Frequent nonrandom chromosome abnormalities in 27 patients with untreated large cell lymphoma and immunoblastic lymphoma.

Fresh tumor samples from 27 patients with large cell lymphoma, either previously untreated (26 patients) or minimally treated (one patient), were processed for cytogenetic studies. Cytogenetic abnormalities were observed in all patients, most commonly in chromosomes 1, 3, 7, 12, 14, 17, and 18. Nine chromosomal breakpoints appeared frequently: 14q32 in 14 instances; 18q21 in seven; 9p13-21, 17p11-13, and 3q21-23 in six each; 1p11-21 in five instances; 1p36 in four; and 2p21-23 in three. The most common structural abnormalities were t(14;18)(q32;q21) in seven patients (26%) and 17p- in six (22%). The presence of 17p- was associated with a significantly higher proliferative capacity as manifested by the percentage of S phase = 22% versus 11% for cases without 17p-(P less than 0.05). Trisomy 12, typical of small lymphocytic lymphoma, was seen in five patients in this series, all of whom had diffuse large cell lymphoma; frequently, it appeared simultaneously with t(14;18). The two patients with immunoblastic lymphoma of B-cell type had an abnormality involving chromosome 2p21-23. Deletions in the long arm of chromosome 6, previously described as typical of diffuse large cell lymphoma and B-cell immunoblastic lymphoma were observed infrequently in this series. However, this abnormality has been present in 50% of patients with large cell lymphoma previously exposed to therapy, suggesting that it may be related to effects of chemotherapy or to clonal evolution.

Multilineage involvement of Philadelphia chromosome positive acute lymphoblastic leukemia.

Acute lymphocytic leukemia (ALL) is considered a clonal disease restricted to the lymphoid compartment. The Philadelphia chromosome (Ph) is found in a subset of ALL with poor prognosis. Here we present the largest series of Ph+ ALL analyzed for involvement of the myeloid compartment. For the first time at a single cell level the presence of Ph in lineages other than lymphoid is demonstrated. Granulocytes from nine patients diagnosed with BCR-ABL + ALL (eight Ph+, one Ph-) were purified using two layer density gradient separation. They were further identified by the morphology of DAPI-stained nuclei and studied for the presence of the Ph by fluorescence in situ hybridization (FISH) using a BCR-ABL dual-color probe. Ph was demonstrated in 30 to 93% of granulocytes in all patients. FISH identified major and minor BCR gene breakpoints (M-bcr and m-bcr). In one patient, with CD19+/34+/33-/2-/3-/7-/10- lymphoblasts, involvement of B cells (CD19+), T cells (CD3+), myeloid (CD13+), erythroid (glycophorin A+) cells was found by FISH following fluorescence-activated cell sorting (FACS). The diagnosis of ALL as opposed to lymphoblastic transformation of CML was established based on clinical and laboratory data including Western blot results demonstrating the presence of p190/m-bcr in five of the nine cases studied. Results suggest that Ph+ ALL originates from a pluripotent stem cell.

Quantitation of minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes in complete remission by molecular cytogenetics of progenitor cells.

Detection of karyotypic clonal abnormalities are prognostically useful in patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS), but cytogenetic methods are not sensitive enough to detect low numbers of residual leukemic cells in patients who have achieved complete remission (CR). Fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) were used to investigate the frequency and presence of minimal residual disease (MRD) in AML and MDS patients (n = 28) with monosomy of chromosomes 7, 17 and 18 and trisomy of chromosomes 6, 8, 9 and 10 in CR. MRD was detected in all patients with monosomy 7 (n = 10) and followed by relapse in eight patients after 4.8 +/- 3.1 months. In contrast, persistent leukemic cells occurred in 11/12 patients with trisomy 8, but only three of them relapsed after 7.7 +/- 4.0 months. Cox regression analysis showed that cytogenetic class and levels of clonal cells at CR were related to time to relapse (P = 0.001). The level of MRD identified patients at high and low risk of relapse. High absolute levels of proliferating residual leukemic cells correlated with monosomy 7 and high risk of relapse.

S phase determination in intact colonic crypts by histone H3 messenger RNA in situ hybridization and confocal microscopy.

Proliferating cells have a restricted three-dimensional spatial distribution within the crypt, which is the proliferative unit of the colon. Accurate quantitative and spatial analyses of S phase cells in the colon have therefore been limited by histological techniques. To overcome these limitations, S phase cells in microdissected intact colonic crypts of control, modified-starved, and refed rats were labeled by histone H3 in situ hybridization and analyzed by confocal microscopy. High-resolution digital images of the crypt cell nuclei stained with cyanine nucleic acid and of the labeled S phase cells were produced from confocal microscopic optical crypt sections. The S phase labeling index (LI) per whole crypt significantly (P < 0.001) discriminated the proliferative differences between control, modified-starved, and refed rats and correlated (r = 0.92) with the LI determined from histological crypt sections of the same rats. The variance component of the LI attributable to differences between whole crypts, 0.44 (95% confidence interval, 0.38-0.51), was considerably smaller than that attributable to differences between histological crypt sections, 6.07 (95% confidence interval, 5.18-6.96). Confocal microscopy and histone H3 in situ hybridization of intact three-dimensional crypts enables precise in vitro quantitation and spatial analysis of the total and S phase crypt cells.

Specific chromosome anomalies and predisposition to human breast, renal cell, and colorectal carcinoma.

Specific chromosome abnormalities in three human solid tumors of adulthood--renal cell carcinoma, breast tumor, and colon carcinoma--are described. Two of the neoplasms are associated with a reciprocal translocation involving chromosome #3 (breakpoint at band p13-14 with 6, 8, 11, and 16) in renal cell carcinomas and chromosome #1 (breakpoint at band q21 with chromosomes #3, #5, #10, #11, and #12) in breast carcinomas. Most of these chromosomal rearrangements have been seen as tumor-specific acquired changes in tumor cells, as well as some constitutionally present in normal tissues of patients. In a limited number of colorectal carcinoma samples a deletion in the short arm of a chromosome #12 is implicated as a specific abnormality. The expression of fragile sites in these specific chromosomal regions in the normal peripheral blood cultures might identify an individual predisposed to develop a particular type of neoplasm.

Ph-positive chronic myeloid leukemia with near-haploid conversion in vivo and establishment of a continuously growing cell line with similar cytogenetic pattern.

Blast cells from a 39-year-old man in the blastic phase of chronic myeloid leukemia, with a benign phase of 15 years duration, as well as a cell line arising from this cell population, were studied. Cellular morphology, cytochemical staining pattern, and absence of terminal deoxynucleotidyl transferase showed the blast cells to be of myeloid character. Cytogenetic studies revealed the presence of two near-haploid cell populations with +8 and +8, +15, respectively, both of them containing the translocation t(9;22) in the original tumor cell sample. The cell line derived from this patient's leukemic cell sample contained both near-haploid and hyperdiploid clones, the hyperdiploid clones being multiples of the near-haploid clone(s). All of the clones carried the t(9;22) in the form of a Philadelphia chromosome.

Monosomy 21, partial duplication of chromosome 11, and structural abnormality of chromosome 1q21 in a case of lymphoma developing in a transplant recipient: characteristic abnormalities of secondary lymphoma?

Cytogenetic analysis by QFQ-banding of direct preparation of a testicular mass from a patient with secondary lymphoma revealed a modal chromosome number of 45,XY, including structural and numerical anomalies. The most consistent anomalies were the monosomy 21, duplication of the long arm of #11, and structural anomaly associated with chromosome #1 in band q21.

Methodologic advances in the cytogenetic analysis of human solid tumors.

The major obstacle to successful cytogenetic analysis of human solid tumors is the acquisition of sufficient numbers of good quality metaphases for detailed cytogenetic analysis. At present, no single methodologic approach has been proven to provide successful chromosomal analysis of all human solid tumors. The technical aspects of cell culture, chromosome harvesting, and chromosome banding were the focus of considerable discussion during the First Workshop on Chromosomes in Solid Tumors. This report provides summaries of several technical protocols, emanating from several different laboratories, which have contributed to successful chromosome analysis of a variety of human solid tumors.

Chromosomal abnormalities in adult non-endemic Burkitt's lymphoma and leukemia: 22 new reports and a review of 148 cases from the literature.

Tumour cell karyotypes from patients with Burkitt lymphoma (BL) or Burkitt's type leukemia (ALL3) were studied for correlation with survival, bone marrow and cerebral spinal fluid involvement (CSF), human immunodeficiency virus (HIV) serology, and for recurrent cytogenetic abnormalities. The records of 22 patients with BL from our institution and of 148 cases of BL and ALL3 reported in the literature with karyotypes were evaluated for clinical and cytological features. Overall survival was only 28 per cent and 88 per cent of deaths occurred within the first nine months after diagnosis. Those who survived at least 18 months were unlikely to relapse. Age and gender did not significantly affect survival. Patients presenting with advanced Ann Arbor stage, bone marrow or CSF involvement had lower survival rates. The association of translocations involving chromosome band 8q24 with this disease is confirmed. Sixty-two per cent of karyotypes had t(8;14)(q24;q32) translocations; the recognized variant translocations t(8;22)(q24;q11) and t(2;8)(p12;q24) affected 12 per cent and 9 per cent respectively. Seventeen per cent had abnormal karyotypes but no classic translocation. Patients with variant translocations had the poorest survival rates, and those with the classic t(8;14)(q24;q32) did the best. Despite a small sample size, the variant translocation t(8;22)(q24;q11) appeared to occur at an increased frequency in the patients with AIDS. In the entire group, recurrent involvement of chromosome regions 1q2, 6q11-14 and 17p1 suggests that alteration of genes at these loci, B Cell Growth Factor (BCGF) at 1q2 and p53 on 17p, may contribute to the development and progression of this tumour. Similarly, the frequent trisomies of chromosomes 7, 8, 12 and 18 may indicate an effect on tumour cell growth due to increased gene dosage. Trisomy 12 was found in eight tumours, five from patients with AIDS, suggesting that chromosome 12 has a site or gene whose allelic dosage is selected for in AIDS related lymphoma cells. Cytogenetic studies of adult Burkitt lymphoma and leukemia suggest several likely loci for gene alterations that in conjunction with myc translocations can lead to tumorigenesis.

Establishment and characterization of human B-cell lymphoma cell lines using B-cell growth factor.

B-cell non-Hodgkin's lymphomas (NHL-B) have been difficult to establish in long-term cell culture using standard techniques. We report the establishment of five representative cell lines from high grade NHL-B using B-cell growth factor (BCGF). The five NHL-B cell lines display the morphologic, immunophenotypic, genotypic, and biologic characteristics of the lymphoma cells present in the original diagnostic specimen. The cell lines showed at least a sevenfold dose-dependent increase in proliferation in vitro over background in the presence of BCGF. Other putative B-cell growth-stimulating cytokines showed no significant proliferative activity or were inhibitory in some cases. NHL-B cell lines secreted growth factor(s) into culture supernatants that mediated at least a fivefold dose-dependent increase in cell proliferation in autochthonous lymphoma cells and a 10-fold or greater stimulation in growth factor-dependent normal B cell lines in vitro. The cell lines show monoclonal rearrangements of IgH genes and nonrandom chromosomal abnormalities characteristic of NHL-B, while the expression of Epstein-Barr virus associated antigen (EBNA-I) is present in two of the five cell lines. The studies show that lineage-specific growth factors may be used to establish neoplastic B cell lines in vitro, which are important experimental systems for cellular and molecular studies in the NHL-B.

Plasma cell karyotype in multiple myeloma.

Karyotypic abnormalities were studied in multiple myeloma and were correlated with clinical features. Among 115 evaluable patients, 46% had an abnormal karyotype. Trisomy 3, 5, 9, and 15 and monosomy 13 and 16 were the most common clonal abnormalities. Translocations described previously in other B cell malignancies occurred in nine patients, including four with t(8;14)(q24;q32) translocations. The association of all t(8;14) abnormalities with IgA protein type suggested a pathogenetic relationship between a specific karyotypic abnormality and myeloma protein type. Hypodiploidy occurred mainly in patients with only Bence Jones protein, was associated with resistance to therapy, and justified the early consideration of investigational therapies.

Transformation of chronic lymphocytic leukemia to lymphoma of true histiocytic type.

BACKGROUND: Chronic lymphocytic leukemia (CLL) may evolve into large cell lymphoma (Richter's syndrome), prolymphocytic leukemia, acute lymphoblastic leukemia, and myeloma. METHODS: A patient with CLL that transformed into a lymphoma of true histiocytic type is described, and the literature on the association of these two disorders is reviewed. RESULTS: Lymphomas of true histiocytic type developing as an aggressive terminal phase of CLL previously have been reported in nine patients. Fever and rapidly increasing lymphadenopathy and splenomegaly were the most common signs and symptoms. As with de novo lymphoma of true histiocytic type, extranodal involvement of the soft tissue, gastrointestinal tract, kidneys, bone marrow, liver, and lungs was documented among the 10 patients with lymphoma of true histiocytic type transformed from CLL. The median interval between diagnosis of CLL and the evolution to lymphoma of true histiocytic type was 25.5 months. Patients with lymphomas of true histiocytic type after CLL were treated with fludarabine and various other combination chemotherapy regimens with only short-lived responses. The median time to death after transformation was only 33 days (range, 10 days to 5 months). CONCLUSION: Lymphomas of true histiocytic type appear to represent an additional, though uncommon, form of transformation in CLL. Although their presentation is reminiscent of other intermediate to high grade lymphomas, they can be distinguished based on their morphologic and immunophenotypic features. In the patients described in this study to date, transformation of CLL to lymphomas of true histiocytic type is a poor prognostic sign, with survival generally of only days to weeks.

Fine-needle aspiration evaluation of lymphoproliferative lesions in human immunodeficiency virus-positive patients. A multiparameter approach.

Forty-six fine-needle aspirates of lymphoproliferative lesions from 31 human immunodeficiency virus (HIV)-positive patients were reviewed using cytomorphologic, immunocytochemical, flow cytometric (FCM), cytogenetic, and molecular studies. There were 29 lymphomas (15 small non-cleaved cell [SNCL], 11 large cell [LCL], one small lymphocytic, and two Hodgkin's), 14 reactive hyperplasias, and three "atypical lymphoid proliferations." The reactive hyperplasias were characteristically polymorphic and polyclonal lymphoid populations; six of seven were diploid on FCM, the seventh was hypodiploid. Higher proliferative indices (mean, 11.6%) and higher RNA indices (mean, 1.2) characterized this subgroup compared with published reactive lymphoid hyperplasias from patients without HIV positivity. Aspirates of SNCL showed monotonous populations of intermediate-sized cells except in one patient where a giant cell syncytial variant occurred. Nine of 13 SNCL aspirates showed light chain restriction. JH rearrangement revealed B-cell lineage in one aspirate in which immunocytochemical study was negative for Kappa, lambda, B1, and Leu-4. Nine of 12 SNCL were diploid; the mean proliferative index was 25.6% and the mean RNA index 2.3. Chromosomal translocations involving the c-myc locus were demonstrated in five of seven SNCL aspirates karyotyped. Five of eight LCL showed light chain restriction the remaining three showed null cell phenotype. Large cell lymphomas were diploid on tetraploid with the mean proliferative index of 22.0% and mean RNA index of 2.2. One of two LCL aspirates karyotyped demonstrated c-myc translocation. Despite the multiparameter approach, a definitive diagnosis could not be reached in three aspirates.

The clinical relevance of t(14;18)/BCL-2 rearrangement and DEL 6q in diffuse large cell lymphoma and immunoblastic lymphoma.

BACKGROUND: t(14;18)/bcl-2 gene rearrangement (R) is claimed to impart a worse rate of complete remission and disease-free survival in diffuse large cell lymphoma (DLCL). DEL 6q has also been associated with poor outcome. DESIGN: Retrospective study of 54 patients with either diffuse large cell or immunoblastic lymphoma who had cytogenetics and/or molecular studies performed. RESULTS: Patient characteristics, complete remission rate, and time to treatment failure (TTF) were similar at three year follow-up for groups with and without t(14;18)/BCL-2R. Survival was worse for the former but the difference was not statistically significant. For DEL 6q, patient characteristics and survival rates were similar at three year follow-up for patients with and without the abnormality. TTF was worse for the former but this was not statistically significant. CONCLUSION: This study, with equal or greater number of patients with t(14;18) than previous reports, fails to show a worse prognosis for patients with the t(14;18) chromosomal abnormality. A definite association will await further accrual of patients and a meaningful multivariate analysis.

Variation in the expression of aphidicolin-induced fragile sites in human lymphocyte cultures.

A correlation between specific fragile sites and cancer breakpoints has been suggested raising the question of fragile site expression as a predisposing factor in the occurrence of cancer in some persons. Before addressing the question of increased fragility among patients at high risk for cancer, we analyzed the variability of aphidicolin-induced fragile sites among nine normal persons and also among repeated samples from three of these individuals. Considerable variation in both the frequency and location of these fragile sites was observed and the data strongly suggest the significant variation of 6 of the 16 selected sites to be primarily due to sampling differences. These findings indicate that the use of fragile sites as a screening tool for patients at high risk of cancer should be carefully monitored relative to the variation inherent in both culture and individual expression.

Chromosome 17- and p53 changes in lymphoma.

The clinical course of lymphoma patients in whom rearrangements or deletions of the short arm of chromosome 17 (17p) were evident by cytogenetics was rapidly progressive with a short survival. The gene for the protein designated p53 resides in 17p. We studied four lymphoma cell lines derived from human tumours, and 25 tumour samples of patients with lymphomas, for any evidence of p53 genomic changes by Southern blot technique. The four cell lines and four of the 25 tumour samples showed numerical changes of chromosome 17 or structural abnormalities of 17p (translocations or deletions). Allelic loss of the p53 gene was found in two of the four cell lines, and one of these in addition showed a rearrangement of the 3' end of the gene. Of the four tumours known to have chromosome 17 abnormality, one specimen showed allelic loss of the p53 gene. None of the remaining tumour samples showed any significant change. These studies suggest that acquisition of changes in the short arm of chromosome 17, which may be interrelated with the p53 gene, may carry a poor prognosis in patients with non-Hodgkin's lymphoma.

Minimal residual disease in acute myelogenous leukaemia and myelodysplastic syndromes: a follow-up of patients in clinical remission.

The majority of patients with acute myelogenous leukaemia (AML) and myelodysplastic syndromes (MDS) relapse, especially those with unfavourable cytogenetics. This study was designed to investigate the presence and frequency of minimal residual disease (MRD) in patients with AML or MDS (n=35) and numerical abnormalities of chromosomes 6, 7, 8, 9, 10, 17 and 18 in clinical remission by using a combination of fluorescence activated cell sorting (FACS), fluorescence in-situ hybridization (FISH) and labelling with bromodeoxyuridine (BUdR). The technique enables the detection of as few as three leukaemic cells in 10(5) normal cells. MRD was detected in 33/35 patients in complete remission (CR). 16 patients relapsed (8/11 with monosomy 7, 4/17 with trisomy 8, and 4/7 with other cytogenetic abnormalities) after a median of 4.8 months (range 3-13). Levels of MRD (P=0.007) and proliferation index (P=0.011) were significantly higher in patients with monosomy 7 than in patients with trisomy 8 or other cytogenetic abnormalities. The percentage of cells in S-phase, the number of abnormal cells and cytogenetic class were related to time to relapse (P=0.001) with S-phase being the single most important prognostic factor (P=0.0001). We conclude that the combination of FACS/FISH/BUdR, which determines the number, phenotype and proliferation rate of very rare leukaemic cells in patients with AML or MDS in clinical remission, provides information that is useful in the identification of patients with high and low likelihood of relapse.

Analysis of p53 gene deletions in patients with non-Hodgkin's lymphoma by dual-colour fluorescence in-situ hybridization.

The most common tumour suppressor gene altered in human cancers is p53, which is located on the short arm of chromosome 17. Structural abnormalities of the short arm and loss of chromosome 17 have been reported to confer resistance to chemotherapy in patients with non-Hodgkin's lymphoma (NHL). Therefore we studied the incidence and prognostic value of p53 deletions in patients with NHL by fluorescence in-situ hybridization using a 40 kb cosmid probe. Specimens obtained from 79 patients with NHL were studied. 46 patients were untreated, and 33 were previously treated. 40 tumours had indolent and 39 had aggressive histologies. p53 deletions were observed in 14 specimens (18%) in 32-90% of the cells. No statistically significant difference in the incidence of p53 deletion was observed between indolent and aggressive NHLs or between untreated and previously treated patients. However, p53 deletions were observed in three of four patients with transformed lymphoma. In the untreated patients, p53 deletion had no effect on response to therapy, time to treatment failure, or survival. We conclude that p53 deletions are uncommon in NHL, and may be frequent in patients with transformed lymphoma. In this study, p53 deletions did not influence treatment outcome or prognosis of NHL. Because monosomy 17 and 17p abnormalities have been reported to confer poor prognosis in NHL, other tumour suppressor genes on 17p should therefore be studied.

Polysomy of chromosome 12 in 60 patients with non-Hodgkin's lymphoma assessed by fluorescence in situ hybridization: differences between follicular and diffuse large cell lymphoma.

Sixty consecutive evaluable specimens from patients with non-Hodgkin's lymphoma (NHL) were studied for the incidence of polysomy of chromosome 12 by fluorescence in situ hybridization (FISH) with probes for the repetitive DNA sequence in the centromeric region of chromosome 12. Thirty-six samples were from follicular lymphomas (FLs), and twenty-four were from diffuse large cell lymphomas (DLCLs). Fifty-two specimens (86%) were obtained by fine-needle aspiration of a diseased node, seven (11.6%) were from involved bone marrows, and one specimen was from a pleural effusion. Twelve of the thirty-six (33%) cases with FL had trisomy 12 in 3-41% of the cells (median, 10%) (normal controls had three signals in 1.4 +/- 0.7% of cells). Trisomy 12 was found in 62% of the patients who had had FL for more than 5 years. Nine of the twenty-four cases (37%) with DLCL had more than two copies of chromosome 12 in 4-92% of the cells (median, 78%), and all nine cases were of B-cell phenotype. Unlike FL cells, some DLCL cells had 4-6 copies of chromosome 12. In previously untreated patients, 54% of DLCLs and 26% of FLs had subpopulations of cells containing more than two copies of chromosome 12 (P = 0.04). Only 2/7 cases of DLCL with polysomy 12 had rearrangement of the BCL2 gene, indicating that the majority of DLCL cases with polysomy 12 did not result from histologic transformation of low grade follicular lymphomas. These data demonstrate that FISH of interphase cells is a sensitive method for detecting numerical abnormalities of chromosome 12 in NHL.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of human immunodeficiency virus hybridizing sequences in the peripheral blood of a patient with systemic lupus erythematosus.

Systemic lupus erythematosus, a multisystemic disorder, is considered a prototype of the autoimmune diseases. Although its cause remains unknown, a viral etiology has been proposed. We report that a rapid and sensitive messenger RNA in situ hybridization technique detected hybridizing sequences to the human immunodeficiency virus type in the peripheral blood cells of a woman with systemic lupus erythematosus in whom the presence of acquired immuno-deficiency syndrome was reasonably excluded.