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).

Simultaneous presence of t(11;14) and a variant Burkitt's translocation in the terminal phase of a mantle cell lymphoma.

Little is known about the clinical significance of secondary chromosome aberrations in lymphomas with t(11;14)(q13;q32), the characteristic change of mantle cell lymphomas. Here we present a patient with mantle cell lymphoma, who showed a variant Burkitt's translocation t(2;8)(p12;q24) in addition to t(11;14) during the progression of the disease. An involvement of chromosome 8q24, the localization of the c-myc gene, has so far been described in only four patients, who seemed to have a fatal clinical course. Although no blastic transformation occurred in our patient, no remission could be induce by intensified treatment and survival was only 5 months. This case demonstrates that secondary chromosome aberrations can determine the clinical course of patients, even if morphologic and immunophenotypic findings fail to predict the poor outcome.

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.

Organization of the monocyte/macrophage system of normal human skin.

Monocytes and macrophages are known to be important for a variety of functions; however, whereas epidermal Langerhans cells have been studied in great detail, few data are available for the dermal monocyte/macrophage system. Therefore we investigated the density, distribution, and phenotype of dermal macrophages in normal human skin using a panel of monoclonal antibodies for single and double labeling. We demonstrate here that within normal human dermis macrophages reside with a remarkable density. Principally, these cells exhibit the phenotype of the phagocytic macrophage system (CD11c+, KiM8+), whereas members of the immune phagocyte system (CD11c+, KiM8-) are absent from normal dermis with the exception of a few Langerhans cells in the papillary body. Within the dermal phagocytic macrophage system we uncover an unexpected phenotypical and morphologic heterogeneity, which correlates with the tissue localization. This study provides a basis for investigating the participation and change of the dermal macrophage system in cutaneous disorders.

Cell trafficking in positive and negative patch-test reactions: demonstration of a stereotypic migration pathway.

The cellular and molecular events taking place during epidermal antigen exposure in sensitized individuals are principally well understood. Epidermal Langerhans cells (LC) are supposed to take up, process, and express a given foreign substance on their cell surface. The antigen is then recognized by T cells bearing the appropriate T-cell receptor (TCR). Because LC do not bear variable antigen (Ag)-specific binding sites, one could postulate that the epidermal exposure of any substance should activate LC and other cells of the skin immune system. To test this hypothesis, we analyzed immunophenotypically the cellular trafficking events in positive (n = 5) and negative epicutaneous patch-test reactions (n = 10), using a panel of monoclonal antibodies against CD1a, CD11c (Ki-M1, LeuM5), CD68 (Ki-M6), Ki-M8, and CD3 (Leu4). We can demonstrate that irrespective of whether or not an antigen will be responded to by the immune system (i.e., positive or negative test reaction), epidermal antigen exposure causes a decrease of LC density in the epidermis and simultaneously causes an increase of LC in the dermis. Moreover, monocytes and T cells immigrate into the dermis both in positive and negative patch-test reactions. As is to be expected, the degree of this cellular traffic is more pronounced in positive test reactions, which may be due to amplification mechanisms caused by antigen recognition of sensitized T cells. This finding demonstrates that human skin contains cell migration programs that ensure that any foreign substance will be accessible to the skin immune and phagocytic system.

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.

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.

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.

Combined immunophenotyping and karyotyping in peripheral T cell lymphomas demonstrating different clonal and nonclonal chromosome aberrations in T helper cells.

Combined immunophenotyping and karyotyping was performed in seven cases of peripheral T cell lymphoma with complex aberrant clones. Various lymphocytic cell populations entered mitosis, whereas all aberrant cells belonged to the T helper/inducer cell population. Lymphomas with the same recurrent chromosome aberrations, i.e. inversion inv(14)(q11q32.1) and isochromosome i(8)(q10), had a very similar immunophenotype. The aberrant cells in these cases expressed CD3+, CD4+, CD7+, CD45RO+. The immunophenotypic similarity is underlined in one case of T prolymphocytic leukemia, in whom the aberrant cells lost the CD8 antigen originally present, during cultivation with PHA. In one case of Sézary's syndrome, two or possibly even three different clones as well as nonclonal aberrations were identified within the T (helper/inducer) cell population, providing further evidence that chromosomal instability is a characteristic feature of cutaneous T cell lymphoma.

Fluorescence in situ hybridization as a diagnostic tool in malignant lymphomas.

Primary and secondary chromosomal abnormalities play an important role in the characterization of biological, pathological, and clinical subgroups of malignant lymphomas. The introduction of fluorescence in situ hybridization (FISH) and the combination of immunophenotyping plus FISH to the diagnosis of lymphatic neoplasms allows the fast and sensitive detection of specific chromosomal changes and provides new insights into the genetic basis of lymphomagenesis. This article reviews the possibilities and limitations of molecular cytogenetic techniques in comparison to cytogenetic and molecular genetic methods and discusses their clinicopathological impact for non-Hodgkin's lymphoma and Hodgkin's disease.

Kappa- and lambda-positive cells in centroblastic-centrocytic lymphoma (follicular lymphoma) may share a secondary chromosome aberration: reflections on early lymphoma development.

The technique of simultaneous fluorescence immunophenotyping and interphase cytogenetics (FICTION) was applied in four cases of centroblastic-centrocytic (working formulation: follicular) lymphoma. Our aim in this study was to establish whether secondary chromosome aberrations known from a prior cytogenetic analysis were detectable in both kappa- and lambda-expressing tumor cells from the same lymphoma patient. One of the cases did indeed contain kappa- and lambda-positive tumor cells with trisomy 8. On the basis of our results, we reflect on the events that take place during early development of centroblastic-centrocytic lymphoma.

Cytogenetics and molecular cytogenetics in Hodgkin's disease.

For about 20 years we have known from cytogenetic studies that there is a clonal cell population in Hodgkin's disease. Most karyotypes are complexly aberrant and chromosome numbers typically lie in the hyperploid range. Some chromosome regions seem to be preferentially involved, but a chromosome aberration specific for Hodgkin's disease has not yet been determined. Although the existence of a clonal cell population is evident from these cytogenetic studies, there is still an ongoing debate, whether in all cases the pathognomonic Hodgkin and Reed-Sternberg cells belong to one single aberrant clone and thus represent a monoclonal proliferation. This article reviews the current knowledge on cytogenetics in Hodgkin's disease. Moreover, our recent data from simultaneous immunophenotyping and interphase cytogenetics (FICTION) are introduced into the passionate discussion on the monoclonality of the Hodgkin and Reed-Sternberg cells.

Detection of aberrant clones in nearly all cases of angioimmunoblastic lymphadenopathy with dysproteinemia-type T-cell lymphoma by combined interphase and metaphase cytogenetics.

Trisomy 3, trisomy 5, and an X additional chromosome are the most frequent chromosome aberrations in angioimmunoblastic lymphadenopathy with proteinemia (AILD)-type T-cell lymphomas. To evaluate the frequency of +3 and +X clones, fluorescence in situ hybridization studies with centromere-specific probes for chromosome 3 and X were done in 41 patients with peripheral T-cell lymphomas (PTL). With this interphase cytogenetic approach, 32 of 41 patients (78%) showed +3 clones, and 14 patients (34%) +X clones. These frequencies far exceeded those observed with metaphase cytogenetics (+3, 41%; +X, 20%). Summing up the results of metaphase and interphase cytogenetics, aberrant clones were found in 37 of 41 patients with PTL (90%) and 32 of 36 patients with AILD-type T-cell lymphoma (89%). Although AILD-type T-cell lymphoma is considered a neoplastic disease, it is an exception in that it shows a high frequency of cytogenetically unrelated clones and single cells that cannot be derived from a common cell of origin because of their completely different karyotypes. In five patients, double hybridization with centromere-specific probes for chromosomes 3 and X showed that these aberrations occurred in different cells. When the results of metaphase and interphase cytogenetics were combined, 17 of 36 patients with AILD-type T-cell lymphoma (47%) had unrelated clones. This high frequency of oligoclonal proliferations may be caused by increased genetic instability and an immune defect resulting in impaired elimination of aberrant cells.

Clarification of dubious karyotypes in Hodgkin's disease by simultaneous fluorescence immunophenotyping and interphase cytogenetics (FICTION).

Cytogenetic studies on Hodgkin's disease (HD) typically reveal very complex karyotypes with a variety of numerical and structural abnormalities. The confusing thing is that about 10% of cases contain relatively discrete chromosome aberrations, for example a simple trisomy or loss of one single chromosome. Whether these karyotypes really correspond to Hodgkin and Reed-Sternberg (HRS) cells is uncertain. They could, for example, represent early stages in the evolution of the karyotype of the pathognomonic HRS cells. On the other hand, they could be artificial events that occur during the cytogenetic procedure. In our experience, isolated loss of the Y chromosome is the most frequent finding of this type. This aberration is usually considered to be a preparation artifact. However, if one takes into account that in HD up to 50% of male cases with complex karyotypes also lack the Y chromosome, a possible relation to HRS cells must be considered. The technique of simultaneous fluorescence immunophenotyping and interphase cytogenetic analysis (referred to as FICTION) is a powerful tool for studying the nature of cytogenetically abnormal cells. With the FICTION technique we studied four cases of HD in which the chromosome analysis had shown only the loss of the Y chromosome. Our aim was to clarify whether these karyotypes corresponded to the CD30-positive HRS cells. In two cases we found that HRS cells actually lacked the Y chromosome. There was strong evidence, however, that the HRS cells additionally had other chromosome aberrations and thus could not correspond to the cytogenetically determined karyotypes.

Rapid immunophenotypic characterization of chromosomally aberrant cells by the new FICTION method.

Recently, we have presented a new technique for immunophenotyping cells that have numerical chromosome aberrations. We referred to this method as "Fluorescence-Immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms" (FICTION). We present here an advanced FICTION method with three-color staining and improved sensitivity.

Numerical chromosome aberrations are present within the CD30+ Hodgkin and Reed-Sternberg cells in 100% of analyzed cases of Hodgkin's disease.

In Hodgkin's disease, cytogenetically aberrant clones have been demonstrated in a minority of cases studied. In the remaining cases, only normal metaphases have been found, but it is questionable whether normal karyotypes actually correspond to the pathognomonic Hodgkin and Reed-Sternberg (HRS) cells. Numerical aberrations could be studied by fluorescence in situ hybridization (FISH). However, in Hodgkin's disease, the percentage of tumor cells is mostly below the detection limit of FISH, which is near 1%. With the technique of simultaneous fluorescence immunophenotyping and interphase cytogenetic analysis (FICTION), this problem can be overcome. By FICTION, hybridization signals can selectively be evaluated within the CD30a+ cell population. We have studied 30 cytogenetically analyzed cases of Hodgkin's disease by means of FICTION. In all cases, we found numerical chromosome aberrations within the majority of CD30+ HRS cells. In cases with complex and hyperdiploid karyotypes, the cytogenetic results agreed with the FICTION data. There was considerable variability in the chromosome numbers, demonstrating that karyotype instability is an in vivo phenomenon of HRS cells. Lymphocytes never displayed numerical chromosome changes. Our results indicate that HRS cells regularly exhibit numerical chromosome aberrations and that the chromosome numbers are always in the hyperploid range.