Detection of the t(14;18) chromosomal translocation by interphase cytogenetics with yeast-artificial-chromosome probes in follicular lymphoma and nonneoplastic lymphoproliferation.

PURPOSE: The aim of this study was to establish a fluorescence in situ hybridization (FISH) technique for the detection of t(14;18)(q32;q21), characteristic for follicular lymphoma (Kiel classification: centroblastic centrocytic [cb-cc] lymphoma). MATERIALS AND METHODS: After the FISH system had been established, parallel studies of lymph node biopsy specimens from 30 patients with cb-cc lymphoma and from 32 patients with nonneoplastic lymphoproliferation were performed by means of chromosome analysis, polymerase chain reaction (PCR), and FISH analysis. Two differently labeled yeast-artificial-chromosome (YAC) probes that contained the entire bcl-2 gene and the C-region of the immunoglobulin H (IgH) gene, respectively, were used to detect t(14;18) by FISH. RESULTS: The presence of the translocation is indicated by a red (Cy3)/green (fluorescien isothiocyanate [FITC]) double signal, which corresponds to the IgH/bcl-2 fusion gene, whereas in normal cells the signals are separate. Control studies showed that the double signal is visible in less than 1% of normal cells. FISH analysis was able to identify the t(14;18) in all cases of cb-cc lymphoma we studied. All bcl-2 breakpoints can be detected. Combined immunophenotyping and interphase cytogenetics demonstrated that t(14;18) was restricted to CD22+ B lymphocytes and never occurred in CD3+ T lymphocytes. In four of 32 cases of nonneoplastic lymphoproliferation, t(14;18) was also detected. CONCLUSION: FISH turned out to be the most sensitive method to detect t(14;18). Our FISH results confirm PCR data from other groups that found evidence for the presence of t(14;18) in nonneoplastic lymphoproliferation. It needs to be determined whether, in morphologically nonneoplastic processes, t(14;18) is associated with an increased risk for the development of non-Hodgkin's lymphoma.

Combined immunophenotyping and interphase cytogenetics on cryostat sections by the new FICTION method.

A technique is described to detect tumor cells with certain chromosome aberrations within cryostat sections and to characterize these individual cells by immunophenotyping. This new method is called fluorescence-immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms (FICTION).

Molecular cytogenetic detection of chromosomal breakpoints in T-cell receptor gene loci.

Chromosomal aberrations with breakpoints in T-cell receptor (TCR) gene loci are recurrent in several T-cell malignancies. Although the importance of interphase cytogenetics has been extensively shown in B-cell lymphomas, hardly any molecular cytogenetic tools are available for recurrent changes in T-cell disorders. Thus, we have established fluorescence in situ hybridization (FISH)-based break-apart assays for the TCRA/D (14q11), TCRB (7q34) and TCRG (7p14) genes and the TCL cluster (14q32). The assays were validated in normal controls as well as in 43 T-cell malignancies with cytogenetically proven 14q11, 7q34-35 or 7p13-21 aberrations. Breakpoints in TCRA/D, TCRB and TCRG could be diagnosed by these assays in 32/33 T-cell neoplasms with chromosome 14q11, 3/6 with 7q34-35 and 1/7 with 7p13-21 alterations, respectively. Application of the new FISH assays to a series of 24 angioimmunoblastic and 12 cutaneous T-cell lymphomas confirmed the cytogenetic evidence of lack of breakpoints in the TCRA/D or TCRB locus. Simultaneous detection of TCRA/D or TCRB breaks was achieved in a multicolor approach, which was further combined with detection of the T-cell-specific CD3 antigen in a multicolor FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasm) assay. These new FISH and FICTION assays provide sensitive, rapid and accurate tools for the diagnosis and biological characterization of T-cell malignancies.

Cytogenetic findings and results of combined immunophenotyping and karyotyping in Hodgkin's disease.

Cytogenetic studies were performed in 21 cases of Hodgkin's disease. Fourteen cases revealed chromosomally aberrant clones which could be fully described in 12 cases. Two cases showed different unrelated clones and five cases only single cell aberrations. Recurrent breakpoints were 1p13/21 (six cases), 7q32/34 (five cases), 2p16/21 and 19p13 (four cases each), 4q25/28, 6q15/21 and 12q22/23 (three cases each). In two cases, a translocation between band 19q13 and band 14q11 or 14q32 was found. This finding may indicate that an unknown oncogene in 19p13 is activated by juxtaposition next to a T-cell receptor or immunoglobulin gene in 14q11 or 14q32, respectively. In eight cases each, total or partial monosomy 4 or 6 was present suggesting that tumor suppressor genes in 4q or 6q play a role in tumor development in Hodgkin's disease. Moreover, the aberrant clones lacked the Y-chromosome in men and the second X-chromosome in women in eight out of nine and in two out of three cases, respectively. Although different cell populations, especially T cells, showed mitotic activity in unstimulated short term culture, combined immunophenotyping and karyotyping unequivocally demonstrated that CD30 and CD15 positive Hodgkin and Sternberg-Reed cells represented the chromosomally aberrant clones.

Large scale production and purification of human IL-2 from buffy coat lymphocytes stimulated with 12-O-tetradecanoylphorbol 13-acetate and calcium ionophore A23187.

Methods for the production of high titers of interleukin-2 (IL-2) from human buffy coat lymphocytes, and subsequent purification of the IL-2 are described. 50 buffy coats containing 1 X 10(11) leukocytes were first depleted of erythrocytes by batchwise leukapheresis using a Haemonetics model 15 blood wash centrifuge. Further lymphocyte enrichment was achieved using a one-step sedimentation in the presence of hydroxyethyl starch, which produced suspensions of more than 90% lymphocytes. This degree of lymphocyte purity was important since phagocytes were inhibitory to 12-O-tetradecanoylphorbol 13-acetate/calcium ionophore (TPA/A23187)-induced IL-2 production when their concentration exceeded 15% of the total cells. Cell culture was performed in stirred fermenters. Using TPA/A23187 induction, up to 500 micrograms of IL-2 per liter were produced. The IL-2 was purified by absorption from the supernatants onto controlled pore glass and elution with 50% ethylene glycol, followed by Fractogel chromatography, and then preparative high-performance liquid chromatography (HPLC) using an RP-6 column and elution with a gradient of n-propanol. A final HPLC rechromatography step using an analytical RP-6 column gave a homogeneous preparation with specific activity of 1.2 X 10(7) U/mg and a recovery from the starting supernatant of 22%.

Discrimination of distinct subpopulations within a tumor with combined double immunophenotyping and interphase cytogenetics.

We describe a method that enables detection and immunophenotypical characterization of distinct subpopulations within a cytogenetically defined tumor clone. Coexisting normal cells do not hinder microscopic evaluation because they can be distinguished from cytogenetically aberrant tumor cells. This is also true when normal and neoplastic cells cannot be clearly distinguished by cytology or immunohistochemistry, i.e., if both constituents have similar immunophenotypes and morphology. The method is based on fluorescence double staining for two different antigens combined with interphase cytogenetic analysis. It is referred to as "Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)." In a case of follicular lymphoma we demonstrate that FICTION can differentiate bcl-2-positive malignant and non-malignant cells and can verify the presence of bcl-2-positive but cytogenetically inconspicuous T-lymphocytes.

Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells.

In immunocytochemical studies, the phenotypic evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypical variability, such as bone marrow. A method that identifies mitotic tumor cells by chromosomal aberrations and permits the subsequent immunophenotypical analysis was a first progress, demonstrated by Teerenhovi et al. However, the results are usually hampered by the low number of analyzable mitoses. We demonstrate here a method that simultaneously combines immunophenotyping and in situ hybridization with centromere-specific probes. Using our method, numerically aberrant tumor cells can be identified by interphase cytogenetics and subsequently analyzed immunophenotypically. Since all interphase cells can be analyzed, we are not limited by the number and banding quality of analyzable mitoses.

Common fragile sites in couples with recurrent spontaneous abortions.

Recently, increased spontaneous chromosome instability was reported in couples with recurrent spontaneous abortions but without constitutional chromosome aberrations. Therefore, we investigated the frequency and distribution of aphidicolin-induced common fragile sites in couples with recurrent spontaneous abortions and no children and in age-related control couples. The breakage rate was significantly elevated in the abortion-prone couples; the women in the abortion couples had an especially higher breakage rate than the control women. Breakpoints cluster to those chromosome regions where common fragile sites have been localized. No preference of a particular common fragile site was demonstrated in the abortion couples. Our findings appear to support the hypothesis of increased chromosome instability in at least some couples with recurrent spontaneous abortions. As long as the nature of aphidicolin-induced common fragile sites is not completely understood, the significance of these findings remains unclear.

Meiotic segregation in familial reciprocal translocation t(8q;22q).

We studied the chromosomes of a mentally retarded boy with minor anomalies and of his parents using a G-band stained high-resolution chromosome method. This documented dup (8q24.1 = to 8qter) and dup(22pter = to 22q11.2) in the boy due to a maternal balanced reciprocal translocation of chromosomes 8 and 22 and 3:1 disjunction during meiosis I. The karyotype of the boy is 47,XY, +der(22) (22pter = to 22q11.2::8q24.1 = to 8qter). The der(22) was involved in satellite associations and stained positively with AgNO3 in mother and child. The case is compared to similar cases in the literature and the function of the small acrocentric marker chromosome during meiosis is discussed.

Intralymphatic interleukin 2 treatment in patients with acquired immunodeficiency syndrome: preliminary experience in three cases.

Patients with opportunistic infections during the course of acquired immunodeficiency syndrome (AIDS) were analyzed for cellular immune functions and found to be severely immunocompromised. In particular, interleukin 2 (IL 2) production appeared to be defect not only qualitatively but also quantitatively. In some of these patients, exogenous IL 2 improved immune response in vitro. Intralymphatically administered highly purified natural IL 2 was given repeatedly (over a time period of ten days) to three of these patients. In two cases, such a treatment course was repeated later. Clinical response - at least in some patients - appeared to be of temporary benefit. Shortly after termination of IL 2 application in two patients an increase of lectin responsiveness as well as improved reactivity in skin testing was noted, encouraging further exploration of IL 2 as an immunostimulatory drug in AIDS patients.

Cytogenetic findings in T-zone lymphoma.

Five 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.

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.

Inv(14)(q11q32) in one of four different clones in a case of angioimmunoblastic lymphadenopathy.

A case of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) with four cytogenetically different cell clones (49,XX,+5,+19,+21/47,XX,+X/46,XX,inv (14)(q11q32)/45,X,-X) is reported. To our knowledge, this is the first case of AILD with an inv(14)(q11q32), thus probably involving the T-cell receptor alpha-chain gene. The cytogenetic findings are discussed with respect to the possible progression of AILD to malignant lymphoma.

Translocation (X;8)(q2?6;q21.3) in a case of systemic mastocytosis.

Mastocytosis is a rare disease which occasionally progresses into mast cell leukemia or other myeloid neoplasms. Here we report on a patient with systemic mastocytosis who was found to have a clone with t(X;8)(q2?6;q21.3) and two copies of der(8)t(X;8). In accordance with these results, interphase cytogenetic analysis revealed that 93% of bone marrow cells contained three centromeric regions of chromosome 8. We suggest that the t(X;8) and the duplication of the translocation chromosome 8 may play a role in the progression of the diseases.

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.

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.

Rationalization of in situ hybridization: testing up to 16 different probes on a single slide.

Although particularly interested in tumor research, investigators in many tumor cytogenetics laboratories cannot afford extensive molecular/interphase cytogenetics studies in addition to routine cytogenetics analyses, primarily because many slides must be stained to examine a few cases using a panel of centromeric probes. Moreover, fluorescence in situ hybridization (FISH) techniques mostly require freshly prepared reagents, such as hybridization mixtures or antibody solutions. These technical requirements are very time-consuming, thus limiting their use in routine screening. We report a significantly economical method for rapid performance of interphase cytogenetics in great numbers of cases. The major advantage if its superior efficiency: up to 16 different probes may be used on one single slide. Moreover, the method is significantly cheaper than other techniques owing to minimal probe consumption. The technique is also best suited if great numbers of new probes, e.g., polymerase chain reaction (PCR)-generated YAC-probes, must be tested for their applicability in FISH.

Cytogenetic and molecular characterization of simultaneous chronic and acute myelocytic leukemia.

We describe a patient initially diagnosed with a chronic myeloproliferative disorder in the accelerated phase. Cytogenetic analysis showed the presence of two independent clones. One clone contained a typical Philadelphia (Ph) chromosome due to t(9;22)(q34;q11), as the sole abnormality which was proven molecularly to result in the b2a2-BCR/ABL fusion. The other clone displayed a complex karyotype with several structural and numerical aberrations including trisomy 11 and 22 but lacking a t(9;22) or any other structural abnormalities involving chromosomes 9 and 22. Fluorescence in situ hybridization demonstrated that the t(9;22) was present only in cells with two copies of chromosomes 11 and 22. In contrast, cells with trisomies 11 and 22 lacked evidence for a BCR/ABL fusion. Based on the genetic findings, simultaneous chronic and acute myelocytic leukemias were diagnosed rather than a blastic phase of a chronic myelocytic leukemia.

Detection of translocations involving the HOX11/TCL3-locus in 10q24 by interphase fluorescence in situ hybridization.

The t(10;14)(q24;q11) and its variant t(7;10)(q35;q24), which are recurrent in acute T-cell leukemia, lead to activation of the HOX11/TCL3-gene in chromosomal region 10q24 by juxtaposing this gene to one of the T-cell receptor loci. In the present study, we established a diagnostic assay for detecting these translocations by interphase fluorescence in situ hybridization (FISH). BAC clones flanking the HOX11/TCL3-locus were obtained from a fingerprinted BAC-contig of chromosomal region 10q24. BAC clones located proximal and distal of the HOX11/TCL3-locus were differently labeled and applied to interphase-FISH in seven normal controls and eight T-cell neoplasms with t(10;14)(q24;q11) or t(7;10)(q35;q24). In over 1600 nuclei of controls, a considerable split defined as separation of each one signal for the proximal and distal probe by more than three times the signal diameter was observed in only one cell. In contrast, all T-cell neoplasms with t(10;14) or t(7;10) contained at least 47% of nuclei with a signal split indicating a breakpoint in the HOX11/TCL3-locus. Thus, the established double-color FISH approach provides a new reliable and routinely applicable tool for diagnosing breakpoints in the HOX11/TCL3-locus.