Clonal Chromosomal Abnormalities Research Articles

Current Status of Allogeneic Hematopoietic Cell Transplantation for MDS

Hematopoietic cell transplantation (HCT) offers potentially curative therapy for patients with myelodysplastic syndromes (MDS). However, as most patients with MDS are in the 7th or 8th decade of life, only few of these were transplanted in the past, using high-dose conditioning regimens. The development of reduced-intensity conditioning has allowed to offer HCT also to older patients and those with clinically relevant comorbid conditions. Dependent upon disease status and the type of clonal chromosomal abnormalities present at the time of HCT, some 25%-75% of patients will be cured of their disease and survive long-term. Recent results with HLA matched unrelated donors are comparable to those with HLA genotypically identical siblings. The increasing use of cord blood and HLA haploidentical donors is expected to make HCT available to a growing number of patients. However, post-transplant relapse and graft-versus-host disease remain problems requiring further investigation.

Metaphase Harvest and Cytogenetic Analysis of Malignant Hematological Specimens

AbstractCytogenetic analysis of malignant hematological disease is an important methodology used by clinicians and researchers, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance. The Basic Protocol describes culture, harvest, and preparation of chromosome spreads from bone marrow aspirates. Three alternate protocols describe adaptations of the Basic Protocol for preparation of specimens from chronic lymphocytic leukemia (CLL) blood/bone marrow, plasma cell disorders, mainly bone marrow multiple myeloma (MM), and “solid” hematological samples including lymph nodes and splenic tissue. The modifications involve slightly different culture and harvest methods that are necessary to assure optimal results. Curr. Protoc. Hum. Genet. 73:10.2.1‐10.2.15. © 2012 by John Wiley & Sons, Inc.

MED12 mutations in uterine fibroids—their relationship to cytogenetic subgroups

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex 12 (MED12) have been detected in a majority of fibroids but no information is available whether or not they co-occur with cytogenetic subtypes as, e.g., rearrangements of the genes encoding high mobility group AT-hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients, we were not only able to confirm the frequent occurrence of MED12 mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of HMGA2 reflected by clonal chromosome abnormalities affecting 12q14~15 or by mutations affecting exon 2 of MED12. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED12 mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED12 mutations expressed significantly higher levels of the gene encoding wingless-type MMTV integration site family, member 4 (WNT4). Based on these findings and data from the literature, we hypothesize that estrogen and the mutated MED12 cooperate in activating the Wnt pathway which in turn activates β-catenin known to cause leiomyoma-like lesions in a mouse model. The occurrence of a "fibroid-type mutation" in a rare histologic subtype of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

A Case of Therapy-Related Acute Myeloid Leukemia Following 5-Fluorouracil Chemotherapy

A Case of Therapy-Related Acute Myeloid Leukemia Following 5-Fluorouracil Chemotherapy

T(1;3)(p36;p21) as the Sole Clonal Abnormality in Refractory Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with a multitude of molecular genetic aberrations and variable clinical outcome. Clonal chromosomal abnormalities have been identified in over 50% of AML cases, and have been regarded as one of the most important prognostic markers. We present a case of a 56-year-old Hispanic man with AML with minimal differentiation. Morphologically, the bone marrow was hypercellular with trilineage hypoplasia and 80% blasts. Flow cytometry analysis showed that the blasts were of myeloid immunophenotype. Conventional cytogenetic analysis showed t(1;3)(p36;p21) as the sole cytogenetic abnormality in 5 of 20 metaphases analyzed. The patient received daunorubicin and cytarabine, and achieved first remission. He relapsed 4 months later, and was treated with fludarabine, cytarabine, idarubicin, and G-CSF, and consolidated with high-dose cytarabine. He then received matched related stem cell transplantation. However, the disease relapsed again, and the patient died 11 months after initial diagnosis. To our best knowledge, this is the first report of t(1;3)(p36;p21) as the sole cytogenetic abnormality.

Novel Chromosomal Aberration as Evidence of Clonal Evolution in a Case of Relapsed Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with a multitude of molecular genetic aberrations and variable clinical outcome. Clonal chromosomal abnormalities have been identified in over 50% of AML cases, and have been regarded as one of the most important prognostic markers. We present a case of a 28-year-old Caucasian woman with AML without maturation, diploid karyotype, that was resistant to multiple chemotherapies and relapsed after matched unrelated stem cell transplantation. Conventional cytogenetic analysis performed on bone marrow specimens revealed 46,XX,t(2;16)(p21;p11.2),t(11;14)(p13;p11.2). The t(11;14)(p13;p11.2) was confirmed by fluorescence in situ hybridization using a whole chromosome paint probe for chromosome 11. Morphologically, the bone marrow was hypercellular with trilineage hypoplasia and 84% blasts. Flow cytometry analysis showed that the blasts were of myeloid immunophenotype. Molecular studies detected internal tandem duplication of the FLT3 gene and a mutation in exon 12 of the NPM1 gene. The patient then received monotherapy with AC220, achieved a brief remission, and died of relapsed disease 23 months after initial diagnosis. This is the first report of this novel clonal chromosome aberration as evidence of clonal evolution in AML. [N A J Med Sci. 2012;5(1):48-50.]

Pathogenesis of aplastic anaemia and paroxysmal nocturnal haemoglobinuria: the concept of clonality in stem cell disease

Molecular technologies have been applied not only to the investigations of haematological malignancies but also bone marrow failure syndromes, including aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and single lineage cytopenias, often associated with T cell large granular lymphocyte leukaemia (T-LGL). One of the main diagnostic problems is the distinction of hypoplastic clonal processes consistent with the diagnosis of myelodysplasdtic syndrome (MDS) from true idiopathic AA. Due to the paucity of the marrow, pathomorpho-logical diagnosis may be difficult and metaphase cytogenetics may not yield informative results, precluding proper diagnostic case assignment. Single nucleotide polymorphisms arrays (SNP-A) do not require cell division and may help to detect clonal chromosomal abnormalities. Systematic application of this technology has shown that chromosomal defects consistent with the diagnosis of MDS can be detected in a proportion of otherwise typical AA patients, initially or throughout the clinical course. At presentation, clonal abnormalities, in particular non-recurrent/random defects may be transient and reflect oligoclonality of the stem cell compartment rather than true malignant clonal process. Using SNP-A, submicroscopic chromosomal microdeletions can be detected; we have found recurrent microdeletions involving HLA and PIG-A loci on chromosomes 6 and X, respectively. These lesions likely evolve as a result of immune selection pressure and facilitate escape of the immune-resistant haematopoietic clones. Manifest evolution of MDS in the course of AA can be expected in up to 20% of patients with AA. The most common abnormality is monosomy-7, present in most cases of MDS derived from antedescent AA. In this context, monosomy-7 is associated with a distinct mutational pattern that includes presence of CBL mutations and RUNX1, while common mutations such as TET2, U2AF1, SRSF2 and ASXL1 are frequent in primary MDS with monosomy-7 or deletion7q. In contrast, childhood cases of juvenile myelomonocytic leukemia with monosomy-7 have a similar mutational pattern as seen in AA. Whole genome sequencing technologies including exome sequencing have led to identification of new mutations, not only in MDS but also in T-LGL which is characterised by frequent somatic activating mutation in STAT3. By ultra-deep sequencing of T cell receptor (TCR) VB chains, one can also characterise TCR repertoire and identify oligoclonal T cell mediated processes. The presence of STAT3 mutations distinguish clonal processes from semi-reactive oligoclonal T cell expansions.

Allogeneic Hematopoietic Cell Transplantation for Myelodysplastic Syndrome: Current Status

Hematopoietic cell transplantation (HCT) offers potentially curative therapy for patients with myelodysplastic syndrome (MDS). However, as the majority of patients with MDS is in the 7th or 8th decade of life, only few of these patients were transplanted following high-dose conditioning regimens. The development of reduced-intensity conditioning has allowed to apply HCT also to older patients and those with clinically relevant comorbid conditions. Dependent upon disease status and the type of clonal chromosomal abnormalities present at the time of HCT, some 25-75% of patients will be cured of their disease and survive long term. Recent results with HLA-matched unrelated donors are comparable to those with HLA genotypically identical siblings. The increasing use of cord blood and HLA-haploidentical donors is expected to make HCT available to a growing number of patients. However, post-transplant relapse and graft-versus-host disease remain problems requiring further instigations.

Spectral Karyotyping (SKY) Reveals a New Subset of MDS Patients with Clonal Chromosomal Abnormalities Not Detected by G-Banding Analysis

Abstract 1718Cytogenetic findings in bone marrow cells of MDS patients are essential for a correct diagnosis and classification of the disease and constitute one of the most important independent prognostic factors. The classical cytogenetic analysis, however, often cannot be fully resolved by G-banding because of the presence of marker chromosomes, rings or unidentified material attached to chromosomes. Spectral karyotyping (SKY) has proven to be an important tool for the interpretation of complex karyotypes or identification of suitable abnormalities in hematological malignancies. By using SKY analysis in combination with G-banding were identified new clonal chromosomal abnormalities “masked” by the limited resolution of classical cytogenetic. As a consequence changes in IPSS score were observed. Bone marrow samples of 46 (forty-six) MDS patients were incubated in RPMI 1640 with 20% fetal calf serum for 72h at 37°C. Chromosome preparations were obtained by using standard procedures and the subsequent cytogenetic analysis and interpretation were made according to ISCN 2009. The patients studied were classified as refractory anemia (RA) and refractory anemia with ringed sideroblast (RARS), with less than 5% blast. Slides for SKY were prepared by using the same fixed chromosome preparations, stored at −20°C, as employed for G-banding analysis. Chromosome labeling was performed with the SKY fluorescent labeling kit (Applied Spectral Imaging, Migdal HaEmek, Israel) according to the manufacturer’s protocol. A minimum of twenty metaphases were analyzed using the SkyView 5.5 software (ASI, Carlsbad, CA, USA). In a group of 46 subjects studied, the cytogenetic analysis (G-banding) showed chromosomal aberrations in 13 patients (54.2%) and normal karyotype was observed in 11 subjects (45.8%). The abnormalities observed were dup(1)(q21q32), inv(3)(q21q26), t(3;3)(q21;q26), +4, del(5)(q31), −7, del(7)(q22q36), +8, add(17)(p12), +i(17)(q10), del(20)(q11). The group with normal cytogenetic, SKY analysis revealed “masked” chromosomal abnormalities in 6 patients, being t(7;9)(q36;q34), ins(1;6)(q21;?), t(11;12)(p15;q24.1), ins(3;5)(p21;?), t(8;16)(q23;?) and ins(6;11)(q21;?). Among 13 cases studied with previous chromosomal abnormalities by G-banding analysis, SKY identified additional abnormalities in 8 patients. Some abnormalities found include t(6;9)(q27;q22), t(12;17)(p13;p12) and t(8;11)(p12;q12). For both groups with normal and altered karyotypes, the profile of masked chromosomal abnormalities seen were insertions and translocations involving small segments of chromosomes. In the majority of the cases the frequency of abnormal clones was less than 50%. However, in all patients the abnormalities identified by SKY were classified as clonal. All abnormalities identified were confirmed by FISH, by using a set of probes. SKY analysis has proved to be a promising and reliable method for identification of additional and complex chromosomal abnormalities usually present in a great number of human neoplasias. The contribution for the prognostic information of these new chromosomal abnormalities identified beyond the limited resolution of G-banding in MDS will require a detail analysis of the patients’ evolution.Financial Support: FAPESP (Proc. 07/52462-7) Disclosures:No relevant conflicts of interest to declare.

Frequency and Prognosis of Clonal Chromosomal Abnormalities Following Stem Cell Transplantation (SCT) in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS)

Abstract 4897 Objective.To study the frequency, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML and MDS. Patients and methods.One hundred thirty patients were studied between 2000 and 2010. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2009. Results.Clonal abnormalities were observed in 36/130 patients (28%) with a median follow-up was 11 months (range 3–131). Initial diagnosis (OMS 2008): AML with maduration (8 patients), acute erythroid leukaemia (4), acute monocytic leukaemia (3), AML with multilineage dysplasia (3), AML with myelodysplasia-related changes (3), AML with inv(16)(p13q22) (2), acute monoblastic leukaemia (2), AML with minimal differentation (2), AML without maduration (1), acute myelomonocytic leukaemia (1), refractory anaemia with excess blasts (4), chronic myelomonocytic leukaemia (2) and refractory cytopenia with multilineage dysplasia (1). Treatment before SCT: idarubicin, cytarabine, etoposide and mitoxantrone (25), idarubicin, cytarabine and etoposide (6), idarubicin, cytarabine, etoposide and gemtuzumab (2), FLAG-ida (2) and azacitidine (1). SCT type: autologous (23 patients), allogeneic of reduced intensity (7), allogenic (4), umbilical cord blood (1) and syngeneic transplant (1). Conditioning regimen: TBI and cyclophosphamide (24 cases), fludarabine and busulfan (7), busulfan and cyclophosphamide (4) and thymoglobulin, thiotepa, fludarabine and busulfan (1). The median time between SCT and the appearance of clonal abnormalities was 6.5 months (range 2–51). At the time of clonal abnormality detection, 32 patients were in cytological and/or clinical relapse and 4 in cytological remission. In 15 cases the initial clone reappeared, 2 showed the initial abnormalities with an acquired abnormality and 19 presented de novo clonal abnormalities (53%). The most frequent clonal chromosomal abnormalities observed were: complex karyotype (22%), estructural abnormalities with afection of chromosomes 1, 4, 6, 7, 10, 11, 12, 16 and 17 (22%), +21 (5%) and +11 (3%). The median survival from the appearance of clonal abnormalities was 2.5 months (range 1–100). There were no differences when we compared the survival of patients with de novo clonal abnormalities with that of patients with initial abnormalities (including those with acquired abnormality). Furtheremore there were no differences when we compared the median survival of relapsed patients with normal Karyotype with that of patients with clonal chromosomal abnormalities following SCT (2.5 months [range 1–25] and 2.5 months [range 1–100] respectively). At the time of the analysis only 1 patients are alive and in complete remission (100 months). Conclusions.1. The appearance of clonal abnormalities following SCT in patients with AML and MDS is frequent and the majority of cases are detected at the time of relapse. Half of cases presented de novo clonal abnormalities and the majority were not related with prior irradiation exposition, alkylating agents neither topoisomerase II inhibitors. The prognosis of patients with acquired clonal abnormalities after SCT is poor. Disclosures:No relevant conflicts of interest to declare.

Therapy-Related Myeloid Neoplasms (t-MN) in 71 Patients Following Radiation Therapy (RT) Only,

Abstract 3522Exposure to ionizing radiation has long been associated with the development of secondary malignant neoplasms including therapy-related myeloid neoplasms (t-MNs). We examined the clinical, epidemiologic, and cytogenetic characteristics of 71 consecutive cases of t-MN presenting to the University of Chicago between 1972–2011 in patients (pts) who received radiation therapy (RT) alone for a malignant or non-malignant disease; one-third had been treated for their primary disease at our institution. No subsequent chemotherapy had been given for the primary disease. 64 (90%) pts had had a primary solid tumor, 5 had had a primary hematologic malignancy (4 Hodgkin lymphoma), and 2 had received RT for non-malignant disorders (acne; hydatiform mole). Prostate and testicular cancers were the predominant primary tumors (n=27; 38%). Breast (n=15; 21%) and gynecological cancers (n=13; 18%, including ovarian, cervical, endometrial, and vaginal cancers) were also common. There were 36 women and 35 men; 50 white (70%), 10 African American (14%), 1 Asian American (1%), and 10 with unrecorded race/ethnicity. The median age at primary diagnosis was 64 years (range, 0–83 yrs). Pts with solid tumors were older at primary diagnosis (median, 66 yrs; range, 0–83) compared to pts with primary hematologic malignancies (median, 48 yrs; range, 32–67) or no malignancy (median, 22 yrs) (p= 0.01). Median latency from RT to the first confirmed diagnosis of t-MN overall was 67 months (interquartile range (IQR), 27–120 mos). Patients with a hematologic malignancy had shorter latency intervals (median, 25 mos; IQR, 18–50) compared to pts with solid tumors (median, 68 mos; IQR, 31–117) (p=0.017, Kruskal-Wallis test). Younger age at primary diagnosis correlated with longer latency intervals (p=0.0013, Spearman’s rank correlation test). However, this apparent association may be attributed in part to competing risk factors such as medical comorbidities and death in older pts prior to the development of t-MN. Gender was not associated with latency intervals. 59 pts had one or more clonal cytogenetic abnormality (83%) at diagnosis of t-MN; 12 (17%) had no detectable abnormality. Most common (38 pts, 54%) were translocation, loss, or deletion of chromosome 5 (n=19), 7 (n=8), or both (n=11). Ten patients (14%) had recurring balanced translocations previously reported in t-MN; 4 with inv(16) or t(16;16), 4 with t(15;17), and 2 with t(21q22). Eleven pts (15%) had other clonal abnormalities. No significant correlations were found between cytogenetics and age or type of primary diagnosis. Only 6 pts currently remain alive: 2 with inv(16), 1 with t(16;16), 1 with t(15;17), 1 with independent -7 and del(11q) clones, and 1 with a normal karyotype. The median time from diagnosis of t-MN to death was 10 months (95% confidence interval (CI), 7–14 mos). Pts with normal cytogenetics or recurring balanced translocations survived longer (log rank, p=0.0009). t-MN with clonal abnormalities of chromosome 5, 7, or both had a poor median survival of 7 mos (95% CI, 3–12 mos) compared to other cytogenetic groups. There was no association between age at diagnosis of t-MN and overall survival. Treatments given after the development of t-MN were individualized and variable, ranging from supportive care only to intensive chemotherapy and subsequent hematopoietic cell transplantation. We reviewed treatment records for 38 pts: 18 received low intensity treatment with best supportive care or participated in a phase I clinical trial; 20 underwent high intensity induction chemotherapy with or without hematopoietic cell transplantation. Pts in the low intensity treatment group had a median survival of 8 mos (95% CI, 3–11 mos) compared to pts in the high intensity treatment group who had a median survival of 14 mos (95% CI, 5–30 mos) (log rank, p=0.12). We conclude that t-MN following RT alone bears striking clinical and cytogenetic similarities to alkylator-associated t-MN with frequent clonal abnormalities of chromosomes 5 and 7, relatively long latency, and poor outcomes even with intensive therapy. However, some pts who develop t-MN after RT alone have recurring, balanced chromosomal translocations or normal karyotypes, and they have a better response to anti-leukemia treatment and longer survival. Disclosures:No relevant conflicts of interest to declare.

Significance of Bone Marrow Karyotype and Morphology in Patients with Inherited Bone Marrow Failure Syndromes,

Abstract 3431Recurring clonal cytogenetic abnormalities have been described in patients with Fanconi anemia (FA) and Shwachman-Diamond syndrome (SDS). In FA, gains of 3q and monosomy 7 (−7) imply progression to myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). In SDS, isochromosome 7q and deletion (del) 20q are usually benign. Dyskeratosis congenita (DC) and Diamond-Blackfan anemia (DBA) do not have unique clones. We report here the types and frequencies of cytogenetic clones and their association with morphologic MDS or AML in the major inherited bone marrow failure syndromes (IBMFS), in a prospective/ retrospective study of patients with FA, SDS, DC and DBA enrolled in the NCI IBMFS cohort from 2002–2010. Bone marrow (BM) morphology and cytogenetics (G-banding; selected FISH, CGH, SKY) performed at our institute and all outside cytogenetics reports were centrally reviewed. Cytogenetic abnormalities were defined and karyotypes designated according to ISCN (2009). Two independent blinded hematopathologists reviewed BM morphology. Diagnosis of morphologic MDS was based on a modification of WHO 2008 and required ≥10% dysplasia in 2 cell lineages. Data analysis was both cross-sectional and longitudinal. P values are global comparing all 4 disorders using Fisher’s exact test.ParameterAll IBMFSFASDSDCDBAP valueTotal number (N)12835113646–N with clone ever2817 (49%)4 (36%)4 (11%)3 (7%)<0.01N with MDS ever105 (14%)3 (27%)1 (3%)1 (2%)0.01N with clone + MDS75 (14%)2 (18%)00<0.01N with clone alone2112 (34%)2 (18%)4 (11%)3 (7%)<0.01N with MDS alone301 (9%)1 (3%)1 (2%)0.3N with clone at 1st BM179 (26%)4 (36%)3 (8%)1 (2%)<0.01N with clones at follow-up118012<0.01N with follow-up BMs591791716–Median follow-up in years3 (0–19)6 (1–16)2 (1–6)3 (0–19)2 (0–10)–More FA and SDS patients had clones and/or MDS compared with DC or DBA (Table). MDS was always associated with clones in FA but not in the other IBMFS.In FA, bone marrow transplant (BMT) or death occurred with similar frequencies in those with or without clones. Among 17 patients with clones, follow-up cytogenetics were unavailable in 5; of these, 2 with clone alone [one with del 7q and 18p and one with t(3;6)(q?25;p?21)] progressed to AML, while one with clone and MDS died from other causes. Recurring abnormalities in 12 FA patients with clones followed for up to 16 years, included gains of 1q in 4, −7 or del 7q in 3, and deletions of 6p, 13q, 18p and 20q in 2 patients each; only one had gain of 3q. These patients showed fluctuation or disappearance of clones, new appearance of clones, stable clone, or clonal evolution. Progression to MDS occurred with gain of 1q and 6p deletion, gain of 3q, or −7 in 3 patients, respectively; one patient with MDS had clonal persistence. No disease progression was seen in 5 FA patients with clone alone.All 5 SDS patients with clones and/or MDS are alive with no disease progression. The 4 with a clone had stable persistent del 20q as a sole abnormality; 2 had MDS and 2 did not. One had MDS with a normal karyotype.Four DC patients had abnormal clones including 2 with gain of 1q only. One patient with 1q gain died from pulmonary fibrosis. Three others are alive; 2 with stable clones at 7 and 19 years' follow-up, respectively. One additional DC patient has morphologic MDS but no clone.All 3 DBA patients with clones had del 16q, 2 alone and 1 with del 9p; none had MDS. The clones were transient in 2, disappearing within 1–2 years; the third was recently identified. None of these had disease progression. One patient with morphologic MDS alone died from complications of iron overload.This study shows that clonal chromosome abnormalities occur more frequently in FA and SDS than in DC and DBA. Gain of 3q in FA was not as common here as reported by others. This is the first comprehensive study of clones and MDS in DC and DBA. Strengths of this study include the large number of patients, and central review of cytogenetics and morphology. It is unbiased compared with FA literature reports that include many patients referred for BMT. Limitations include a relatively small number of patients with each diagnosis and short follow-up in most. The study demonstrates that clones may fluctuate or disappear, and may not per se portend a bad prognosis. Progression to clinically significant MDS or AML may be related to the severity of cytopenias and not to clone alone, and warrants more extensive long-term studies. Disclosures:No relevant conflicts of interest to declare.

Diagnosis and Treatment of Acquired Aplastic Anaemia in Adults: 142 Cases from a Multicentre, Prospective Cohort Study in Shanghai, China

To improve diagnosis and therapy for aplastic anaemia (AA) in Shanghai, clinical and laboratory data for patients with AA (n = 142) and hypocellular myelodysplastic syndrome (MDS; n = 22) were comparatively analysed (follow-up 2 - 6 years). Red blood cell distribution width and absolute lymphocyte and reticulocyte counts were significantly different between the two groups. AA was diagnosed in 54.2% of patients using a single bone marrow aspirate smear plus peripheral haemogram results, and in 95.1% using an additional bone marrow biopsy; 4.9% required multiple-site bone marrow examination. Clonal chromosomal abnormalities occurred in 3.9% and 31.8% of patients with AA and MDS, respectively. In patients with severe AA, 12.0% received antithymocyte globulin (ATG) + cyclosporin A (CSA; effectiveness rate 77.8%; 5-year survival 74.1%), 45.3% received CSA + androgen therapy (effectiveness rate 58.8%; 5-year survival 76.5%) and 26.7% received androgen monotherapy (effectiveness rate 25.0%). Multivariate analysis of prognostic factors indicated that therapy regimen and blood platelet count affected survival. Peripheral blood smears, bone marrow spicule classification and biopsy are important diagnostic factors. Standardization of evidence-based therapy and promotion of ATG + CSA would improve general therapeutic effects in AA.

Cytogenetic Analysis of CpG-Oligonucleotide DSP30 plus Interleukin-2-Stimulated Canine B-Cell Lymphoma Cells Reveals the Loss of One X Chromosome as the Sole Abnormality

Human and canine lymphoid neoplasms are characterized by non-random cytogenetic abnormalities. However, due to the low mitotic activity of the B cells, cytogenetic analyses of B-cell lymphoid proliferations are difficult to perform. In the present study we stimulated canine B-cell lymphoma cells with the immunostimulatory CpG-oligonucleotide DSP30 in combination with interleukin-2 (IL-2) and obtained an adequate number of metaphases. Cytogenetic analyses revealed the loss of one X chromosome as the sole cytogenetic aberration. Chromosome analysis of the corresponding blood showed a normal female karyotype. Monosomy X as the sole clonal chromosomal abnormality is found in human hematopoietic malignancies as well, thus the dog may serve as a promising animal model.

IDENTIFICATION OF E6A2 BCR-ABL FUSION IN A PHILADELPHIA-POSITIVE CML WITH MARKED BASOPHILIA: IMPLICATIONS FOR TREATMENT STRATEGY

This is a case report of a 51 year old male with marked splenomegaly, basophilia, severe thrombocytopenia, anemia and high SFKL phosphorylation downstream of Bcr-Abl, investigated for association of the e6a2 BCR-ABL fusion gene and marked basophilia. The treatment strategy implications in patients with Philadelphia positive CML are described. RT-PCR and sequencing were carried out on the peripheral blood leukocytes to detect the type of BCR-ABL transcript. The BCR-ABL mutational status was assessed using sequencing of the RT-PCR products. The in vitro test of sensitivity to TKIs was based on detecting inhibited phosphorylation of the Crkl and Phospho-Src family kinases (SFK, Tyr416) using immunodetection. The cytogenetics revealed 90% of Ph+ (Philadelphia) cells in the bone marrow aspirate with no additional clonal chromosomal abnormalities at diagnosis. This correlated with an accelerated phase of the CML. Sequencing analysis of reverse transcribed and PCR amplified BCR-ABL transcript revealed a rare e6a2 fusion, with no evidence for Bcr-Abl kinase domain mutation. Western blot analysis showed high phosphorylation (activation) of Crkl and the Src family of kinases (P-SFK). In vitro test of sensitivity of the patients' leukemic cells to imatinib demonstrated sensitivity of Bcr-Abl tyrosine kinase to imatinib, as assessed by a decrease in phosphorylated Crkl and the disappearance of P-SFK, suggesting that P-Src reflects only the Bcr-Abl-dependent Src activity. The initial treatment strategy was reduced imatinib and search for an unrelated hematopoietic stem cell donor (according to the ELN recommendations). The patient was allografted with peripheral stem cells from an HLA- identical male donor but on day +70 graft failure occurred. He was allografted again with the peripheral stem cells from an HLA-identical female donor, engrafted on day +15 and showed 100% donor chimerism with no evidence of the e6a2 BCR-ABL fusion transcript on day +30. The clinical disease course in patients with the rare e6a2 BCR-ABL transcript variant is aggressive. This may be the result of increased kinase activity due to partial loss of the guanine exchange factor/dbl-like domain which mediates the interaction with several Ras-like G-proteins involved in cell proliferation, signal transduction, and cytoskeletal organization. For the above reasons, these patients should receive stem cell transplant immediately after a short course of treatment with imatinib/ dual Src/Abl kinase inhibitor or they should be registered in clinical trials with experimental agents.

127 Spectral karyotyping (SKY) reveals a new subset of MDS patients with clonal chromosomal abnormalities not detected by G-banding analysis

127 Spectral karyotyping (SKY) reveals a new subset of MDS patients with clonal chromosomal abnormalities not detected by G-banding analysis

Short tandem repeat and human leukocyte antigen mutations or losses confound engraftment and typing analysis in hematopoietic stem cell transplants

Clonal chromosomal abnormalities are often found in the tumor cells of patients with malignancies. These abnormalities can cause downregulation of human leukocyte antigen (HLA) and instability of short tandem repeat (STR) DNA sequences, confounding HLA typing and/or engraftment analysis in hematopoietic stem cell transplants (HSCT). We describe here the abnormalities observed during testing of 600 HSCT patients. HLA molecular typing was performed by reference strand conformational analyses and/or sequence-based typing. STR testing was performed with 10 to 16 STR primer sets, following 1 to 4 informative loci in each patient. Eight patients exhibited either loss of heterozygosity (4 STR, 3 HLA) or STR length mutation ( n = 1), and 5 of the 8 exhibited correlative cytogenetic abnormalities. Diagnoses were acute myelogenous leukemia (AML; n = 7) or myelofibrosis (MFIB: n = 1), yielding an 11% incidence of these chromosomal abnormalities among the subset of 72 AML/MFIB HSCT patients. These results highlight some of the problems encountered and the possibility for interpretive errors that can arise when analyzing molecular typing and engraftment data, particularly among AML/MFIB patients.

NPM1 gene deletions in myelodysplastic syndromes with 5q- and complex karyotype

The deletion of 5q (5q-) is a frequent clonal chromosomal abnormality detected in patients with myelodysplastic syndromes (MDS). MDS with 5q- as a sole chromosome alteration is characterized by a more favorable prognosis when compared to other forms of MDS.[1][1] By contrast, when detected in

Molecular Genetics and Cytogenetics in Cancer

This special issue of Genetics Research International focuses on molecular genetics and cytogenetics in cancer. The hunt for informative disease-related genetic biomarkers may become so valuable for the differential diagnosis, prognosis, and disease monitoring of cancer patients that numerous researchers are committed to this challenge. Indeed, a colossal effort of current molecular medicine to progress in the understanding of the hallmarks of cancer is focusing on the field of biomarker discovery through genetic and cytogenetic profiling. Ambitious initiatives such as the Cancer Genome Atlas (http://cancergenome.nih.gov/) or the Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/cgap.html) from the US National Institutes of Health (NIH) are devoted to provide collaborative platforms to better comprehend and fight the malignant signature of cancer. The fast development of high-throughput technologies is bringing to biomedical research more affordable and high-resolution ways to explore whole genomes, chromosomes, genes, proteins, RNA, and metabolites. In consequence, numerous genomic datasets accumulate in international repositories, such as GEO from the US National Center for Biotechnology Information (NCBI), and around 500,000 clonal chromosomal abnormalities belonging to more than 60,000 human neoplasms are described in the latest catalogue of chromosome aberrations in cancer. However, translational application of biomarker discovery still has to face its ultimate challenge: incorporation into routine clinical practice. We are under the pressure to combine the multidisciplinary efforts of basic, clinical, and computational research fields to identify meaningful genetic profiles that can be used as powerful tools for diagnosis, prognosis, and treatment. Every patient who suffers from cancer hopes that our research will deliver alternatives. For scientists, the identification of the specific genetic abnormalities responsible for tumor diversity and heterogeneity is crucial to shed light on the molecular mechanisms of cancer and the identification of druggable targets and functional pathways. For clinicians, the appropriate molecular categorization of patients would allow to enroll them in tailored—more adequate and less toxic—treatments, improving healthcare and optimizing medical costs. Ultimately, any effort to (i) frame the important unsolved problems, (ii) to review what is known about the molecular pathways involved in the pathogenesis of distinct types of cancer, (iii) to catalog recurrent genetic alterations that could serve as biomarkers, (iv) to suggest standardization of routine protocols of diagnosis and molecular detection, and (v) to rank available or potential drugs and their targets, is decisive to help molecular medicine in its challenge against cancer. This is the scope of this special issue where the biographies of some of the most aggressive hematological and solid tumors are discussed. The journal Genetics Research International is serving here as a forum for discussion and review of major genetic risks involved in breast cancer metastasis to the brain as well as in the evolution of myxoid soft-tissue sarcomas and parathyroid tumors. The importance of applying genetic protocols in the risk-based stratification of multiple myeloma is also reviewed here, and new evidences supporting genetic aberrations in a rare variant of T-cell lymphomas known as Sezary syndrome are presented. Finally, myeloid malignancies are revealed as a paradigm to discuss the role of dicentric chromosomes as instigators of genomic instability and suggest a model for their implication in the formation of unbalanced translocations in malignancy. Breaking the barriers of cancer is a difficult task that is continuously challenging the research community. We, the editors of this special issue, are hopeful that the ideas presented here can inspire the critical thinking of the specialized readers and contribute to the common efforts to better understand and fight the multiple faces of cancer. Jose Maria Sayagues Sergio Roa Norma C. gutierrez Ilana Zalcberg Renault

Biological evaluation of CpG stimulation of normal human B‐cells: implications for B‐cell biology and cytogenetic analysis of CLL B‐cells

Biological evaluation of CpG stimulation of normal human B‐cells: implications for B‐cell biology and cytogenetic analysis of CLL B‐cells