Angela Goodacre

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.

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 patients 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.

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.

Chromosome 17p 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‐Hodgkins lymphoma.

Ratio of bcl-xshort to bcl-xlong is different in good- and poor- prognosis subsets of acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous collection of leukemic disorders ranging from chemotherapy-sensitive subsets [inversion 16 and t(8;21)], which often can be cured with cytosine arabinoside alone, to the most resistant subsets, which can survive even supralethal levels of combination alkylator chemotherapy (cytogenetic subsets monosomy 5 and monosomy 7).Materials and MethodsTo analyze the expression of BCL-2 family genes, which are expressed in these subsets of AML, we used PCR sequence amplification reactions that are dependent on oligonucleotide primers representing the BH1 and BH2 homology domains to generate the unique regions between BH1 and BH2. These primers are conserved among all members of the BCL-2 gene family and are separated by a 150 nucleotide region sequence between the BH1 and BH2 domains. The PCR products unique to each BCL-2 family member were cloned directionally into sequencing vectors. The identity of the insert of each clone was determined by slot-blots of the DNA amplified from individual colonies and by hybridization with radioactive probes specific to the bcl-2, bcl-x, or bax genes.ResultsWe found that bcl-2 is the predominant member expressed in AML samples with a poor prognosis (−5, −7), whereas the transcripts of bcl-x are higher than those of bcl-2 in the AML samples with a good prognosis [invl6, t(8;21)]. No significant difference in bax expression was detected between AML subsets of good and bad prognosis. The ratio of bcl-xlong, which inhibits apoptosis, to bcl-xshort, which promotes apopto-sis, was determined by amplification with a pair of primers specific to bcl-x followed by separation of the PCR product on agarose gels. Bcl-xlong and bcl-xshort appeared as bands of different molecular mass on a molecular weight gel and were visualized by ethidium bromide staining or Southern blot analysis with a bcl-x-specific probe.ConclusionsWe found that the ratio of bcl-x long to bcl-x short was higher in the AML patients with a poor prognosis. These experiments showed that the levels of BCL-2 family members in the leukemia cells of good- and poor-prognosis subsets are different. In addition, novel members of the BCL-2 family were isolated from the cells of AML patients of either prognosis.

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.

Transformation of chronic lymphocytic leukemia to lymphoma of true histiocytic type

Background. Chronic lymphocytic leukemia (CLL) may evolve into large cell lymphoma (Richters syndrome), prolymphocytic leukemia, acute lymphoblastic leukemia, and myeloma.

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‐Hodgkins 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.

Cell Kinetic Analysis of Intact Rat Colonic Crypts by Confocal Microscopy and Immunofluorescence

BACKGROUND & AIMS Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5-bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision.

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 Hodgkins), 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.