A 24-month prospective study on the efficacy and safety of two different monthly regimens of vitamin D supplementation in pre-menopausal women with systemic lupus erythematosus.

BACKGROUND: Low vitamin D (vit.D) serum levels are common in patients with systemic lupus erythematosus (SLE) and seem to correlate with higher disease activity. We investigated the effects of different regimens of vit.D supplementation in SLE patients with inactive disease. METHODS: This 24-month prospective study included 34 SLE women who were randomized to receive, together with their ongoing treatment, a standard regimen (SR) of cholecalcipherol (25,000 UI monthly) or an intensive regimen (IR) (300,000 UI initial bolus followed by 50,000 UI monthly) for one year and then were switched to the other regimen in the second year. Patients were seen quarterly for assessment of 25-OH vit.D levels, disease activity, SLE serology and bone metabolism markers. RESULTS: By intra-patient comparison, only the IR was found able to significantly raise vit.D serum levels. After 12 months, values above 30 ng/ml were found in 75% of patients in IR while in only 28% in SR. No significant differences in disease activity and SLE serology were found at any time point between SR and IR. No changes in the mineral metabolism were observed. CONCLUSIONS: The IR was safe and effective in obtaining sufficient levels of vit.D in most SLE patients. However, both regimens of supplementation did not differently affect disease activity nor SLE serology.

Clinical importance of interphase cytogenetics detecting occult chromosome lesions in myelodysplastic syndromes with normal karyotype.

At diagnosis, approximately half of myelodysplastic (MDS) patients presents a normal karyotype by conventional cytogenetic analysis (CCA). Fluorescent in situ hybridization (FISH) is more sensitive than CCA allowing for the detection of minor clones and of submicroscopic lesions. We have analyzed by FISH 101 MDS patients with normal karyotype for the occurrence of the abnormalities which are most frequently observed in MDS (ie -5/5q-, -7/7q-, +8, 17p-). In 18 patients, 15 to 32% of interphase cells were found to carry one FISH abnormality. Six patients presented trisomy 8, five had del(5)(q31), five del(7)(q31), one monosomy 7 and one del(17)(p13). FISH abnormalities were more frequently observed among patients with an increased percentage of bone marrow blasts (P = 0.001). FISH abnormalities were also associated with a higher rate of progression into AML (13/18 vs 12/83, P < 0.001) and were predictive for a worse prognosis (P < 0.001). Multivariate analysis indicated that FISH positivity and IPSS risk group were independent predictors for a poor survival (P = 0.0057 and 0.0123, respectively) and for leukemic transformation (P = 0.0006 and 0.035, respectively). Leukemic transformation in FISH-positive patients was associated in all cases with an expansion of the abnormal clone. Our data demonstrated that a significant proportion of MDS patients with normal karyotype presented, if analyzed by FISH, clones of cytogenetically abnormal cells which played a determinant role in the progression of the disease. The presence of FISH abnormalities identified a group of MDS patients with normal karyotype characterized by an inferior prognosis.

Assessment of distribution of CD34 epitope classes in fresh and cryopreserved peripheral blood progenitor cells and acute myeloid leukemic blasts.

BACKGROUND AND OBJECTIVE: So far several reports have described changes in the expression of surface antigens in progenitor cells and blasts following cryopreservation. However, there are no data on the effects of cryopreservation on the expression of the three CD34 epitope classes, and on their relationship with the clonogenic capacity of PBPC collected by leukapheresis. DESIGN AND METHODS: In order to analyze the effects of freezing/thawing procedures (Eth 80C storage for 3 months) and use of dimethylsulfoxide (DMSO) on the immunophenotype profile and colony production of peripheral blood progenitor cells (PBPC) in apheresis products derived from 20 patients with stage 0-III non-Hodgkin's lymphoma (nHL), a flow cytometry study was undertaken using different CD34 monoclonal antibodies (MoAbs) capable of recognizing the 3 epitope classes of CD34 molecule (class III: HPCA-2/FITC, HPCA-2/PE, 581/FITC, 581/PE; class II: Q-Bend 10/PE; class I: ICH3/PE, BI3C5-PE, Immu-133-PE). CD34 epitope expression was also analyzed in thawed CD34+ blasts obtained from 14 patients with acute myeloid leukemia (AML), who were analyzed using a larger number (#17) of CD34 epitope class I, II, and III reactive MoAbs. RESULTS: Under our experimental conditions it was found that class III and class II CD34 epitopes (differentially resistant to enzymatic cleavage with neuraminidase, chymopapain and glycoprotease) are better preserved than class I epitope Eth sensitive to degradation Eth after cell exposure to cryoprotectant DMSO and the freezing- thawing procedures. Results further showed a concomitant decrease in class I CD34+ counts and in BFU-E colony production. A significant increase in CD34 antigen expression levels (i.e. antibody binding capacity, ABC) by cryopreserved cells stained with CD34 epitope class III, and class II reactive MoAbs was also documented, while no changes after cryopreservation were noted using class I-reactive MoAbs. The slight increase in the percentage of CD34+ cells detected after frozen storage was correlated to a concomitant decrease in the number of more mature myeloid cells (CD15+, CD13+, CD33+). Compared to pre-cryopreservation values, a slight reduction in class I CD34 epitope expression was also found in thawed CD34+ AML blasts. INTERPRETATION AND CONCLUSIONS: As far as the reduction of class I CD34 epitope is concerned, it may be hypothesized that the freezing procedure, use of DMSO, and/or lysis methodology may either damage a CD34 subset, or induce distinct alterations of the CD34 glycoprotein, possibly determining a reduction in their immunoreactivity with some CD34 MoAbs. In conclusion, this study has shown that exposure to the cryoprotectant DMSO and the freezing/thawing procedures modifies the distribution of CD34 epitopes as well as the clonogenic capacity of PBPCs from nHL patients, and CD34+ blasts from AML. These findings need to considered when selecting CD34 MoAbs for enumeration and positive selection of stem/progenitor cells for research and clinical purposes.

Cytogenetic profile of lymphoma of follicle mantle lineage: correlation with clinicobiologic features.

Conventional chromosome analysis (CCA) and interphase fluorescence in situ hybridization (FISH) was performed in 42 patients with mantle-cell lymphoma (MCL), with BCL1 rearrangement. The t(11;14)(q13;q32) or 11q abnormalities were detected by CCA in 34 cases, 20 of which had additional aberrations. A normal karyotype was observed in 8 cases. Probes detecting the chromosome aberrations that were observed in at least 3 cases by CCA, ie, +12, 13q14 deletion, and 17p deletion, were used for interphase FISH analysis. FISH detected total or partial +12, 13q14 deletion and 17p- in 28.5%, 52.4%, and 26% of the cases, respectively. The presence of these anomalies was not a function of karyotype complexity. Based on the results of CCA/FISH, three groups of increasing karyotype complexity were recognized: group 1, including 11 patients without detectable aberrations in addition to BCL1 rearrangement; group 2, including 14 patients with 1 to 2 additional anomalies; and group 3, including 17 patients with three or more additional anomalies. Clinical parameters associated with shorter survival were male sex (P =.006) and primary lymph-node involvement compared with primary bone marrow involvement (P =.015). Trisomy 12 was the only single cytogenetic parameter predictive of a poor prognosis (P =.006) and the best prognostic indicator was the derived measure of karyotype complexity (P <.0001), which maintained statistical significance in multivariate analysis (P<.0001). We arrived at the following conclusions: 13q14 deletion occurs at a high incidence in MCL; 17p deletion and total/partial +12 are relatively frequent events in MCL, the latter aberration being associated with a shorter survival; and the degree of karyotype complexity has a strong impact on prognosis in this neoplasia.

5' region and exon 7 mutations of the TP53 gene in two cases of B-cell prolymphocytic leukemia.

We previously found that cases of typical B-chronic lymphocytic leukemia (CLL), atypical B-CLL with t(11;14) and mantle cell lymphomas characterized by rapid progression of the disease and resistance to therapy, had mutations of the TP53 gene. In this paper, abnormalities of the TP53 gene were investigated in two cases of prolymphocytic leukemia, one with t(11;14)(q13;q32), evolving from atypical CLL (patient 1), and one presenting as a de novo condition (patient 2). TP53 DNA was investigated by Southern blot and PCR-SSCP analysis, and TP53 expression was investigated by Northern blot analysis and immunocytochemistry. C-MYC and BCL-1/PRAD1 gene expression were also investigated. Restriction enzyme analysis of TP53 DNA in patient 1 showed alteration of fragments including exon I and intron I, and, in both patients, a specific loss of TP53 DNA. In patient 2, PCR direct sequencing showed in exon VII a 9 bp deletion including codons 252-254. In patient 1, TP53 RNA and protein were not found, indicating that the unusual 5' rearrangement has affected TP53 gene expression. By contrast, patient 2 exhibited detectable TP53 RNA and protein. Detectable but weak BCL-1/PRAD1 RNA was present in both patients, whereas C-MYC RNA expression was clearly present only in case 1. The presence of TP53 hemizygous mutations in both patients suggests that TP53 abnormalities may be important in the pathogenesis of prolymphocytic leukemia (PLL), and may possibly account for the frequent resistance to therapy observed in this disease.

Fluorescence in situ hybridization for the detection and monitoring of the Ph-positive clone in chronic myelogenous leukemia: comparison with metaphase banding analysis.

In order to analyze the efficiency of interphase FISH for the detection and monitoring of Ph+ cells in chronic myelogenous leukemia (CML) under interferon (IFN) treatment, the following experiments were performed: (1) 98 specimens derived from 32 patients were analyzed in parallel by dual-color FISH and by conventional chromosome analysis (CCA). A 300/200 kb BCR/ABL probe was used in all tests and a smaller 35.5/39 kb probe was tested in parallel in 22 BM samples; (2) 30 BM samples were prepared by direct harvest and by 24-h culture and were analyzed in parallel; (3) PB and BM samples obtained simultaneously from 11 patients were analyzed. The cut-off point for the recognition of BCR/ABL fusion was set at 2.4%, calculated as the mean percent of false positivity in 11 controls plus 3 s.d. A very close correlation was observed (r=0.994, r2=0.988, P < 0.0001) between the percentages of Ph+ cells as assessed by CCA and by interphase FISH in 98 samples (26 at diagnosis). There was a moderate overestimation of the frequency of Ph+ cells by FISH with respect to CCA, that was more evident at low-to-medium values of Ph positivity. Seven specimens without Ph+ metaphases (17-50 cells analyzed) were shown to carry 2.5-8% interphase cells with BCR/ABL fusion. Similar percentages of BCR/ABL+ nuclei were recorded in 22 samples hybridized using the 300/200 kb and the 35.5/39 kb probe-sets (variation range: 0-5%, mean 2.3%). A very good correlation between the frequency of Ph+ interphase cells was observed when analyzing in parallel BM preparations after direct harvest and after 24-h culture. Underestimation of the percentage of BCR/ABL+ cells was noted to occur in 2/11 PB samples, compared to BM samples, the remaining nine cases showing superimposable results at either sites. We arrived at the following conclusions: (1) dual-color FISH enables an accurate detection and monitoring of the size of the Ph-positive clone in CML at diagnosis and after IFN-therapy; (2) FISH is more accurate than CCA, especially at low levels of Ph-positive cells; (3) testing of directly harvested BM samples is feasible and accurate, giving the opportunity to perform centralized FISH analysis in the context of multicentre trials; (4) the percentage of BCR/ABL+ PB cells usually, though not invariably, reflects the frequency of mutated cells in the BM.

Exposure to myelotoxic agents and myelodysplasia: case-control study and correlation with clinicobiological findings.

To better define the role of exposure to myelotoxic agents in the genesis of myelodysplastic syndrome (MDS), we carried out (a) a case-control study for the determination of the relative risk (RR) of developing MDS, including 178 consecutive patients and 178 sex- and age-matched controls: (b) a study of clinicobiological features in MDS arising after occupational exposure to myelotoxic agents and in MDS in 'non-exposed' patients. The definition of the 'exposure' status was based on a predetermined questionnaire, with calculation of an 'exposure' index (hours/day x days/year x years). Cumulative exposure to pesticides or to organic solvents, for >2400 h, was recorded in 48 and 25 MDS patients, respectively, compared to 27 and four controls (P<0.00001; RR 3.74; 95% confidence interval 2.02-5.37). Older age and an excess of refractory anaemia with ringed sideroblasts and refractory anaemia with excess of blasts was noted among 'exposed' MDS-patients (group 1), compared to non-exposed MDS-patients (group 2). 68.3% patients in group 1 had clonal chromosome changes, compared with 43.2% patients in group 2. Complex karyotypes, -7/7q-, -5/5q-, +8, 7p and 17p aberrations were seen more frequently in group 1, whereas a normal karyotype, isolated 5q- or 20q- occurred more frequently in group 2. The association of exposure to myelotoxic agents with older age at presentation and with unfavourable chromosome changes accounted for the shorter survival observed in 'exposed' patients. These data show that occupational exposure to pesticides and organic solvents in our region resulted in an increased RR of developing MDS and that a distinct cytogenetic profile was associated with MDS in 'exposed' patients. These findings provide strong indirect evidence that these agents may play a role in the pathogenesis of MDS, preferentially targeting some of the chromosome regions which are frequently involved in therapy-related myeloid neoplasias.

Detection and monitoring of trisomy 8 by fluorescence in situ hybridization in acute myeloid leukemia: a multicentric study.

BACKGROUND AND OBJECTIVE: The role of fluorescence in situ hybridization (FISH) in the detection and monitoring of trisomy 8 (+8) in acute myelogenous leukemia (AML) has not been defined exactly. This multicentric study was performed in order to: i) analyze the sensitivity of interphase FISH with respect to conventional chromosome analysis (CCA) in detecting +8; ii) compare the results of FISH and CCA in the quantitation of the frequency of +8-positive cells; iii) analyze the possible role of FISH in the cytogenetic follow-up of patients with +8. DESIGN AND METHODS: One hundred and ninety-eight nonconsecutive patients with a diagnosis of AML seen at five centers over a 3-year period were studied by CCA and FISH with a chromosome 8-specific centromeric probe. Two hundred interphase cells were scored in each test and the cut-off for the recognition of +8 was set at 3%. An irrelevant pericentromeric probe was used as negative control in those cases with an apparently normal karyotype and trisomy 8 in interphase cells. FISH studies were conducted at diagnosis and, in 14 cases with +8, on 1.5 occasions during follow-up. RESULTS: Karyotype aberrations were seen in 121 cases (61.1%), with +8 being present in 38 of them (16 as the sole aberration). Interphase FISH detected +8 in 37/38 cases; in a patient with 1/10 metaphases with +8, 2.3% interphase cells with 3 signals were seen. Fourteen additional cases with occult +8 were detected by FISH, which showed 4-22% interphase cells with three signals; 6 patients had an abnormal karyotype without +8, 3 had a normal karyotype, 5 had no analyzable mitoses. In 24 cases with > 15 analyzable metaphases, percent variations between CCA and FISH in the estimation of the size of the trisomic clone ranged between 0.4% and 51%, median value 22%. Underestimation of the percent of trisomy 8 by FISH occurred in all 10 cases with > 90% +8 metaphases. In 7/14 cases investigated sequentially, FISH detected 5-35% trisomic cells in the BM after induction therapy (4 CR, 3 PR); 4 cases relapsed with +8 at 8-15 months. The absence of +8 in remission marrows was documented in the remaining 7 cases, 4 of which relapsed at 20-32 months. INTERPRETATION AND CONCLUSIONS: It is concluded that FISH was a valuable method in this multicentric study since it showed greater sensitivity than CCA in detecting minor clones with +8, in patients with both normal and abnormal karyotypes. The role of FISH in the cytogenetic follow-up of trisomies in AML patients may be promising.

A randomized double-blind placebo-controlled study with subcutaneous recombinant human erythropoietin in patients with low-risk myelodysplastic syndromes.

To evaluate the effect of recombinant human erythropoietin (rHuEpo) on the haemoglobin level and transfusion requirement in low-risk myelodysplastic syndromes (MDS), 87 patients were enrolled in a randomized double-blind placebo-controlled study, 44 patients were assigned to epoetin alpha (150 U/kg/d s.c. for 8 weeks) and 43 to placebo arms. MDS types were homogenous in both groups: refractory anaemia (RA) 47.7-48.8%. refractory anaemia with ringed sideroblasts (RAS) 20.5-25.6%, refractory anaemia with excess of blasts (RAEB) (blasts < 10%) 31.8-25.6%, 14/38 evaluable patients responded to epoetin alpha versus 4/37 to placebo (P=0.007). 50% of RA responded to epoetin alpha versus 5.9% to placebo (P=0.0072), RAS 37.5% v 18.2% (P=0.6) and RAEB 16.7% v 11.1% (P=1.00). 60% of non-pretransfused patients responded to epoetin alpha (Hb 8.35< or = 0.73 to 10.07+/-1.87 g/dl), whereas a slight decrease was observed in the placebo group (8.4+/-0.66 to 8.19+/-0.92 g/dl) (P=0.0004). Percentage of transfused patients was similar in both arms. Basal erythropoietin (Epo) serum levels > 200 mU/l predicted for a non-response. At week 4 sTfR levels were increased > 50% in responders (P=0.013), whereas an increase < 18% predicted for non-response (P=0.006). Leucocyte and platelet counts were not influenced by epoetin alpha treatment. Adverse events occurred in 31.8% of the rHuEpo-treated versus 42.99%) of the placebo-treated patients (P=0.2), and seven patients did not complete the course. In conclusion, rHuEpo was effective in the treatment of low-risk MDS. RA subtype, no transfusions prior to rHuEpo therapy, and low basal Epo levels were associated with higher probability of response. Soluble transferrin receptor level at the fourth week was an early predictor of response.

Chromosome aberrations in atypical chronic lymphocytic leukemia: a cytogenetic and interphase cytogenetic study.

To define better the chromosomal profile of atypical chronic lymphocytic leukemia (aCLL), cytogenetic and interphase cytogenetic studies were performed in 43 cases, using mitogen-stimulated cultures and DNA probes detecting the two most frequently occurring aberrations in CLL, ie +12 and 13q14 deletions. All cases showed monoclonal CD5/CD19-positive lymphocytosis, with more than 10% large lymphocytes and/or prolymphocytes in peripheral blood smears and reactivity with FMC7, or bright expression of surface immunoglobulins in a fraction of the cases. Karyotype aberrations were detected in 27 of 43 cases (62.8%). Recurrent chromosome changes were +12 (nine cases), 13q14 aberrations (five cases), 11q anomalies (three cases), 6q21-q23 abnormalities and 4q anomalies with different breakpoints (two cases each). Additional chromosome changes were seen in four cases with +12, in three cases with 13q14 anomalies, in two cases with 11q anomalies, in one case with 6q and 4q anomalies. Trisomy 12 was associated with 13q14 anomalies in three cases, one of which also had an 11q abnormality; other associations, found in one case each, were: 13q14 deletion with a 6q anomaly, 11q anomaly with 13q- and 7q-, a 6q anomaly with 7q- and +12. Interphase cytogenetics confirmed the results of chromosome banding analysis and showed that six patients with normal karyotype or no mitosis in fact had concomitant +12 and 13q14 deletion in four cases and isolated +12 or 13q14 deletion in one case each, with a resultant 76% overall incidence of cytogenetic abnormalities. The presence of +12, 13q14 deletions, 11q, and 6q21-q23 anomalies in 19 cases was associated with a 2-month median interval between diagnosis and start of treatment, as compared with a 24-month median interval in 14 cases with normal karyotype or non-recurrent chromosome changes (P = 0.003). We conclude that aCLL is characterized by a relatively high incidence of chromosome anomalies, with recurrent chromosome changes, involving chromosomes 12, 13q14, 6q21q23, 11q, and, possibly, 4q. The presence of complex karyotypes, with concomitant abnormalities of 13q, +12, 6q, 11q, suggests that the development of sequential chromosome changes, rather than any single specific anomaly, may underlie leukemogenesis in this cytologic subset of CLL, partially accounting for the relatively aggressive clinical course.

[Role of computerized tomography in the diagnosis of bone disease in multiple myeloma]. / Ruolo della tomografia computerizzata nella diagnosi della malattia ossea del mieloma multiplo.

PURPOSE: To assess the role of CT in the diagnosis and management of multiple myeloma (MM) and to investigate if CT findings can influence the clinical approach, prognosis and treatment. STUDY DESIGN AND PATIENTS: We reviewed the findings relative to 273 MM patients submitted to CT June, 1994, to December, 1996. The patients were 143 men and 130 women (mean age: 65 years): 143 were stage I, 38 stage II and 92 stage III according to Durie and Salmon's clinical classification. All patients were submitted to blood tests, spinal radiography and CT, the latter with serial 5-mm scans on several vertebral bodies. The CT unit was a Philips Tomoscan SR 7000. RESULTS: CT showed lysis foci in some vertebral bodies (4 cases) where conventional radiography had shown only aspecific osteopenia. CT also depicted vertebral arch and process involvement in 3 cases with the vertebral pedicle sign. Moreover, CT proved superior to radiography in showing the spread of myelomatous masses into the soft tissues in a case with solitary permeative lesion in the left pubic bone, which facilitated subsequent biopsy. As for extraosseous localizations, CT demonstrated thoracic soft tissue (1 woman) and pelvic (1 man) involvement by myelomatous masses penetrating into surrounding tissues. In our series, only a case of osteosclerotic bone myeloma was observed in the pelvis, associated with lytic abnormalities. DISCUSSION AND CONCLUSIONS: The role of CT in the diagnosis and management of MM has not been assessed, because this technique demonstrates tumor extent more accurately than radiography but CT findings do not seem to improve the clinical approach and therapeutic management of the disease. Nevertheless, we recommend CT for some myelomatous conditions, namely: a) in the patients with focal bone pain but normal skeletal radiographs; b) in the patients with M protein, bone marrow plasmocytosis and back pain, but with an inconclusive MM diagnosis; c) to assess bone spread in the regions which are anatomically complex or difficult to study with radiography and to depict soft tissue involvement; d) for bone biopsy.

Detection of numerical aberrations in hematologic neoplasias by fluorescence in situ hybridization.

BACKGROUND AND OBJECTIVE: Over the last 5 years, fluorescence in situ hybridization (FISH) techniques have had an important impact on molecular cytogenetic diagnosis, providing a better understanding of the role of numerical aberrations in hemopoietic neoplasms. The objective of this article is to analyze the clinical applications of FISH in the management of hemopoietic malignancies. EVIDENCE AND INFORMATION SOURCES: The material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline, and personal published and unpublished data. STATE OF ART: FISH technology has the advantage of being relatively simple, fast and flexible. Published data and ongoing prospective studies show that, under well-controlled experimental conditions, interphase FISH is more sensitive than conventional metaphase analysis in the detection of numerical abnormalities. Due to the relatively high rate of false positive results, FISH cannot be used for the study of minimal residual disease. However, since molecular strategies for the detection of small-sized aneuploid clones have not been developed yet, FISH represents a useful adjunct to conventional cytogenetics, especially for the quantitation of the size of abnormal clones during the course of the disease and to monitor XX/XY chimerism following sex mis-matched bone marrow transplantation. Different approaches to the study of multiple cell-lineage involvement by chromosome changes have been developed that take advantage of FISH techniques by: a) simultaneous FISH and membrane immunophenotyping of cytologic and histologic preparations; b) two-step analysis based on assessment of the morphology of cells on panoptical stains, with subsequent hybridization and relocation of previously identified cells; c) FISH analysis of enriched cell fractions obtained by cell sorting or by separation of bone marrow cells on a density gradient, and d) study of single hemopoietic colonies grown in semisolid media. PERSPECTIVES: New molecular cytogenetic techniques, such as dual color FISH comparative genomic hybridization, are at hand that will greatly improve the diagnostic power of cytogenetics and make FISH increasingly useful in research laboratories as well as in clinical practice.

Interleukin-3 plus interleukin-6 may improve chromosomal analysis of multiple myeloma: cytologic and cytogenetic evidence in thirty-four patients.

To better define the role of interleukin-3 (IL-3) and IL-6 in the cytogenetic analysis of multiple myeloma (MM), we performed concomitant chromosome and cytologic studies in 34 patients. In each case, 10-30 x 10(6) bone marrow cells were incubated in two independent cultures consisting of conventional cytogenetic medium with and without IL-3 plus IL-6 added for 72 hours. 1-ml aliquots of each culture were aspirated at 24, 48, and 72 hours and exposed to colcemid for 6 hours. Cytospin preparations were then made and mitotic cells were counted and identified as plasma cells or as nonmalignant cells based on their reactivity with an appropriate anti kappa/lambda serum. Slides for conventional cytogenetic analysis were prepared at 72 hours. A greater than two-fold increase of mitotic plasma cells was observed in cytospin preparations from stimulated cultures versus unstimulated cultures in 15 of 34 cases, whereas a less than 2-fold increase, no variation or no mitosis was recorded in 19 cases. Comparison of the number of mitotic plasma cells in stimulated cultures at 24, 48, and 72 hours showed a decreased mitotic activity at 72 hours. Clonal abnormalities were detected by conventional cytogenetic analysis in 19 of 34 cases (55.8%). Recurrent clonal aberrations involved chromosome 13 (4 cases), chromosomes 1p, and 14q (3 cases); chromosomes 3p, 6q, 7q, and 9q (2 cases). We conclude that IL-3 + IL-6 may increase the number of dividing plasma cells in cytogenetic cultures and that a 2-day culture with these cytokines may facilitate the detection of chromosome abnormalities in MM.

Experience with poorly myelosuppressive chemotherapy schedules for advanced myeloma. The Cooperative Group of Study and Treatment of Multiple Myeloma.

In a multicentre study, 83 patients with advanced and previously uniformly treated multiple myeloma (MM) were randomised between cyclophosphamide (600 mg m-2) and epirubicin (70 mg m-2), administered every 3 weeks for three courses and both associated with prednisone and interferon-alpha2b. Both regimens were administered on an outpatient basis and had low haematological toxicity. Clinical results were similar. Overall response rate (43%) and median response and survival (5.9 and 14.1 months respectively) compare well with those obtained with more aggressive chemotherapy schedules.

Richter's syndrome in a case of atypical chronic lymphocytic leukaemia with the t(11;14)(q13;q32): role for a p53 exon 7 gene mutation.

Clinicobiological, histological, cytogenetic and molecular genetic studies were performed in a case of atypical B-cell chronic lymphocytic leukaemia (B-CLL) with the t(11;14)(q13;q32) evolving into Richter's syndrome (RS) in order (a) to determine the clonal relationship between the cell of origin for B-CLL and RS, and (b) to analyse genetic events underlying the disease progression in this patient. After 4 years following diagnosis, a rapid deterioration of the clinical picture occurred, concomitant with the appearance of large lymphoid blasts in peripheral blood (PB), bone marrow (BM) and ascites samples. A diagnosis of RS was made and cytogenetic analysis revealed karyotype evolution with trisomy 7 and del(17p) in addition to t(11;14). Fluorescence in situ hybridization showed 78% lymphoid blast cells obtained from ascites sample to be trisomic using a chromosome-7-specific pericentromeric probe. Whereas no rearrangement of the c-myc proto-oncogene was detected at disease progression, direct sequencing of p53 gene exon 5-9 revealed an exon 7 missense point mutation. This abnormality was not present in the CLL phase. Immunological staining with the monoclonal antibody PAb-1801, detecting the p53 protein product, revealed a negative pattern in the CLL phase, whereas 24% positivity was documented in representative samples obtained at RS. It is concluded that RS was cytogenetically related with B-CLL in this patient, suggesting the occurrence of a bona fide transformation and that the mutation of p53 exon 7, in association with the development of 17p deletion, possibly played a role in the development of RS.

Atypical chronic lymphocytic leukaemia with t(11;14)(q13;q32): karyotype evolution and prolymphocytic transformation.

In order to define better the cytological and clinical features of atypical B-cell chronic lymphocytic leukaemia (B-CLL) with t(11:14)(q13;q32), sequential morphologic immunological and cytogenetic studies were performed in seven patients belonging to a series of 72 consecutive cases presenting with a diagnosis of CLL or atypical CLL according to the FAB criteria. Cytologic diagnosis in these seven patients with t(11;14) was typical CLL in two cases presenting with < 10% large lymphocytes (LL) and prolymphocytes (PL) and atypical CLL in five cases in which LL and PL comprised between 10% and 55%. The diagnosis was supported by histologic findings on bone marrow biopsy (five cases) or splenectomy specimens (two cases). A progressive increase of peripheral LL and PL was observed, resulting in a switch of FAB diagnosis over a 6-60-month period from typical CLL into atypical CLL in two cases and from atypical CLL into prolymphocytic leukaemia in five cases. Immunophenotyping showed a mature B-cell phenotype with CD19, CD22, CD24 positivity and CD10 negativity in all patients. A bright-staining pattern for surface immunoglobulins (SIg) was detected in 6/7 cases, CD5 positivity in 6/7 cases, and CD23 positivity in 1/7 cases. The FMC-7 monoclonal antibody was positive in > 40% cells in 5/6 cases. Chromosome changes in addition to t(11;14) were seen in five cases; in two cases unbalanced translocations involving the 3q21 chromosome region, resulting in partial trisomy for the long arm of chromosome 3, were detected early in the course of the disease. Karyotype evolution that was associated with disease progression occurred in 3/6 assessable patients. Comparison of these findings with similar data from 65 B-CLL patients without t(11:14) showed that atypical morphology, switch of FAB diagnosis during the course of the disease, and karyotype evolution were more frequently seen in cases with t(11;14) (5/7 v 15/65 cases, P = 0.015, 7/7 v 7/65 cases, P < 0.0001, and 3/6 v 5/45 assessable cases, P = 0.04, respectively). The frequency of positivity for CD23 and bright SIg staining differed significantly in the two groups. It is concluded that t(11;14) identifies a cytologically atypical subset of B-CLL, characterized by frequent cytologic and cytogenetic evolution and by a distinct immunological profile, sharing some biological features with mantle cell lymphoma.

Trisomy 12 in chronic lymphocytic leukemia and hairy cell leukemia: a cytogenetic and interphase cytogenetic study.

Fluorescent in situ hybridization (FISH) with a chromosome 12-specific pericentromeric probe was performed in 42 patients with B-cell chronic lymphocytic leukemia (CLL) and in 10 patients with hairy cell leukemia (HCL). In all cases, a normal karyotype in more than 10 metaphase cells was obtained by conventional chromosome study. FISH documented that 6/42 patients with CLL in fact had trisomy 12 in 15-49% interphase cells. Sequential FISH studies were performed in 2 cases, showing an increase of percentage of trisomic cells over a 2-month to 4-year period. Two out of 10 patients with HCL, one of whom had morphologic features consistent with a diagnosis of HCL variant, showed 5.5 and 10% interphase nuclei with three fluorescent signals, a finding suggestive of the presence of trisomy 12. Combined immunophenotyping and FISH staining in these patients with HCL documented that trisomic cells were CD11c-positive, CD13-negative, and CD2-negative. We conclude that FISH is a sensitive technique allowing for the detection of trisomy 12 in a fraction of cytogenetically normal patients affected with CLL and HCL.

Lineage switch and multilineage involvement in two cases of pH chromosome-positive acute leukemia: evidence for a stem cell disease.

Philadelphia chromosome-positive acute leukemias (Ph+ AL) show variable cytologic features, possibly reflecting heterogeneous stem cell involvement. Morphologic, immunologic and cytogenetic studies were performed in two cases of Ph+ acute lymphoblastic leukemia (ALL) in order to better delineate the clinicobiological features of this cytogenetic subset of AL. Sequential cytoimmunologic studies in patient 1 documented a lineage switch from pro-B ALL with a minor myeloid component at diagnosis to minimally differentiated acute myeloid leukemia (AML) at relapse. In this patient the major breakpoint cluster region (M-bcr) was in a rearranged configuration and all metaphase cells showed t(9;22)(q34;q11), both at diagnosis and at relapse. In patient 2 a diagnosis of Ph+ early T-cell ALL with minor myeloid component was made. In this patient the M-bcr was in a germline configuration. Cytogenetic studies documented the presence of the Ph chromosome in all metaphases from a lymphoid cell population obtained by fine-needle aspiration of an enlarged lymph node, and from a bone marrow cell fraction enriched in granulocyte precursors. This finding suggests multilineage involvement in this patient. Lineage switch and multilineage involvement in two patients suggest that a pluripotent stem cell may be affected rather frequently in patients with Ph+ AL. These findings show that biologically Ph+ AL may resemble chronic myelogenous leukemia blast crisis, since it may originate from an undifferentiated stem cell carrying the t(9;22) translocation.

A prospective, controlled, nonrandomized study on prophylactic parenteral dichloromethylene bisphosphonate (clodronate) in multiple-myeloma.

Bone resorption by osteoclasts causes neoplastic bone disease, which is a significant cause of death in multiple myeloma (MM). Counteracting bone resorption with prophylactic bisphosphonates has delayed bane disease, and this is expected to improve survival. Between January, 1987 and March, 1990, 341 evaluable previously untreated, consecutive patients with MM entered a prospective, multicenter study in which cytostatic therapy was randomized. The first 148 patients recruited were not planned for prophylaxis and the following 193 were scheduled to receive parenteral, prophylactic clodronate. Clodronate was administered at a dose of 600-1000 mg/4-6 weeks and was started at diagnosis and continued throughout survival time. Data on clodronate prophylaxis were evaluated on both an intention-to-treat and a compliance analysis basis. The rate of response and the duration of response were independent of clodronate prophylaxis. Progression of skeletal disease occurred less often in patients who received the drug than in those who were not given prophylaxis (50.5 vs 34.8%; p<.02 by compliance analysis). Survival was longer for patients on clodronate prophylaxis than for those who were not planned for (p<.02 by intention to-treat-analysis) or for those who did not receive clodronate prophylaxis (p<.009 by compliance analysis). Local pain associated with i.m. administration was the only significant side effect of clodronate. Parenteral clodronate prophylaxis prolongs survival in MM, probably because it allows better control of bone disease and reduces deaths related to it.