Werner Grote

Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci.

Many B‐cell malignancies bear chromosomal translocations juxtaposing immunoglobulin (IG) genes with oncogenes, resulting in deregulated expression of the latter. Translocations affecting the IG heavy chain (IGH) locus in chromosomal region 14q32 are most prevalent. However, variant translocations involving the IG kappa (IGK) locus in 2p12 or the IG lambda (IGL) locus in 22q11 occur recurrently in B‐cell neoplasias. No routine methods for the detection of all breakpoints involving IG light chain loci independently of the translocation partner have been described. For this reason, we have designed 2 novel interphase fluorescence in situ hybridization (FISH) assays using differentially labeled probes flanking the IGK and IGL locus, respectively. Based on extensive control studies, the diagnostic thresholds for the detection of breakpoints were set at 0.3% for IGK and 1.4% for IGL. Fifteen cases of B‐cell malignancies with cytogenetically detectable chromosomal abnormalities in 2p11‐14 were investigated with the FISH assay for IGK. Breakpoints affecting the IGK locus were detected in 7 cases including all 4 variant Burkitts translocations t(2;8)(p12;q24) and a variant BCL2‐associated translocation t(2;18)(p12;q21). Other translocation partners were chromosome bands 7q21 and 16q24. Ten cases with abnormalities in 22q11‐12 were investigated with the FISH assay for IGL. Breakpoints in the IGL locus were diagnosed in 7 cases including both variant Burkitts translocations t(8;22)(q24;q11) and a t(3;22)(q27;q11) involving the BCL6 locus. Other translocation partners were 2p13‐14, 4q13 and 16p12. Our results show that these FISH assays provide flexible, simple and reliable tools in the diagnosis and characterization of genetic changes in B‐cell malignancies.

Multicolor-FICTION: Expanding the Possibilities of Combined Morphologic, Immunophenotypic, and Genetic Single Cell Analyses

Phenotypic and genotypic analyses of cells are increasingly essential for understanding pathogenetic mechanisms as well as for diagnosing and classifying malignancies and other diseases. We report a novel multicolor approach based on the FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms) technique, which enables the simultaneous detection of morphological, immunophenotypic, and genetic characteristics of single cells. As prerequisite, multicolor interphase fluorescence in situ hybridization assays for B-cell non-Hodgkins lymphoma and anaplastic large-cell lymphoma have been developed. These assays allow the simultaneous detection of the most frequent primary chromosomal aberrations in these neoplasms, such as t(8;14), t(11;14), t(14;18), and t(3;14), and the various rearrangements of the ALK gene, respectively. To establish the multicolor FICTION technique, these assays were combined with the immunophenotypic detection of lineage- or tumor-specific antigens, namely CD20 and ALK, respectively. For evaluation of multicolor FICTION experiments, image acquisition was performed by automatic sequential capturing of multiple focal planes. Thus, three-dimensional information was obtained. The multicolor FICTION assays were applied to well-characterized lymphoma samples, proving the performance, validity, and diagnostic power of the technique. Future multicolor FICTION applications include the detection of preneoplastic lesions, early stage and minimal residual diseases, or micrometastases.

Stepwise development of chromosomal abnormalities in angioimmunoblastic lymphadenopathy

Cytogenetic studies of lymphoproliferative diseases, such as angioimmunoblastic lymphadenopathy (AILD), may provide a clue to the understanding of tumor development. Angioimmunoblastic lymphadenopathy may evolve from a nonmalignant lymphoproliferation into a peripheral T-cell lymphoma or even into a high-grade B-cell lymphoma and thus offers the chance to observe cytogenetic changes during lymphoma development. We report the cytogenetic findings in 24 cases of AILD. They are discussed together with 18 previously published cases from the same series. A striking feature was that unrelated chromosome abnormalities, both clonal and nonclonal, were frequently observed. Eighteen of 25 cases with aberrant clones show trisomy 3 (a characteristic chromosome abnormality in peripheral T-cell lymphoma), trisomy 5, or both. This finding provides cytogenetic evidence that these cases are definitely peripheral T-cell lymphomas. From the results of the 42 cases, hypotheses of stepwise evolution of the chromosome abnormalities in AILD are deduced: the first step is the appearance of chromosome abnormalities in different cells because of a genetic instability. At this time, clonal proliferation of T cells was already demonstrated by the rearrangement of T-cell receptor genes. As a second step, chromosomally aberrant clones become established. A cytogenetically detectable monoclonal proliferation represents the third step.

Cytogenetic findings in T-zone lymphoma

SummaryFive cases of T-zone lymphoma were investigated with histologic, immunologic, and cytogenetic methods. The chromosome analyses were performed on lymphoma cells prepared immediately after removal of the lymph nodes. The chromosomes involved in structural rearrangements were nos. 1, 2, 3, 4, 14, and Y. Numbers 3, 5, 6, and 13 were lost by some tumors, and nos. 3 and 9 were gained. Chromosome 3 was involved most often in structural and numerical aberrations, whereas 14q+ markers occurred in only one case. The importance of multidisciplinary studies is pointed out.

Recurrent chromosome abnormalities in peripheral T-cell lymphomas

Cytogenetic findings in 45 cases of peripheral T-cell lymphomas (PTL) diagnosed according to the updated Kiel classification are reported. Recurrent numerical chromosome aberrations comprised -X, -Y, -13, +X, +3, +5 and +7. Recurrent structural aberrations included t/del(1)(p31-32), t(2;5)(p23;q35), dup(5)(q23q31-32), t/dup(6q), t/del(6q), trisomy 7q, and trisomy 8q, mostly due to i(8)(q10), and changes in 14q11 and 14q32.1, mostly due to inv(14)(q11q32.1), t/del(13)(q14), t(6;7)(q13;q13), and t(13;17)(q11-13;p11). All deletions in 6q involved band 6q21 and all partial trisomies of 7q led to an amplification of band 7q21. Further studies are needed to ascertain whether these cytogenetic findings in PTL are of clinical and prognostic significance.

Secondary acute leukaemias with 11q23 rearrangement : clinical, cytogenetic, FISH and FICTION studies

Three patients with secondary acute leukaemia after treatment with topoisomerase II inhibitor agents are described. Two patients had acute myeloid leukaemia (AML), FAB M5a, one had pro‐B‐acute lymphoblastic leukaemia (ALL). The interval between initiation of chemotherapy and the onset of secondary acute leukaemia was 19–20 months. 11q23 rearrangements were detected in all cases. They were due to translocations t(11;19) (q23;p13.3), t(11;16)(q23;p13) and t(4;11)(q21;q23), respectively. Fluorescence in situ hybridization (FISH) with Yeast Artificial Chromosome (YAC) probe 13HH4 spanning the ALL‐1 gene on 11q23 confirmed that in each case the ALL‐1 gene had been disrupted by the translocations. The study underlined the relationship between the development of secondary acute leukaemias with 11q23 rearrangement and previous chemotherapy with topoisomerase II inhibitor agents. So far, however, only six adult patients with secondary ALL with t(4;11) after treatment with topoisomerase II inhibitor agents have been reported. ALL with t(4;11) mostly occurs in infants or young children. Our patient received epirubicin continuously for >19 months. This indicates that both myeloid and lymphoid leukaemias with involvement of the ALL‐1 gene can be induced by exogenous agents, especially topoisomerase II inhibitors. Thus they may have a common biological background. This hypothesis was substantiated by means of combined immunophenotyping and FISH (FICTION). In the case of AML M5a with t(11;19), the tumour cells with ALL‐1 rearrangement expressed CD34. Moreover, the pro‐B‐ALL with t(4;11) was CD34 positive. These findings suggest that the cell of origin of secondary AML and ALL with 11q23 rearrangement is an immature haemopoietic progenitor cell.

Detection of chromosome aberrations in paraffin sections of seven gonadal yolk sac tumors of childhood

Yolk sac tumors are the most frequent kind of malignant pediatric germ cell tumor and may have a fundamentally different pathogenesis than adult germ cell tumors. Since few cytogenetic studies have been performed so far, in situ hybridization was applied to interphase cell nuclei of seven gonadal yolk sac tumors of childhood in routine paraffin-embedded tissue sections. The panel of chromosome-specific DNA probes was selected on the basis of their relevance in adult germ cell tumors and consisted of five DNA probes specific for the (peri)centromeric regions of chromosomes 1, 8, 12, 17 and/or X and/ or one DNA probe specific for the subtelomeric region of chromosome 1 (p36.3). As in adult germ cell tumors, all pediatric gonadal yolk sac tumors had an increased incidence of numerical chromosome aberrations. All tumors showed an overrepresentation of at least three chromosomes. Gains of chromosome 12, which is highly specific in adult germ cell tumors, were diagnosed in six pediatric gonadal yolk sac tumors. The DNA indices determined in the paraffin-embedded tumor material correlated well with the in situ hybridization findings. A chromosome was either over- or underrepresented, compared with the corresponding DNA indices, in only a few cases. The short arm of chromosome 1 in adult germ cell tumors is often involved in structural aberrations. In pediatric germ cell tumors, the short arm of chromosome 1 is also a nonrandom site of structural aberrations. Moreover, the presence of a deletion at 1p36.3 in four out of five tumors suggests that the loss of gene(s) in this region is an important event in the pathogenesis of gonadal yolk sac tumors of childhood.

Integration of amplified BCR/ABL fusion genes into the short arm of chromosome 17 as a novel mechanism of disease progression in chronic myeloid leukemia

We describe the cases of two patients with Philadelphia chromosome–positive chronic myeloid leukemia (CML), in whom the extramedullary blastic phase developed during disease progression. The similar clinical presentations of these patients was accompanied by gain of identical secondary chromosome abnormalities, that is, monosomies 9, 14, and 22, and by a clustered amplification of the BCR/ABL fusion gene. The additional copies of the BCR/ABL fusion gene were integrated into the short arm of structurally abnormal chromosomes 17 in both patients. The conformity of these genetic features in two patients with a rare disease manifestation leads us to the assumption that either the clustered amplification of the BCR/ABL fusion gene or the integration of this cluster into the short arm of chromosome 17 or both are associated with extramedullar disease progression in CML. Furthermore, the insertion of amplified BCR/ABL fusion genes into structurally abnormal chromosomes provides a novel mechanism of disease progression in BCR/ABL‐positive CML.

DETECTION OF DELETIONS IN THE SHORT ARM OF CHROMOSOME 3 IN UNCULTURED RENAL CELL CARCINOMAS BY INTERPHASE CYTOGENETICS

PURPOSE Analysis of genetic alterations may facilitate the differential diagnosis of renal cell carcinoma (RCC) subtypes. For genetic classification, deletion of the short arm of chromosome 3 (3p), the hallmark of nonpapillary/clear cell RCC, is a major diagnostic criterion. Because of the limited routine applicability of cytogenetics and molecular genetic techniques we investigated interphase fluorescence in situ hybridization (FISH) for the detection of this aberration in RCC. MATERIALS AND METHODS Using seven chromosome 3 specific probes FISH was performed on isolated nuclei from 26 uncultured sporadic RCC. RESULTS Alterations of chromosome 3 were identified in 19 RCC (73%). Monosomy and/or 3p-deletions were observed in 15 of 19 (79%) non-papillary/clear cell RCC but not in other morphologic subgroups. The median percentage of cells in a specimen containing loss of 3p was 45%. Deletion mapping indicated that large deletions affecting different regions in 3p are predominant. Chromosomal region 3p24 was recurrently involved in all RCC with a deletion in 3p. CONCLUSION Interphase FISH for the detection of loss in 3p provides a sensitive and feasible method for the genetic classification of kidney tumors and the delineation of recurrently deleted regions in 3p.

Interphase cytogenetics on paraffin sections of paediatric extragonadal yolk sac tumours

Germ cell tumours in children are more often extragonadal than in adults and the most frequent type is the yolk sac tumour. Limited cytogenetic data exist on extragonadal yolk sac tumours in children. We applied in situ hybridization (ISH) to interphase cell nuclei of four paediatric extragonadal pure yolk sac tumours and one yolk sac tumour component of a mixed germ cell tumour using paraffin-embedded tissue sections. The panel of chromosome-specific DNA probes was selected on the basis of their relevance in adult germ cell tumours and consisted of five DNA probes specific for the (peri)centromeric regions of chromosomes 1, 8, 12, and/or 17, X and/or one DNA probe specific for the subtelomeric region of chromosome 1 (p36.3). Only one tumour failed to show numerical and structural chromosome aberrations with the DNA probes used. The other four had an increased incidence of numerical chromosome aberrations with an over-representation of at least one chromosome. The DNA indices determined in the paraffin-embedded tumour material correlated well with the in situ hybridization findings. In only a few cases were chromosomes over-represented, when compared with the corresponding DNA indices. Recently, we have shown that the short arm of chromosome 1 is a non-random site of deletion in paediatric gonadal pure yolk sac tumours. The occurrence of similar deletions in one extragonadal pure yolk sac tumour and in one yolk sac tumour component, in conjunction with two further ISH reports, suggests that the loss of gene(s) in this region is an important event in the pathogenesis of paediatric malignant germ cell tumours of nearly all sites.