Myelodysplastic syndromes, from French-American-British to World Health Organization: comparison of classifications on 431 unselected patients from a single institution.

In 1999 a working group of the World Health Organization (WHO) published a revised classification for myelodysplastic syndromes (MDS): RA, RARS, refractory cytopenia with multilineage dysplasia (RC+Dys), RAEB I and II, del (5q) syndrome, and MDS unclassifiable. Chronic myelomonocytic leukemia (CMML) and RAEB-t were excluded. Standard French-American-British (FAB) and new WHO classifications have been compared in a series of patients (n = 431) from a single center, analyzing morphologic, clinical, and cytogenetic data. According to the WHO findings, dysgranulocytopoiesis or dysmegakaryocytopoiesis only were found in 26% of patients with less than 5% medullary blasts. These patients are thus unclassified and should remain in the subgroups RA and RARS. Splitting of heterogeneous RAEB into 2 subgroups according to blast count was supported by a trend to a statistically significant difference in the single-center study population. Patients with CMML whose white blood cell counts are above 13 000/microL may be excluded from the MDS classification, as warranted by WHO, but a redistribution of patients with dysplastic CMML according to medullary blast count leads to more heterogeneity in other WHO subgroups. Although the natural courses of RAEB-T and acute myeloid leukemia (AML) with dysplasia are different, comparable median survival durations after treatment in patients with RAEB-T and AML were in favor of the proposed 20% medullary blast threshold for AML. The homogeneity of subgroups was studied by evaluating prognostic scores. A significant shift into lower IPSS risk groups was evident in the new classification. These data cannot provide evidence for the new WHO proposal, which should not be adopted for routine clinical use at present. Some of its aspects can provide a starting point for further studies involving refined cytogenetics and clinical results.

Dysplastic versus proliferative CMML--a retrospective analysis of 91 patients from a single institution.

In chronic myelomonocytic leukemia (CMML) segregation of two subtypes has been suggested depending on WBC count-myelodysplastic (MD-CMML) and myeloproliferative (MP-CMML). In a retrospective analysis of 91 (60/31) previously untreated CMML patients, we compared the presenting clinical, haematological, laboratory and bone marrow features and examined the clinical impact of this reclassification. LDH values and bone marrow cellularity were significantly increased in MP-CMML. Median survival was significantly longer for patients with MD-CMML, progression rate was higher for MP-CMML. Patients with MD-CMML had longer median preleukemic duration; after transition to AML, MP-CMML patients had longer median survival. In MDS phase anemia was more common in MP-CMML and thrombocytopenia more common in MD-CMML whereas transfusion rates showed no difference. Evaluation of prognostic scoring systems for both groups confirmed that patients' characteristics and outcome could be well compared. Our data suggest that segregation into MD-CMML and MP-CMML is justified.

Amplification of ribosomal RNA genes in acute myeloid leukemia.

Gene amplification is a relatively rare event in acute myeloid leukemia (AML). Double minutes (dmin) and homogeneously staining regions are well established phenomena as cytogenetic correlates of gene amplification. Recently, however, two additional mechanisms leading to gene amplification, i.e., segmental jumping translocations and formation of ring chromosomes, have been described. We report four patients with AML, in whom bone marrow cells exhibited amplifications of ribosomal RNA (rRNA) genes in the form of ring chromosomes or a hsr. In two patients, the MLL gene, and in one patient the CBFA2 gene were shown to be co-amplified with rRNA genes. In two of the four patients, multiple copies of alpha-satellite sequences of the centromeres 13 and 21, respectively, were also demonstrated. In three of the four patients, the clinical course was very aggressive, leading to death within 2-8 months. In these three patients, complex karyotype abnormalities were found, whereas the karyotype of Patient 4 was characterized only by supernumerary ring 21 chromosomes of different sizes and a trisomy 8 in half of the metaphases. Modes of origin and clinical significance of the amplification of rRNA genes are discussed.

Clinicopathogenetic significance of chromosomal abnormalities in patients with blastic peripheral B-cell lymphoma. Kiel-Wien-Lymphoma Study Group.

So far, reproducible histomorphologic and immunological criteria to distinguish clinicopathologic subtypes of blastic peripheral B-cell non-Hodgkin's lymphoma (BBCL), especially centroblastic (cb) and immunoblastic (ib) lymphomas, for daily diagnostic use are still lacking. Therefore, we correlated the cytogenetic findings in 126 patients with BBCL with histopathologic diagnoses. Subclassification of cb and ib lymphomas relied on the criteria defined in the updated Kiel classification; these subtypes are also listed in the Revised European-American Lymphoma (REAL) classification and in a preliminary report on the newly established World Health Organization classification, to investigate their clinical significance. Moreover, we performed a multivariate analysis to compare the prognostic significance of cytogenetic findings with the International Index. There were significant differences in the frequency of chromosome aberrations between different BBCL subtypes: t(8;14) was predominantly present in Burkitt's lymphomas, t(14;18) in centroblastic lymphomas, deletions in 8q and 14q, changes of 4q and losses of chromosome 10 in immunoblastic lymphomas; t(11;14) was restricted to blastoid mantle cell lymphomas and associated with a poor prognosis. In cb lymphomas, deletions in 1q42-qter, duplications in 1q23-32, trisomy 5, and changes of 15q were identified as independent prognostic factors. In ib lymphomas, changes of 7q and 8q had stronger impact on survival than the International Index. These findings underline that Burkitt's, cb, ib, and blastoid mantle-cell lymphoma are biologically distinct and clinically relevant entities and that cytogenetic findings can be helpful to subtype BBCL.

Cross-validation of prognostic scores in myelodysplastic syndromes on 386 patients from a single institution confirms importance of cytogenetics.

In myelodysplastic syndromes (MDS) different prognostic risk analysis systems based on clinical and morphological data are used for predicting survival. Data on diagnostic and prognostic relevance of karyotype aberrations have prompted the development of scores including cytogenetics. The aim of this study was to assess and compare the explanatory power of different scoring systems and to assess the additional explanatory power of cytogenetics by evaluating the clinical and laboratory data of MDS patients from a single institution. Data of 386 MDS patients was available, with cytogenetic analysis at time of diagnosis in 256. Clinical/morphological scores: Bournemouth, modified Bournemouth and Düsseldorf; and scores including cytogenetics: Lausanne-Bournemouth, Lille and the International Prognostic Scoring System (IPSS), were calculated and their predictive power was compared for both overall survival and preleukaemic duration. Each of the scores had significant correlation on both endpoints. Calculating the prognostic value of different cytogenetic aberrations we found that differentiating between evidence for no aberration, single aberrations excluding chromosomes 7 and 8, aberrations on chromosomes 5, 7 or 8 and complex aberrations was important. These data were incorporated in a 'prognostic index cytogenetics' (pi score). Cytogenetic scores significantly improved the prognostic value of the best clinical/morphological score in regard to both overall survival and preleukaemic duration. In conclusion, our data further stress the importance of cytogenetics for predicting prognosis in MDS.

Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32).

Mucosa-associated lymphoid tissue (MALT) lymphomas most frequently involve the gastrointestinal tract and are the most common subset of extranodal non-Hodgkin lymphoma (NHL). Here we describe overexpression of BCL10, a novel apoptotic signalling gene that encodes an amino-terminal caspase recruitment domain (CARD), in MALT lymphomas due to the recurrent t(1;14)(p22;q32). BCL10 cDNAs from t(1;14)-positive MALT tumours contained a variety of mutations, most resulting in truncations either in or carboxy terminal to the CARD. Wild-type BCL10 activated NF-kappaB but induced apoptosis of MCF7 and 293 cells. CARD-truncation mutants were unable to induce cell death or activate NF-kappaB, whereas mutants with C-terminal truncations retained NF-kappaB activation but did not induce apoptosis. Mutant BCL10 overexpression might have a twofold lymphomagenic effect: loss of BCL10 pro-apoptosis may confer a survival advantage to MALT B-cells, and constitutive NF-kappaB activation may provide both anti-apoptotic and proliferative signals mediated via its transcriptional targets.

Cytogenetic analysis and fluorescence in situ hybridization in a case of IgD multiple myeloma.

Immunoglobulin D multiple myeloma (IgD MM) is a subentity of MM occurring in fewer than 2% of patients with distinct clinical pattern, dismal prognosis, and very little information about genetic abnormalities. The karyotype and the results of fluorescent interphase in situ hybridization analysis of a 62-year-old female patient with IgD MM are presented and show a complex hypodiploid karyotype with loss of an X chromosome and monosomy 13--very well known adverse prognostic factors in MM--but, in addition, several deletions of chromosomes 1, 6, 11, and 12, as well as translocations involving chromosomes 4, 9, 10, 15, 16, and 21 that underline the singularity of IgD MM.

Correlation of cytogenetic, molecular cytogenetic, and clinical findings in 59 patients with ANLL or MDS and abnormalities of the short arm of chromosome 12.

Abnormalities of the short arm of chromosome 12 (12p) are found in about 5% of acute nonlymphocytic leukaemias (ANLL) and myelodysplastic syndromes (MDS). They are described to be characteristic of secondary leukaemias, especially after prior mutagenic exposure, and to be associated with a poor prognosis. In our series of 59 patients with 12p abnormalities and ANLL or MDS, exposure to genotoxic agents was proven only in five patients, but in 13/44 patients ANLL evolved from an MDS. Patients with a small deletion del(12)(p11.2p13) having a mild clinical course were distinguished from those with a large del(12)(p11.2), additional chromosomal anomalies, and a poor clinical course. Among the 31 patients with translocations or dicentric chromosomes involving 12p, a group of eight with t/dic(12;13) was the most frequent and was associated with a poor prognosis. The clinical outcome was adverse in the majority of patients with complex karyotype abnormalities, but in some patients a milder clinical course seems likely. A new, hitherto undescribed, abnormality in an MDS case with a duplication dup(12)(p11.2p13) was the amplification of the signal of the yeast artificial chromosome (YAC) clone 964c10 (D12S736). In 38 cases with deletions or unbalanced translocations/dicentrics one YAC signal was lost. Five patients with balanced translocations demonstrated breakpoints within the YAC, containing the ETV6 (TEL) gene. The breakpoints were telomeric to the YAC 964c10 in seven cases and centromeric in one patient.

Cytogenetic findings in 175 patients indicate that items of the Kiel classification should not be disregarded in the REAL classification of lymphoid neoplasms.

Cytogenetics have proved to be a valuable tool for classifying systemic lymphatic neoplasms, as this technique allows different stem line aberrations and clonal developments to be distinguished. This study was designed to analyze how far groups defined according to common cytogenetic features correlated with their position in either the Kiel (KC) or the REAL classification. Cytogenetic analyses were performed on material from 175 patients with lymphoid neoplasms (LN). Samples were prepared from peripheral blood and bone marrow in acute lymphoblastic leukemia (ALL), from bone marrow in multiple myeloma (MM), and from lymph node biopsies in lymphomas. The results of this study support the inclusion of ALL, MM, and extranodal lymphomas into a comprehensive classification, because their chromosomal aberrations were always characteristic for LN. From the cytogenetic point of view, a subgroup of ALL appears as a leukemic manifestation of lymphoblastic lymphoma. MM have structural aberrations of chromosomes 1, 11, and 14 and secondary aberrations of chromosomes 3, 6, 7, 12, 13, and 18, all of which are characteristic for lymphatic disease. The groups with follicle center cell lymphoma and mantle cell lymphoma correlate well with our results both in the low-grade subtype and in the blastic variant type, the majority of cases demonstrating t(14; 18) and its variants and t(11; 14), respectively. In contrast, the group of diffuse large B-cell (DLB) lymphomas proved to be heterogeneous on the basis of our cytogenetic results. Accordingly, we would suggest keeping the immunoblastic lymphoma (IB) subtype defined by the KC. IB demonstrates no stem line aberration in common with any other group and seems to be characterized by stem line aberrations involving chromosomes 3 and 6. As some DLB lymphomas have a t(14;18) or variant translocations involving chromosome 18, they should either be separated as a subgroup or included into the group of follicle center lymphomas.

Variant forms of Philadelphia translocation in two patients with chronic myeloid leukemia.

During a 4-year period (December 1990-December 1994), among other diagnoses one hundred cases of chronic myeloid leukemia (CML) were analyzed in our department. We focused our attention on two cases with a variant form of Philadelphia translocation. Cytogenetic and molecular genetic studies were performed to resolve the status of BCR and ABL in the bone marrow or peripheral blood cells of the two CML patients with complex translocations involving chromosomes 3, 9, 22 and 9, 12, 22 respectively. In the first case the presence of Ph chromosome was detected cytogenetically, BCR-ABL translocation was detected by Southern hybridization. In the second case, only the PCR method showed BCR-ABL rearrangement. The second case, with a random variant form of Ph translocation, could be detected using different methods of clinical molecular genetics.

Multiple myeloma: high incidence of chromosomal aneuploidy as detected by interphase fluorescence in situ hybridization.

Because metaphase cytogenetic studies in multiple myeloma (MM) are hampered by a low proliferative activity of myeloma cells in vitro, interphase cytogenetics by means of fluorescence in situ hybridization (FISH) should improve the detection of chromosomal abnormalities in MM. We therefore investigated chromosomal aneuploidy in 36 patients with MM using interphase FISH and alpha-satellite DNA probes for chromosomes 1, 3, 7, 8, 11, 12, 16, 17, 18, and X. By FISH, myeloma cells from 32 patients (88.9%) were aneuploid for at least one of the chromosomes examined. In 24 patients (66%), aberrations of > or = 3 chromosomes were observed. Aneuploidy was predominantly characterized by a gain of chromosome numbers, with involvement of chromosomes 3, 7, and 11 occurring in > 50% of patients. Loss of a centromeric signal suggesting monosomy was most frequently observed for chromosomes 17 (22.2% of patients) and X (monosomic in 42.3% of female patients, but loss of chromosome X was never observed in males, P < 0.05). Dual-color FISH studies provided evidence for marked heterogeneity of aneuploid cells in 8 patients (22.8%). Occurrence of chromosomal aneuploidy was independent of stage and pretreatment status. Gain of chromosome 3 was significantly correlated with an IgA paraprotein (P < 0.05). In 12 patients, the direct comparison of metaphase cytogenetics and FISH showed that FISH detected aneuploidy of chromosomes in 9 patients that was missed by metaphase analysis. In conclusion, interphase FISH, by which chromosomal aneuploidy was detected in almost 90% of patients with MM, represents an approach for evaluating the clinical significance of specific chromosomal abnormalities in MM.

Phorbol-12,13-dibutyrate improves the quality of cytogenetic preparation in lymphoid malignancies.

In cytogenetic preparation of lymphoid malignancies we investigated the quantitative and qualitative impact of phorbol-12,-13-dibutyrate (P) and of this tumor promoter in combination with the calcium ionophore A23187 (PA). Using parallel cultures of unstimulated and stimulated preparations, the effect was examined in 13 patients with malignant lymphomas and six patients with acute lymphoblastic leukemias (ALL). Focusing on high-quality analyzable metaphases, the best results were found in seven of 13 cases with lymphomas and five of six patients with ALL in the cultures supplemented with phorbol-12,13-dibutyrate. The yield of metaphases of good quality regarding length, spreading, and banding of chromosomes was regularly better in P-stimulated 24-hour culture (p < 0.05), followed by 48-hour cultures stimulated with P alone. Addition of the calcium-ionophore was of no further benefit. The yield of the unstimulated direct harvest was rather poor in nearly all patients investigated. Because no mutagenic effect of P was observed, the use of this mitogen may offer interesting perspectives in cytogenetic analysis of lymphoid malignancies and perhaps also in other tumors with low mitotic indexes.

Karyotype and prognosis in non-Hodgkin lymphoma.

In malignant non-Hodgkin lymphomas (NHL), cytogenetic analysis may provide prognostic information including prediction of histologic evolution and responsiveness to therapy. In this study, we correlate clinical data and chromosomal aberrations in 70 adult patients with newly diagnosed NHL followed for a median of 20 months. Clonal aberrations were detected in 68/70 patients (97%). Besides t(2;5)(p23;q35), observed exclusively in three patients with anaplastic large cell lymphoma, Ki-1 positive, none of the characteristic aberrations observed was specific for a given histological subtype. Aberrations of chromosome 7 (n = 21) occurred in all histological subtypes together with aberrations of chromosome 3 and of the short arm of chromosome 17. They were clinically associated with a high serum lactate dehydrogenase level (LDH) and a trend to short survival. Anomalies of the long arm of chromosome 13 (n = 10) were found in patients with high grade B-cell lymphomas and bulky disease. In t(14;18)(q32;q21) bearing lymphomas (n = 27), distinct patterns of additional aberrations were observed in low grade and high grade lymphomas: trisomy 3 and trisomy 18 occurred concomitantly in high grade lymphomas (n = 6, p < 0.001) as well as aberrations of 1q, 5q, 6q and +der (18)(q21). In conclusion, cytogenetic analysis provides information about the complexity of genetic changes in NHL. These changes act not only as indicators of disease activity, but influence clinical outcome as demonstrated by their stringent correlation to the International Index and might reveal more general rules of tumor growth and spreading.

Prognostic impact of karyotype and immunologic phenotype in 125 adult patients with de novo AML.

One hundred-twenty-five adult patients with de novo acute myeloid leukemia (AML) were treated according to a standard 7 + 3 induction regimen. Karyotype and immunological phenotype of blasts examined prior to treatment were correlated with each other, with response to treatment and duration of survival. The following monoclonal antibodies (mAbs) were used for immunological phenotyping: VIM-D5 (CD15), MY7 (CD13), MY9 (CD33), VIM-2 (CDw65), VIM-13 (CD14), 63D3 (CD14), VID-1 (anti HLA-DR), WT1 (CD7), CLB-Ery3 (antiblood group H antigen), C17-27 (CD61), and an antiserum against TdT. Despite a considerable overlap between the individual groups, patients with specific aberrations as defined by the MIC classification (n = 39) showed distinct, characteristic, myeloid or myelomonocytic immunophenotypes. In M2/t(8;21) there was a significant association with negativity to CD13, in M3/t(15;17) with negativity to CD15 and HLA-DR, whereas in M4/inv(16) expression of blood group H antigen was unexpectedly found. The response to therapy, as well as rate of complete remission as duration of survival, was better in patients with M2/t(8;21), M3/t(15;17), and M4Eo/inv(16) as compared to all other patients and significantly worse in patients with M5a/t/del(11)(q23). In 35 patients with normal karyotype and 16 patients with cytogenetic anomalies not presently associated with FAB subtypes the expected correlations of rather immature myeloid immunologic phenotypes with M1 and M2 morphology and CD14 expression in monoblastic leukemias was found. Remission rate and survival were significantly worse in 19 patients with complex nonrandom aberrations, where blast cell expression of blood group H antigen and of TdT were significantly increased.

Trisomy 4: a specific karyotype anomaly in primary and secondary acute myeloid leukemia.

Trisomy 4 as single karyotype anomaly has recently been proposed as an acute myeloid leukemia (AML) specific aberration. Up to now, 20 cases have been reported in which the single abnormality occurred without additional chromosomal aberrations. Trisomy 4 has been found in both primary and secondary AML, the majority of cases being diagnosed as FAB M4 or M2 subtypes. In the cytogenetic analysis of 305 patients with AML, we found 209 cases with aberrant karyotypes, among them two patients (22a, male, M2; and 69a, male, M4) with trisomy 4 as single aberration. The younger patient achieved complete remission lasting 13 months and survived 22 months whereas the older patient died in aplastic phase due to septicaemia 5 weeks after admission. Trisomy 4 is proposed to be the primary aberration in both these cases of de novo AML. Although in one case, as in two cases reported earlier, cytogenetic results were only available in first relapse, we have no indication that trisomy 4 appeared in a secondary induced leukemia, because the leukemic blasts of the relapse were morphologically identical to first acute phase. In contrast to other specific chromosomal aberrations, results indicate that trisomy 4 has as yet no prognostic relevance concerning the clinical outcome.

Is trisomy 22 in acute myeloid leukemia a primary abnormality or only a secondary change associated with inversion 16?

In an attempt to confirm the existence of acute myeloid leukemia (AML) with trisomy 22, we studied three patients in whom trisomy 22 imposed as the sole karyotype abnormality. After revision of the karyotypes, however, we were able to identify an inv(16) as the important primary abnormality in all of them. Based on this experience, we investigated whether at least some of the 17 AML cases with trisomy 22 reported so far might possibly have been misinterpreted. Interestingly, ten out of 16 evaluable cases were classified as M4, some of them with bone marrow eosinophilia. As in cases with inv(16), only few metaphases contained trisomy 22. Furthermore, in at least two out of the only four published karyotypes of cases with trisomy 22, an inv(16) is evident and in the other two cases it cannot be ruled out. We therefore believe that at least some of the trisomy 22 cases mentioned in the literature are in fact only secondary changes occurring in AML with an inv(16) and suggest that future reports of AML with trisomy 22 as a specific primary abnormality can only be accepted as such if inv(16) has been excluded with appropriate methods.