Involvement of peripheral blood cells in multiple myeloma: chromosome changes are the rule within circulating plasma cells but not within B lymphocytes.

The mononuclear cells in the peripheral blood are implicated in the myeloma process especially with the presence of peripheral blood plasma cells (PBPC) and clonal B lymphocytes found using phenotypic or gene rearrangement techniques. The purpose of this study was to look for aneuploidy in the two main B cell components of the peripheral blood: PBPC and CD20-positive B lymphocytes. Conventional cytogenetics (CC) or DNA content analysis and fluorescence in situ hybridization (FISH) with centromeric probes were performed on bone marrow plasma cells (BMPC) of 21 patients with multiple myeloma and peripheral blood cells were studied as follows: immunostaining to look for PBPC and to assess their number, image analysis cytometry for the determination of their DNA content, and FISH chromosomes analysis. FISH was performed using probes against the chromsomes that were lost or gained in BMPC and was coupled with immunostaining of the relevant light chain or CD20 antigen to study PBPC or B lymphocytes, respectively. Monotypic PBPC were found in 16 patients. Their DNA content was the same or nearly the same as for BMPC and they exhibited the same monosomies or trisomies as those found within their BM counterpart. By contrast, DNA content of mononuclear cells other than PBPC was within normal ranges, and in 13 of 15 patients CD20-positive B lymphocytes failed to show chromosomal changes by FISH analysis. In two patients however, a few CD20+ cells with lymphoid morphology exhibited chromosome changes, hypothesizing that a few cytogenetically abnormal B cells without plasmocytic morphology may circulate. From these data, we conclude that PBPC share the same genetic abnormalities as BMPC and thus belong to the malignant clone, whereas most peripheral blood B lymphocytes are unrelated to the tumor clone.

Interphase fluorescence in situ hybridization (FISH) as a powerful tool for the detection of aneuploidy in multiple myeloma.

Conventional cytogenetic (CC) studies performed in multiple myeloma (MM) are difficult because of the low proliferation rate of plasma cells (PC). The purpose of this study was to compare results obtained by CC and by FISH for the detection of numeric chromosomal changes in patients with MM. PC DNA content, CC and interphase FISH analysis were performed on 29 consecutive patients with MM. Fifteen patients (control group) had known Cytogenetic abnormalities identified by CC. The other 14 patients (study group) had a normal karyotype but an abnormal DNA content. Bone marrow material prepared for CC or cytospin slides were probed with classical satellite III or alpha satellite DNA sequences for chromosomes 3, 7, 8, 9, 11 and 15 (chromosomes 3, 7, 9, 11, 15 probes for hyperdiploid patients and the chromosome 8 probe for hypodiploid patients). In the control group, an unexplained discrepancy between CC and FISH occurred for only one chromosome in one patient. Also in this group, four patients had only one abnormal cell by CC and the numeric changes in these patients were always confirmed by FISH analysis. In the study group, FISH analysis showed an abnormal result in all but one patient. From these data, we conclude that FISH improves the detection of cytogenetic abnormalities in multiple myeloma. Using commercially available DNA probes for the most frequent numeric changes and slides for CC or cytospin slides, we demonstrated abnormal cytogenetics by FISH in 28/29 patients. In further studies, use of FISH could permit a more accurate description of numeric changes and their prognostic value in MM as well as an approach to clonal evolution. It would also be of interest in the study of monoclonal gammopathies of undetermined significance.

Monoclonal gammopathy of undetermined significance: chromosome changes are a common finding within bone marrow plasma cells.

We used two indirect approaches [image analysis (Feulgen staining) and fluorescence in situ hybridization (FISH)] to study bone marrow plasma cells (BMPC) in 28 patients fulfilling criteria for MGUS. 61% of patients were found to be aneuploid after image analysis: three were hypodiploid and 14 were hyperdiploid. 12/14 hyperdiploid patients also revealed abnormalities after FISH: 12-72% of BMPC exhibited trisomy for at least one of chromosomes 3, 7, 9 and 11. These latter chromosomes are the four chromosomes most frequently implicated (in the shape of trisomy) in MM, confirming the tight relationship between both conditions. After a median follow-up of 19 months (12-41 months) no patient developed overt MM. Also, we failed to find any relationship between currently available biological parameters and DNA findings. As literature data give a transformation rate of 20-30% after a follow-up of 20-35 years, it is worth presuming that some aneuploid patients will evolve to MM, whereas others (also with aneuploid bone marrow plasma cells) will never develop cancer. Our findings indicate that numeric abnormalities, as they are shared both by MGUS and MM patients, are certainly an additional or a prerequisite event, but are not related to an overt disease. They also emphasize the importance of cytogenetic study in the pathophysiology of MGUS.

Combined immunophenotyping and in situ hybridization (FICTION): a rapid method to study cell lineage involvement in myelodysplastic syndromes.

We present a study in which we used a recently described method combining fluorescence in situ hybridization (FISH) and immunophenotyping, i.e. FICTION, to assess the involvement of different cell lineages in myelodysplastic syndrome (MDS) with monosomy 7 (-7), trisomy 8 (+8) or loss of Y chromosome (-Y). Blood or marrow smears or cytocentrifuge preparations were stained both by antibodies to granulocytes (CD15), monocytes (CD14), T lymphocytes (CD3), B lymphocytes (CD20) and by probes specific for chromosomes 7, 8 or Y. Of nine cases of MDS with -7, four with +8 and two with -Y studied, none showed lymphocytic involvement by the chromosome abnormality. In contrast, -7, +8 and -Y were found in granulocytes and monocytes in all patients studied, but they involved a variable proportion of those cells. The partial involvement by -7 and +8 seen in some cases suggests that myelopoïesis was only partially clonal in those cases, or that the chromosome abnormality was a secondary event in the MDS process. FICTION therefore appears to be a simple and easily reproducible method that can be used for the assessment of lineage involvement in MDS and other haematological malignancies.

Improved cytogenetics in multiple myeloma: a study of 151 patients including 117 patients at diagnosis.

Between December 1990 and January 1994, bone marrow (BM) samples from 151 patients with multiple myeloma (MM), including 117 patients evaluated at diagnosis, were collected for cytogenetic analysis. A total of 129 patients had assessable metaphases (100 patients at diagnosis). Cytogenetic studies were performed on BM cells after longterm cultures (6 days) with stimulation of cultures by granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF plus interleukin (IL)-6, IL-3 plus IL-6, or GM-CSF plus IL-3 plus IL-6 to improve myeloma cell growth, and 91 patients had an additional unstimulated culture. Sixty-six patients (51%) had cytogenetic abnormalities, including 47 of 100 patients at diagnosis (47%) and 17 of 24 patients at relapse (71%; P = .04). The aberration rate increased with stage (P = .007), BM plasmacytosis (P = .003), beta 2 microglobulin level (P = .001), C-reactive protein (CRP) level (P = .001), and Ki-67 (P = .007). The abnormality detection rate was higher in stimulated than unstimulated cultures, and the difference was statistically significant (P < .01). Hyperdiploidy was observed in 39 patients (30% of patients with an assessable karyotype) and hypodiploidy in 19 patients (15%). Among numeric changes, gains predominantly involved chromosomes 3, 5, 7, 9, 11, 15, 19 and losses, chromosomes 8, 13, 14, and X. The most frequent loss was loss of chromosome 13, observed in 22 patients (15%), including 18 patients at diagnosis (12%). We observed frequent structural changes of chromosomes 1 (15%) and 14 (10%) but also a 5% incidence of 19q13 abnormality and two patients with translocation t(1;16)(p11;p11). By using the proportional hazard univariate model, patients with abnormal karyotypes were demonstrated to have 2.5-fold greater chance of death than patients with normal karyotypes (P < .014). Despite a multivariate approach with the same model, the respective roles of karyotype abnormality, age, stage, and beta 2 microglobulin level could not be clearly ascertained. From these results we conclude that cytogenetic analysis using stimulation of cultures by cytokine(s) may be a promising method to identify about 50% of cytogenetic abnormalities in patients with newly diagnosed MM. Cytogenetic analysis may help to define a high-risk population that would benefit from intensive therapeutic approaches.

Clinical significance of p53 mutations in newly diagnosed Burkitt's lymphoma and acute lymphoblastic leukemia: a report of 48 cases.

PURPOSE: To correlate the presence of p53 mutations and initial characteristics, response to chemotherapy, and survival in newly diagnosed Burkitt's lymphoma (BL) and Burkitt's acute lymphoblastic leukemia (L3 ALL). PATIENTS AND METHODS: Forty-eight patients with newly diagnosed BL or L3 ALL, most of whom were treated with very intensive regimens, including early CNS disease treatment, were studied. Detection of p53 mutations was made by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 of the gene, and mutations were determined by direct sequencing of exons with abnormal SSCP findings. Comparison of outcome between mutated and nonmutated cases was made in all patients and also after excluding five patients who received therapeutic regimens considered as suboptimal and one patient who died of AIDS while in complete remission (CR), as those six patients had no p53 mutations. RESULTS: A point mutation was found in nine patients (19%), and consisted of a missense mutation in seven and a chain-terminating mutation in two. SSCP, sequence, and cytogenetic analysis strongly suggested that eight of nine patients with mutations had retained the normal p53 allele, which had been lost in the remaining patient. These findings were confirmed by fluorescence-in-situ hybridization (FISH) with a p53-specific probe in two patients, including the one who had lost the normal p53 allele. Unexpectedly, mutations were significantly less frequent in patients with disseminated disease, ie, L3 ALL or stage IV BL (four of 35, 11%), than in more localized forms, ie, BL stage I, II, or III (five of 13, 38%) (P = .03). CR rates were similar in mutated (78%) and nonmutated cases (78%). The actuarial disease-free interval (DFI) after 12 months and actuarial survival rates after 24 months were 49% and 66%, respectively, in patients with mutations, and 73% and 48%, respectively, those without mutations. The differences were not significant. CONCLUSION: Our findings suggest that, contrary to what is seen in most other neoplasias, p53 mutations in newly diagnosed BL and L3 ALL are not associated with extensive tumor mass or poor response to intensive therapeutic regimens. It is hypothesized that this difference with most tumors could be due to the fact that p53 mutations in BL and L3 ALL are generally associated with persistence of a normal residual p53 allele, contrary to what is observed in the majority of tumors.

Myelodysplastic syndromes and acute myeloid leukemia with 17p deletion. An entity characterized by specific dysgranulopoïesis and a high incidence of P53 mutations.

We looked for correlations between cytogenetic rearrangements leading to 17p deletion and presence of dysgranulopoïesis and p53 mutations in MDS and AML. Forty-nine (4.3%) of the MDS and AML studied cytogenetically at our institution over a period of 11 years had detectable 17p deletion, through monosomy 17 (14 cases) or rearrangements of chromosome 17 (generally unbalanced translocations between 17p and another chromosome) (35 cases). Most of the patients had additional complex cytogenetic findings, and 10 cases were therapy related. In 70% of the patients with 17p deletion, a particular type of dysgranulopoïesis, combining pseudo-Pelger-Huët anomaly and small vacuolated neutrophils was seen in > 5% marrow neutrophils, whereas 69% of the patients had a p53 mutation, generally in a missense mutation involving exons 5 to 8 of the p53 gene. FISH analysis, performed in eight cases, confirmed loss of one P53 allele in all of them. No DNA fragmentation suggesting increased apoptosis was found in marrow samples. Response to chemotherapy was almost uniformly poor and median survival was only 3 months. Analysis of dysgranulopoïesis and p53 mutations were also made in 'control' groups of MDS and AML without 17p deletion. 'Typical' dysgranulopoïesis, combining pseudo-Pelger-Huët anomaly and small vacuolated neutrophils in > 5% marrow neutrophils, was not seen in any of the 47 MDS and AML without 17p deletion analyzed and without p53 mutation (P = 10(-4) with patients having 17p deletion), and was seen in one of five patients without 17p deletion but with a p53 mutation. Only 3.1% of 256 MDS and AML without 17p deletion had a p53 mutation (P = 10(-4) with patients having 17p deletion). These findings suggest that 17p deletion, in MDS and AML, is strongly correlated to the presence of a particular type of dysgranulopoïesis and to a high incidence of p53 mutations, and that MDS and AML with 17p deletion could constitute a new morphological-cytogenetic-molecular entity in myeloid disorders.

Reassessment of an apparent t(12;17)(p11;p11) as an unbalanced t(17;21)(p11;q11) in a case of B-cell chronic lymphocytic leukemia by fluorescence in situ hybridization.

We report a published case of chronic lymphocytic leukemia (CLL) in which conventional cytogenetic analysis supported the presence of an unbalanced t(12;17)(p11;p11). However, fluorescence in situ hybridization (FISH) analysis, with specific DNA libraries from chromosomes 17 and 21 (chromosome painting), demonstrated that the translocation was actually an unbalanced t(17;21)(p11;q11).

Two cases of t(1;16)(p11;p11) in multiple myeloma: confirmation by chromosome painting.

Two patients with multiple myeloma and an unbalanced translocation, t(1;16)(p11;p11), are reported. The fluorescence in situ hybridization (FISH) technique was used in one patient to confirm the translocation. To our knowledge, t(1;16)(p36;q13) and t(1;16)(q21;p13), but not t(1;16)(p11;p11), had been reported previously in multiple myeloma. Our results suggest that FISH is useful to characterize structural abnormalities and identify marker chromosomes in multiple myeloma where analysis with conventional cytogenetics is often difficult.

Fluorescence in situ hybridization improves the detection of monosomy 7 in myelodysplastic syndromes.

We performed conventional cytogenetic (CC) and interphase fluorescence in situ hybridization (FISH) analysis with an alpha satellite chromosome 7 specific DNA centromeric probe (p alpha 7t1) on bone marrow material prepared for CC in 11 controls and 80 cases of myelodysplastic syndromes (MDS). In controls, a mean of 4.3 +/- 1% of the 700 cells examined showed only one FISH signal for chromosome 7, and the finding of > 6.3% (mean +2 standard deviations) of cells with one FISH signal was considered to indicate the presence of a clone with -7. By CC, clonal -7 was found in 11 patients, whereas two patients had -7 in only one mitose (non-clonal -7). In eight of the 11 cases of clonal -7 by CC, interphase FISH confirmed -7. In the remaining three patients, 5.1%, 6.3% and 18.4% respectively of the cells had one signal. Those three patients had, in addition to -7 by CC, a marker chromosome which was shown to be constituted of chromosome 7 pericentromeric material by FISH analysis on metaphase spreads (metaphase FISH). Of the two patients with non-clonal -7 by CC, one had a -7 clone by interphase FISH whereas the other patient had normal FISH results. Five of the 67 patients with no -7 mitose by CC had clonal -7 by interphase FISH, with one chromosome 7 signal in 14.4 to 39% of the cells examined. At least three mitoses with -7 were found in two of them by metaphase FISH. Three of the five patients were reexamined 12 to 17 months later: CC and metaphase FISH found no -7, whereas interphase FISH still showed a -7 clone. Three of the patients with clonal -7 by CC and by FISH were reexamined in complete hematological remission after intensive therapy. CC found no -7 and interphase FISH was normal in all three patients. Our findings suggest that interphase FISH may improve the detection of -7 in MDS. Conventional cytogenetics should still be performed in parallel to FISH, however, because of possible false negative FISH results when a pericentromeric chromosome 7 marker is present in patients with -7. Larger numbers of cases with minor -7 clones, detectable by FISH only, and longer follow-up in those cases will be necessary to determine the significance of this finding, the evolution of this minor clone, and the outcome of the patients.

Cytogenetic and molecular investigations of an abnormal Y chromosome: evidence for a pseudo-dicentric (Yq) isochromosome.

A derivative Y chromosome was found in a 55-year-old man with Lambert-Eaton paraneoplasic pseudomyastheniform disease. Small testicles, azoospermia were noticed and hormonal level values were as in the Klinefelter syndrome. A 45,X/46,XYp+ mosaïcism was described on peripheral blood lymphocytes. Cytogenetic investigations with R-G-C- and Q-banding have been performed. In situ hybridization with the GMGY 10 DNA probe showed two copies of proximal Yp sequences. Southern blot analyses were performed using the Y DNA probes 27a, 47z, 64a7, 50f2 disclosing specific Yp and Yq sequences from the pseudoautosomal boundary to the Yq proximal portion. The der(Y) has been defined as a dicentric isochromosome for the long arm with one active and one apparently suppressed centromere. The breakpoint leading to the der(Y), has been located in the pairing segment of the Y short arm (i.e. Yp11.32). So the der(Y) was interpreted as a psu dic(Y) (qter-->cen-->p11.32 ::p11.32-->qter). There was thus an almost complete duplication of the Y chromosome.

RFLPs tightly linked with cystic fibrosis: value of probes at the D7S23 locus in prenatal diagnosis.

In a 1:4 risk family, the usefulness of probes at the D7S23 locus for prenatal diagnosis of cystic fibrosis is discussed by comparison with probes at the MET, D7S8, and D7S18 loci that did not allow accuracy in this family.