined by G-banded karyotyping correlate poorly with particular disease features. Surprisingly, survival of patients with treatment-related MDS was equivalent to that of de novo MDS with del(5q) in this series. Morphologic features associated with del(5q) are diverse. Most patients with del(5q) MDS do not meet criteria for WHO-defined 5q-syndrome, and the presence of del(5q) does not appear to modify the clinical phenotype otherwise risk-stratified by the International Prognostic Scoring System (IPSS). Additional important prognostic factors not taken into account by the IPSS include the baseline erythrocyte indices, lymphocyte count, and clonal burden.

Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment

To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics, spindle morphology, and chromatin alignment on metaphase plates. Experimental animal study. University animal laboratory. Eight-week-old B6D2F1 mice. Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr). Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment. Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% +/- 0% and 95% +/- 4%, respectively; mean +/- SE) and survival (95% +/- 2% and 98% +/- 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 37 degrees C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment, spindle morphology and length were not significantly different between the groups, nor was the incidence of aberrant alignment of chromatin on metaphase plates. Immediately after warming of vitrified MII oocytes, beta-tubulin is depolymerized and chromatin remains condensed on the metaphase plate. Within a 2-hour period, beta-tubulin repolymerizes, forming morphologically normal metaphase spindles with properly aligned chromatin.