Percentage Of Abnormal Metaphases Research Articles

159 - Implementation of Diagnostic Flow Cytometry Allows for the Dissection of Age Related Loss of Y Chromosome from Myelodysplastic Syndrome (MDS)

Loss of Y chromosome (-Y) is described as an age related cytogenetic polymorphism as well as an abnormality associated with clonal diseases mainly Myelodysplastic Syndromes (MDS) reported in up to 15% of these patients. However, it often remains an uncertainty dissecting these two conditions especially in patients with mild dysplasia and a low percentage of abnormal metaphases. The aim of our study was to investigate whether flow cytometry (FCM) allows for a valid discrimination between MDS and age related presence of a loss of Y chromosome. Bone marrow aspirates of 46 patients, suspicious for MDS, with a median age of 76 years and a cytogenetically detected -Y as single abnormality (>75% threshold for assignment to MDS; Wiktor et al. 2011) were investigated. According to cytomorphology MDS could be diagnosed in 26 patients (IPSS-R verylow/low=22, intermediate=3, high/very high=1) and CMML in 6 patients. MDS was excluded in 14 patients (11 IDUS, 1 aplastic anemia, and 2 pts. with reactive changes only). Further routine diagnostic procedures included FCM according to WHO and ELNet iMDS-Flow criteria. Thus, FCM was performed using an 8-color antigen panel measured on a FACS-Canto II cytometer. MDS were defined according to the current FCM-scores developed by Wells et al. (normal/mild: 0-1, moderate: 2-3, or severe: > 4; Blood 2003) and Ogata et al. (low: 0-1, high: >1; Haematologica 2009). In case of an intermediate Wells-score only, we demanded cross lineage antigen expression in myeloid progenitors as criteria for MDS/CMML. The median percentage of abnormal metaphases presenting with -Y was 52% (range: 12%-100%). Applying the above mentioned threshold of 75% abnormal metaphases as criteria for the presence of MDS, only 14 patients (30%) could clearly be allocated to MDS. Because of the present diagnostic incertainty we performed a comprehensive FCM investigation, applying Wells- and Ogata-Score. Thus, in 26 patients (63%) MDS was likely applying FCM. Furthermore, 4 patients with a normal/mild Wells-score plus a low Ogata-score were considered as non-MDS. Applying cytomorphology 70% (32/46) of all -Y patients have been diagnosed as MDS. Of note, MDS diagnosis could be made by FCM or cytomorphology in an equal extent in the groups of more vs. less of 75% cytogenetically abnormal metaphases: 64% vs. 63% and 86% vs. 63%, respectively. Finally, we searched for patients meeting FCM plus cytomorphologic MDS criteria. Of importance, even in the group with less abnormal metaphases 50% of the patients showed a concordant FCM and cytomorphologic assignment to MDS or non-MDS vs. 64% concordance in the high cytogenetically aberrant group. Patients with a loss of Y chromosome as single cytogenetic abnormality often meet FCM and cytomorphologic criteria for MDS diagnosis independent from the load of abnormal metaphases. This study demonstrates that FCM can significantly contribute to MDS diagnosis in this specific subgroup of patients with -Y and often only mild dysplasia. Disclosures No relevant conflicts of interest to declare.

Implementation of Diagnostic Flow Cytometry Allows for the Dissection of Age Related Loss of Y Chromosome from Myelodysplastic Syndrome (MDS)

Loss of Y chromosome (-Y) is described as an age related cytogenetic polymorphism as well as an abnormality associated with clonal diseases mainly Myelodysplastic Syndromes (MDS) reported in up to 15% of these patients. However, it often remains an uncertainty dissecting these two conditions especially in patients with mild dysplasia and a low percentage of abnormal metaphases. The aim of our study was to investigate whether flow cytometry (FCM) allows for a valid discrimination between MDS and age related presence of a loss of Y chromosome.Bone marrow aspirates of 46 patients, suspicious for MDS, with a median age of 76 years and a cytogenetically detected -Y as single abnormality (>75% threshold for assignment to MDS; Wiktor et al. 2011) were investigated. According to cytomorphology MDS could be diagnosed in 26 patients (IPSS-R verylow/low=22, intermediate=3, high/very high=1) and CMML in 6 patients. MDS was excluded in 14 patients (11 IDUS, 1 aplastic anemia, and 2 pts. with reactive changes only). Further routine diagnostic procedures included FCM according to WHO and ELNet iMDS-Flow criteria. Thus, FCM was performed using an 8-color antigen panel measured on a FACS-Canto II cytometer. MDS were defined according to the current FCM-scores developed by Wells et al. (normal/mild: 0-1, moderate: 2-3, or severe: > 4; Blood 2003) and Ogata et al. (low: 0-1, high: >1; Haematologica 2009). In case of an intermediate Wells-score only, we demanded cross lineage antigen expression in myeloid progenitors as criteria for MDS/CMML.The median percentage of abnormal metaphases presenting with -Y was 52% (range: 12%-100%). Applying the above mentioned threshold of 75% abnormal metaphases as criteria for the presence of MDS, only 14 patients (30%) could clearly be allocated to MDS. Because of the present diagnostic incertainty we performed a comprehensive FCM investigation, applying Wells- and Ogata-Score. Thus, in 26 patients (63%) MDS was likely applying FCM. Furthermore, 4 patients with a normal/mild Wells-score plus a low Ogata-score were considered as "non-MDS". Applying cytomorphology 70% (32/46) of all -Y patients have been diagnosed as MDS.Of note, MDS diagnosis could be made by FCM or cytomorphology in an equal extent in the groups of more vs. less of 75% cytogenetically abnormal metaphases: 64% vs. 63% and 86% vs. 63%, respectively. Finally, we searched for patients meeting FCM plus cytomorphologic MDS criteria. Of importance, even in the group with less abnormal metaphases 50% of the patients showed a concordant FCM and cytomorphologic assignment to MDS or non-MDS vs. 64% concordance in the high cytogenetically aberrant group.Patients with a loss of Y chromosome as single cytogenetic abnormality often meet FCM and cytomorphologic criteria for MDS diagnosis independent from the load of abnormal metaphases. This study demonstrates that FCM can significantly contribute to MDS diagnosis in this specific subgroup of patients with -Y and often only mild dysplasia. DisclosuresNo relevant conflicts of interest to declare.

The utility of hematopoietic stem cell karyotyping in the diagnosis of de novo myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a clonal hematopoietic stem cell (HSC) disorders. Cytogenetics plays a vital role in pathogenesis, diagnosis, prognosis, and determining treatments in MDS. Cytogenetic studies on CD 34+ cells in Asian MDS patients are limited. The aim of conducting this study was to compare the karyotypic features of CD 34+ cells and their bone marrow (BM) karyotypes. BM samples of 20 primary MDS patients were collected. BM-CD 34+ cells were isolated by CD34 positive selection. Conventional karyotyping was performed on primary BM samples and isolated CD 34+ cells. Fluorescence in situ hybridization (FISH) was performed to confirm del(5q) by using XL 5q31/5q33 locus-specific probe. Chromosomal abnormalities were detected in 41 % (7/17) of BM and 40 % (8/20) of HSC karyotypes. BM and HSC karyotypes were similar (94 %) except in one (BM—normal; CD 34+ cells had del(5q)) where the patient showed characteristic del(5q) phenotype. The abnormalities found were del(5q)—10 % (2/20), del(11q)—5 % (1/20), del(12p)—5 % (1/20), 47,XY,+19—5 % (1/20), hypodiploidy—5 % (1/20), and random loss of chromosomes—10 % (2/20). The percentage of abnormal metaphases counted per case was higher for CD 34+ cell cultures than for BM cultures. Culture failure were less for CD 34+ cells when compared to BM. Sample limitation for BM does not apply for CD 34+ cells if cultures can be maintained. Although the abnormal karyotypes counted were greater using CD 34+ cells than BM, there was no statistically significant difference (p > 0.05). Detection of karyotypic abnormalities could be greater when using CD 34+ cells. All the karyotypic abnormalities reported in this study had been previously known in MDS. Further large scale studies are needed to verify our findings.

Relapse Prediction Post Allogeneic Hematopoietic Cell Transplant for Myelodysplastic Syndromes Is Not Improved with Use of the More Stringent Blast Percentage Categories in the Revised International Prognostic Scoring System

AbstractAbstract 2011▪▪This icon denotes a clinically relevant abstractResidual disease at the time of allogeneic hematopoietic cell transplant (HCT) has been shown to adversely affect outcomes in patients with acute myelogenous leukemia (AML). Assessment of the impact of MDS disease burden at HCT on post-transplant outcomes is less well defined. While blast percentage is easily quantified on morphology and flow cytometry, the blast percentage captures only a fraction of the true MDS clone. Consequently, improved measures of residual disease in MDS patients are needed to improve post HCT outcome prediction. We hypothesized that a more stringent blast percentage cutoff as well as an assessment of recognizable residual cytogenetic disease burden would better predict outcome post HCT. Disease burden at HCT was characterized as reported in the Revised International Prognostic Scoring System (IPSS-R) (<2%, >2-<5%, 5–10% and >10%) and by assessment of cytogenetic residual disease by calculating the percent of metaphases with ongoing cytogenetic abnormalities immediately prior to transplant (% positive metaphases <25%, 25–50%, and > 50%). Metaphase data was unavailable in n=7. Those patients with normal cytogenetics (n=26) were classified in the <25% category.One hundred consecutive patients with MDS undergoing allogeneic HCT at the University of Minnesota between 1995 –2011 were studied. Their median age was 52 (18–69). At diagnosis, the majority of patients had either refractory cytopenia of multilineage dysplasia (RCMD) (25%) or refractory anemia with excess blasts (RAEB) 1 or 2 (49%) and were INT-1 (37%) or INT-2/High risk (60%). Donor type was related donor (52%), matched unrelated (13%) and umbilical cord blood (35%). Half received myeloablative conditioning and Karnofsky Performance Status (KPS) > 80 in 95%. Results:(Table)Both higher blast percentage at HCT and high percentage of residual cytogenetically positive cells correlated with inferior overall survival as well as more frequent NRM. Relapse incidence was similar in those with blasts up to 10%. Percentage of abnormal metaphases was not predictive of relapse. Conditioning intensity was the only factor that impacted the 2 year relapse rate with decreased relapse seen with myeloablative conditioning.Table% of Patients Per StrataStrata2 year Overall Survival (OS)% (CI 95%)P value2 year Non-Relapse Mortality (NRM) % (CI 95%)P value2 year Relapse % (95% CI)P valuePercentage Blasts at HCT49%≤2%46% (32–59%)<0.0133% (20–46%)0.0229% (15–42%)0.8928%>2–<536% (19–53%)46% (27–66%)21% (6–37%)16%5–10%50% (25–71%)38% (14–61%)25% (11–56%)7%>10%0%86% (55–100%)NEPercentage Abnormal Metaphases At HCT42%<25%56% (39–70%)0.0326% (12–39%)0.0631% (16–46%)0.3311%25–50%50% (18–75%)40% (11–69%)10% (0–27%)47%>50%28% (16–42%)51% (35–67%)25% (12–38%Conditioning IntensityMyeloablative50%42% (28–55%)0.9948% (33–63%)0.3314% (4–24%)0.02Reduced Intensity50%39% (26–53%)34% (21–48%)37% (22–51% Conclusion:These data suggest that survival and relapse prediction are not improved by using the more stringent IPSS-R blast cutoff of < 2% versus <5% at HCT. Additionally, our data suggest that use of a disease burden measure as represented by % residual positive metaphases at HCT may serve as another predictor of outcome. Survival and NRM were worse with >10% blasts or > 50% residual positive metaphases. These patients require additional therapy or new strategies to improve post HCT outcomes. A refined measure of disease burden at HCT using both blast count and % residual abnormal metaphases may allow for more comprehensive prediction of post HCT outcomes in MDS. Disclosures:No relevant conflicts of interest to declare.

Pomalidomide Therapy in Myelofibrosis: 2-Year Follow-up of a Randomized Phase 2 Study.

Abstract 1904Poster Board I-927 BACKGROUND:Pomalidomide is an IMiDsó immunomodulatory drug that was created by chemical modification of thalidomide with the intent to reduce toxicity and enhance therapeutic activity. In a phase-2 randomized study of patients with myelofibrosis (MF), treatment with pomalidomide alone or in combination with a short course of prednisone was shown to be well tolerated and active in alleviating anemia ( JCO 2009). Herein, we report observations during the longer-term follow-up of those patients who had responded to a pomalidomide-containing treatment regimen. METHODS:Both primary and post polycythemia vera/essential thrombocythemia MF were included in the current study. Protocol eligibility criteria included absolute neutrophil count of × 109/L, platelet count of 50 × 109/L and hemoglobin level of < 10 g/dL. Patients with prior thalidomide or lenalidomide therapy were excluded from study participation. Response was assessed by the International Working Group for Myelofibrosis Research and Treatment criteria (IWG-MRT) (Blood. 2006). Pomalidomide was administered daily and continued indefinitely in responding patients. Prednisone was given for the first 3 months only, in a tapering dose schedule, starting at 30 mg/day. RESULTS:A total of 84 patients were enrolled from April 2007 through May 2008 and received treatment with pomalidomide alone 2 mg/day (n=22), pomalidomide 2 mg/day + prednisone (n=19), pomalidomide 0.5 mg/day + prednisone (n=22) or prednisone alone (n=21). Short-term toxicity details have previously been published (Tefferi et al. JCO 2009) and were remarkable for the lack of neuropathy and low prevalence of grade 3 or 4 myelosuppression (5–16%).Based on an intent-to-treat analysis, 16 (25%) of the 63 patients receiving pomalidomide alone or with a 3-month course of prednisone responded at a median of 1.8 months (range 0.9–6.9) from treatment initiation; all responses represented improvement in anemia with little or no effect on splenomegaly. Specifically, 12 of the 16 treatment responders became transfusion independent and the remaining 4 had a hemoglobin increase of > 2 g/dL (median best hemoglobin response = 11.9 g/dL; range 8.5–15.9). Responding patients usually displayed increases in platelet count but there was no consistent change in serum lactate dehydrogenase level.As of August 2009, ten (63%) of the 16 treatment responders were still in anemia remission and receiving pomalidomide therapy; median time on treatment was 19 months (range, 14–26) and median response duration was 17 months (range, 13-24). No new side effects emerged during this period. Six patients discontinued treatment because of either loss of response (n=5) or progressive splenomegaly/leukocytosis (n=1); 3 patients with continued anemia remission have had substantial increases in their spleen size, which qualifies as spleen progression per IWG-MRT. Among 9 patients with available cytogenetic information, all 3 with unfavorable karyotype relapsed while none of the 6 with favorable karyotype did.A small number of patients (n=7) had follow-up bone marrow examination at 1 to 2 years, at the discretion of their treating physician; none of these patients displayed any change in bone marrow fibrosis or JAK2V617F mutational status. Suppression of a monosomy 7 clone was documented in two patients. In one, the percentage of abnormal metaphases decreased from 90% to 15% after 11 months of treatment with pomalidomide but increased back to 100% four months later, when the patient relapsed while still on treatment. In the second patient, the percentage of abnormal metaphases was 65% at baseline, remained at 75% after 10 months of pomalidomide therapy, but decreased to 7% after one month of treatment discontinuation for relapse; a repeat examination in this patient after another 3 months (still off therapy) showed monosomy 7 and new additional abnormalities (including 4q21q25 deletion) in 65% of metaphases evaluated. CONCLUSION:Longer-term follow-up of pomalidomide-treated patients with myelofibrosis confirm the drug's favorable side effect profile. Also, the majority of treatment responders maintain their remission, which is exclusively anemia remission, for at least one year and several remain in remission after 2 years of therapy. The drug has limited effect on splenomegaly and progressive splenomegaly might occur in some patients despite continued remission in anemia. The presence of unfavorable cytogenetic abnormalities predicts relapse. Disclosures:Gale:Celgene: Employment.

The extent of chromosomal aberrations induced by chemotherapy in non-human primates depends on the schedule of administration

We utilized a non-human primate model, the rhesus monkey (Macaca mulatta), to quantitate the extent of chromosomal damage in bone marrow cells following chemotherapy. Thiotepa, etoposide, and paclitaxel were chosen as the chemotherapy agents due to their distinct mechanisms of action. Chromosomal aberrations were quantitated using traditional Giemsa stain. We sought to evaluate the extent to which genotoxicity was dependent on the schedule of administration by giving chemotherapy as either a bolus or a 96h continuous infusion. Neutropenia and areas under the concentration curve (AUCs) were monitored to ensure comparable cytotoxicity and dose administered. At least 100 metaphases were scored in each marrow sample by an investigator unaware of the treatment history of the animals. All three drugs produced a statistically significant higher percentage of abnormal metaphases following bolus chemotherapy (p<0.0001, p=0.0015 and p<0.0001 for thiotepa, etoposide and paclitaxel, respectively). We conclude that infusional administration of thiotepa, etoposide and paclitaxel is less genotoxic to normal bone marrow cells than is bolus administration. These results suggest infusional regimens may be considered where there are concerns about long-term genotoxic sequelae, including secondary cancer, teratogenicity, or possibly the development of drug resistance. We believe this approach provides a reproducible model in which drugs and eventually, regimens can be compared.

Cytogenetic Abnormalities Correlate with the Plasma Cell Labeling Index and Extent of Bone Marrow Involvement in Myeloma

Chromosomal abnormalities have biologic and prognostic significance in multiple myeloma, especially among patients with relapsed disease. We report the relationship between chromosomal abnormalities and known prognostic factors such as plasma cell labeling index (PCLI) and bone marrow plasma cell involvement in 75 consecutive patients undergoing autologous stem cell transplantation for relapsed or refractory myeloma. Thirty of 70 patients (43%) had a chromosomally abnormal clone in their bone marrow, and in most cases the karyotype was complex ( >3 abnormalities). Patients with an abnormal clone on cytogenetic analysis had a higher PCLI (median, 1.4) than patients with a normal karyotype (0.2) ( P < 0.001). Bone marrow plasma cell percentage also differed: median 48% versus 20%, respectively ( P < 0.001). The PCLI and bone marrow plasma cell percentage correlated positively with the percentage of abnormal metaphases on conventional cytogenetic analysis: rho 0.60 ( P < 0.001) and 0.46 ( P < 0.001), respectively. We categorized patients into those with 20% or more abnormal metaphases, less than 20% abnormal metaphases, and only normal metaphases. The median PCLI values were 3.3, 1.1, and 0.3, respectively ( P < 0.001). The bone marrow plasma cell percentage median values were 62%, 40%, and 25%, respectively ( P = 0.003). Chromosomal abnormalities may offer a proliferative advantage to the neoplastic plasma cell, thereby leading to an unfavorable outcome.

Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival.

Increased apoptosis in the bone marrow (BM) may contribute to the cytopenias that occur in myelodysplastic syndromes (MDS). The Fas receptor, Fas ligand (FasL) pathway is a major mechanism of apoptosis. Since hematopoietic progenitors can express the Fas receptor, they may be susceptible to apoptosis induced by FasL-expressing cells. We examined FasL expression in the BM of patients with MDS (n = 50), de novo acute myeloid leukemia (AML; n = 10), AML following prior MDS (n = 6), and normal controls (n = 6). Compared to controls, FasL expression was increased in MDS, and was highest in AML. In MDS, FasL expression was seen in myeloid blasts, erythroblasts, maturing myeloid cells, megakaryocytes and dysplastic cells, whereas in AML, intense expression was seen in the blasts. FasL expression correlated with the FAB subtype groups of MDS, and also correlated directly with the percentage of abnormal metaphases on cytogenetic analysis. The FasL expressed in MDS BM inhibited the growth of clonogenic hematopoietic progenitors. This inhibition could be blocked by a soluble recombinant FasFc protein. In MDS, FasL expression in the initial diagnostic BM was higher in patients who were more anemic, correlated directly with red cell transfusion requirements over the subsequent course of the disease, and was predictive of survival. These studies indicate that FasL expression in MDS is of prognostic significance, and suggest that pharmacological blockade of the Fas-FasL pathway may be of clinical benefit.

Suppression of cytogenetic clonal evolution with interferon alfa therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia.

To investigate whether cytogenetic clonal evolution can be suppressed with interferon alfa (IFN-alpha) therapy in patients with chronic myelogenous leukemia (CML). Ninety patients with CML and cytogenetic clonal evolution who received IFN-alpha-based regimens were analyzed. Clonal evolution was defined as the presence of karyotypic abnormalities in addition to the Philadelphia (Ph) chromosome. Patients were evaluated for the suppression of cytogenetic clonal evolution after therapy, the cytogenetic response, and survival. The median age of the population was 39 years (range, 15 to 70 years), median time from diagnosis to clonal evolution 14 months (range, 0 to 145 months), and median percentage of abnormal metaphases 18% (range, 4% to 100%). Fifty six patients (62%) achieved some suppression of cytogenetic clonal evolution; in 41 patients (46%), the suppression was complete. The overall median survival was 51 months, with 43% alive at 5 years. Patients who achieved a complete suppression of cytogenetic clonal evolution had a median survival of 66 months, with 51% alive at 5 years. Characteristics associated with a better response include a lower percentage of abnormal metaphases, time to cytogenetic clonal evolution of 24 months or less, and absence of other features of accelerated disease. A prognostic classification for cytogenetic clonal evolution defined three groups with complete response (CR) rates of 85%, 34%, and 0% (P < .0001) and median survival times of 58, 31, and 30 months, respectively (P=.02). Patients with cytogenetic clonal evolution can respond to IFN-alpha therapy, and this response is associated with longer survival. A previously described prognostic model separates patients into subsets with different probabilities of response to IFN-alpha and survival.

Effect of conditioned media, nutritive elements, and mitotic synchronization on the accuracy of the cytogenetic analysis in acute nonlymphocytic leukemia patients presenting with inv(16)/t(16;16) or t(15;17)

To improve the yield of the cytogenetic analysis in patients with acute nonlymphocytic leukemia (ANLL), six culture conditions for bone marrow or peripheral blood cells were tested in parallel. Two conditioned media (CM), phytohemagglutinin leukocyte PHA-LCM and 5637 CM, nutritive elements (NE), and methotrexate (MTX) cell synchronization were investigated in 14 patients presenting with either inv(16)/ t(16;16) (group 1, n = 9 patients) or t(15;17) (group 2, n = 5). The criteria used to identify the most favorable culture conditions were the mitotic index (MI), the morphological index (MorI), and the percentage of abnormal metaphases. In the presence of PHA-LCM and 5637 CM, the MI were significantly increased in group 2, whereas in the MTX conditions, MI remained very low in both groups. The values of the MorI did not reveal any significant changes in chromosome resolution between the conditions in either group. The addition of NE did not have a positive effect in quantity or quality of metaphases. Because of the variability of the response of leukemic cells to different stimulations in vitro, several culture conditions in parallel are required to ensure a satisfactory yield of the chromosome analysis in ANLL.

Genetic abnormalities in chronic lymphocytic leukemia and their clinical and prognostic implications

Clonal chromosome abnormalities can be detected in approximately 50% of patients with chronic lymphocytic leukemia (CLL). The most common changes are trisomy 12, followed by structural abnormalities of 13q, 11q, 6q, and 14q. By fluorescence in situ hybridization (FISH), these aberrations can be demonstrated even in cases with insufficient mitotic yield or a normal karyotype. The biologic consequences of trisomy 12 are unknown, but a gene dosage effect is suspected and studies on partial trisomy 12 indicate that the region 12q13 to 12q22 might be of particular pathogenetic importance. Trisomy 12 is strongly associated with atypical lymphocyte morphology and seems to be a secondary event in leukemogenesis, as shown by combined immunophenotyping and interphase FISH. Structural abnormalities of 13q frequently involve hetero- and homozygous deletions of a region in 13q14, distal to the retinoblastoma gene, which may be the site of a tumor suppressor gene. In contrast to a normal karyotype or structural changes of 13q, complex karyotypic abnormalities, high percentage of abnormal metaphases, trisomy 12 and structural changes involving the P53 tumor suppressor gene on 17p13 are adverse prognostic indicators. Cytogenetic and molecular findings provide important diagnostic, clinical, and prognostic information which can contribute to treatment decisions and follow-up of CLL patients.

Significance of cytogenetic clonal evolution in chronic myelogenous leukemia.

To describe the incidence and significance of clonal evolution patterns. We analyzed 264 patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) who developed clonal evolution between 1967 and 1993. The median survival time following clonal evolution was 19 months. Factors associated with worse survival (P < .01) were as follows: chromosome 17 abnormality or chromosomal translocations other than Ph, high percentage of abnormal metaphases, longer time to clonal evolution, and presence of other accelerated-phase features. A recursive partitioning technique (CART) identified different risk groups. The best group (37 patients; no chromosome 17 abnormality, abnormal metaphases < 16%, and interval to clonal evolution < or = 24 months) had an estimated median survival time of 54 months. The worst two groups included 27 patients with chromosome 17 abnormalities and > or = 36% abnormal metaphases (estimated median survival time, 6 months), and 22 patients with other accelerated features and > or = 16% abnormal metaphases (estimated median survival time, 7 months). The intermediate group had an estimated median survival time that ranged from 13 to 24 months. Prior interferon therapy evaluated within risk groups showed a significant survival advantage only in the intermediate-risk group. A multivariate analysis showed similar results, and identified the following independent poor prognostic variables: chromosome 17 abnormality, percentage of abnormal metaphases (cutoff, 24%), longer time to clonal evolution (cutoff, 24 months), other accelerated-phase features, and no prior interferon therapy. Patients with none, one, two, three, or more of the first four features had median survivals times of 51, 24, 14, and 7 months, respectively. The prognostic significance of clonal evolution in CML is not uniform and is related to the specific abnormality, time to its development, its predominance in metaphases, and the presence of other accelerated features, and it may be modified by specific therapies.

Cytogenetic and molecular abnormalitiesin chronic lymphocytic leukaemia

Genetic abnormalities are found in 50% of cases of chronic lymphocytic leukaemia (CLL) by cytogenetic analysis and in a higher percentage of patients using molecular techniques. The commonest cytogenetic abnormalities are trisomy 12 and deletions or translocations of the long arm of chromosome 13 usually involving band q14. The genetic consequences of trisomy 12 are unknown but structural abnormalities of chromosome 13q14 frequently involve hetero or homozygous loss of a region distal to the retinoblastoma gene which may be the site of a tumour suppressor gene. Trisomy 12 or loss of one copy of the retinoblastoma gene have been detected by fluorescent in situ hybridisation (FISH) in interphase cells of patients with a normal karyotype. By combining FISH with immunophenotyping, it has been found that trisomy 12 occurs in only 30 to 40% of the malignant clone, suggesting that it is a secondary event in leukaemogenesis. Trisomy 12 is strongly associated with atypical lymphocyte morphology in patients with otherwise typical CLL. Complex karyotypic abnormalities, a high percentage of abnormal metaphases and trisomy 12 but not structural abnormalities of chromosome 13 are associated with a poor prognosis at all stages of the disease. Mutations or deletions of the P53 gene are found in 10 to 15% of patients with advanced CLL and correlate with resistance to treatment and poor survival.

Genotoxic action of an aqueous extract of Heliotropium curassavicum var. argentinum

Heliotropium curassavicum uar. argentinum is widely employed in gout, rheumatism, neuralgias, arteriosclerotic disorders, muscular algias, phlebitis, varix and other illnesses. In order to analyze the genotoxic effect produced in vitro by this medicinal plant, chromosomal aberrations (CA), mitotic index (MI) and anaphase delay (AD) were studied in the CHO cell line, with and without the addition of S9 mix. Prepared according to the Argentine pharmacopeia 0.1, 1, 10 and 100 μg/ml plant decoction (aqueous extract) were assayed. One hundred cells per culture were studied for CA and AD, while MI was calculated for 2000 nuclei. The results revealed a significant increase in the percentage of abnormal metaphases ( p < 0.001) and in total aberrations ( p < 0.001). Both the MI and the AD affected the cell cycle. All results were enhanced by the addition of an S9 fraction. The toxic effect could be associated with pyrrolizidine alkaloids and their N-oxides, which through a process of in vitro metabolism become activated by microsomal oxidation and change into pyrrolic derivatives.

Chromosome aberrations in B-cell chronic lymphocytic leukemia: Pathogenetic and clinical implications

Chromosome analyses were performed on leukemic cells from 102 patients with B-CLL, of whom 84 were untreated. B-cell mitogen-induced CLL cells yielded suitable metaphases in 85 patients, and 55 showed clonal chromosomal aberrations. Trisomy 12 was found in 26 patients. In nine patients the +12 was a single aberration. A 14q+ chromosome or deletions of the long arm of chromosomes 6, 11, or 13 were other recurrent aberrations. Patients with Rai stage I or more had more frequently clonal aberrations than patients with stage 0 disease (p < .02). Patients with clonal aberrations had poorer 5-year survival than those with a normal karyotype (p < .05). Patients with a high percentage of abnormal metaphases in the sample had poorer prognosis than patients with high admixture of normal metaphases (p < .01). Of the specific clonal aberrations those with 14q+ or trisomy 12 tended to have slightly poorer and those with 6q− or structural aberrations involving the long arm of chromosome 13 tended to have better prognosis than patients with other chromosomal aberrations. A complex karyotype tended to be an adverse prognostic sign. Clonal evolution is rare: complex karyotypes are found at diagnosis and clones with single aberrations did not acquire additional chromosome aberrations despite progressive disease and treatment. Nine hundred and seventy-nine published cases are reviewed, and pathogenetic mechanisms, such as oncogenes and gene dosage, are discussed.