Complex Chromosomal Changes Research Articles

Evolution and strain diversity advance exploration of Candida albicans biology.

Fungi were some of the earliest organismal systems used to explore mutational processes and its phenotypic consequences on members of a species. Yeasts that cause significant human disease were quickly incorporated into these investigations to define the genetic and phenotypic drivers of virulence. Among Candida species, Candida albicans has emerged as a model for studying genomic processes of evolution because of its clinical relevance, relatively small genome, and ability to tolerate complex chromosomal changes. Here, we describe major recent findings that used evolution of strains from defined genetic backgrounds to delineate mutational and adaptative processes and include how nascent exploration into naturally occurring variation is contributing to these conceptual frameworks. Ultimately, efforts to discern adaptive mechanisms used by C. albicans will continue to divulge new biology and can better inform treatment regimens for the increasing prevalence of fungal disease.

Abstract 3371: Analysis of telomere length and dysfunction in normal breast tissue from BRCA2 mutation carriers

Abstract The tumor suppressor protein BRCA2 is important for maintaining genomic stability. BRCA2 is essential for homologous recombination repair of DNA double strand breaks and also has a role in protection of stalled replication forks as well as in telomere protection and maintenance. Disruptions in telomere homeostasis can result in excessive telomere shortening and drive chromosome instability, a hallmark of BRCA2-related cancers. Germline mutations in the BRCA2 gene are associated with highly increased risk of breast cancer. A single deleterious founder mutation, BRCA2999del5, has been detected in the gene in the Icelandic population, making it feasible to study the influence of a single mutation at a population level. The BRCA2999del5 mutation is found in 6-7% of female breast cancer patients in Iceland and is associated with complex chromosomal changes in tumor tissue. Furthermore, the BRCA2999del5 mutation is associated with poor prognosis, although mutation carriers seem to differ with respect to age of onset and severity of disease. It is therefore of great interest to identify factors that influence breast cancer risk within the mutation carrier group. The aim of the study was to measure telomere length (TL) and levels of telomere dysfunction in tumor-adjacent normal breast tissue from BRCA2 mutation carriers and non-carriers and investigate their potential relationship with breast cancer risk. The study group consisted of 186 Icelandic breast cancer patients; BRCA2999del5 mutation carriers and sporadic cases, matched with respect to age and year of diagnosis. For TL measurements, paraffin embedded normal breast tissue samples were analyzed by Quantitative Fluorescence in Situ Hybridization. Telomere dysfunction induced foci (TIFs) can be detected where co-localization of telomere and DNA-damage signals occur. For this purpose, the samples were also immunofluorocently stained for the DNA damage repair protein 53BP1. In concordance with previously reported data in the literature, luminal epithelial cells showed the shortest TL and highest number TIFs of the cell types in normal breast tissue. This observation is highly relevant as these are the cells from which most breast cancers originate. No difference was detected in age-adjusted TL between BRCA2 mutation carriers and non-carriers. However, consistent with our recently published data from TL measurements in blood from the same study group, shorter TL is correlated with younger age at breast cancer diagnosis in BRCA2 mutation carriers but not in non-carriers. In conclusion, these results indicate that in BRCA2 mutation carriers, shorter TL in normal breast tissue is associated with earlier breast cancer occurrence. Citation Format: Birna Thorvaldsdottir, Beth A. Cimini, Morgan E. Diolaiti, Katrin Olafsdottir, Jon G. Jonasson, Jorunn E. Eyfjörd. Analysis of telomere length and dysfunction in normal breast tissue from BRCA2 mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3371.

Cytogenetic study on some common bone tumours

This study aimed to detect the consistent chromosomal abnormalities of each type of common bone tumours . Thirty bone tumors specimens were processed for direct cytogenetic preparation , only (9 cases) showed success results for chromosomal preparation by banding technique , the results of cytogenetic study were 1- Osteosarcoma (4) cases revealed multiple numerical and structural changes with complex karyotype and pronounced cell to cell variation . chromosome 17 was the most frequent involved in these chromosomal alteration . Also loss or structural changes of chromosome 13 was found in (2) cases of osteosarcoma loss or gain of sex chromosome were detected in these cases , loss of Y chromosome in (2) cases ; loss of X chrosome in (1) case and gain of X chrosome in another case . 2- Osteochnodroma : reveled the simple numerical change with no structural change in one case . 3- Chondroblastoma : in this tumor structural and numerical abnormalities of chrosome 5 . 4- Chondrosarcoma : showed chromosomal aberration with multiple numerical and structural changes in chromosome (1) was of interest and monsomy 18 was reported in one case . 5- Giant cell tumors : It showed a complex changes and the range of chromosomal number was 50 – 58 with the characteristic telomeric fusion in malignant cases while simple numerical change only in benign giant cell tumor . Conclusion Cytogenetic study of both benign and malignant bone tumors have revealed abnormlities in the number and / or structures of chromosomes X, Y , 1,5,6,11 , 13,17,18 and complex chromosomal changes in malignant types of bone tumors .

Abstract A26: Telomere length in normal and tumor breast tissue from BRCA2 mutation carriers and sporadic breast cancer cases

Abstract Germline mutations in the BRCA genes are associated with highly increased risk of breast and ovarian cancers and to a lesser extent to prostate and pancreatic cancers. Great number of mutations with variable impact on cancer risk and progression are known in these genes worldwide. A single founder mutation has been detected in each of the BRCA genes in the Icelandic population, making it feasible to study the influence of a single mutation at a population level. The BRCA2999del5 mutation is more frequent and can be found in approximately 6-7% of female breast cancer patients and 40% of male breast cancer patients in Iceland. The mutation leads to a non-functional protein product and is associated with complex chromosomal changes in tumor tissue. Furthermore, the BRCA2999del5 mutation has been associated with poor prognosis, both in breast and prostate cancers, although mutation carriers seem to differ with respect to age of onset and severity of disease. Dysfunctional telomere maintenance can lead to excessive telomere shortening which causes chromosome instability. It is therefore a driving force behind cancer progression and is considered a hallmark of many human cancers. Telomere shortening has been shown to be an early and a common molecular alteration in epithelial cancers, including breast cancers. Previous results from our laboratory, amongst others, have shown that BRCA2 is associated with telomere protection and maintenance. Telomere dysfunction induced foci (TIFs) formation was detected in cell lines derived from heterozygous BRCA2999del5 mutation carriers1. Telomere length has been proposed as a possible predictive factor for cancer risk for various cancers. In the case of breast cancer and BRCA mutation carriers, the results have been conflicting. The aim of the study was to investigate if telomere length in normal and tumor breast tissue is correlated with breast cancer progression and survival in a well-defined group of BRCA2 mutation carriers and sporadic breast cancer cases. The study group consisted of breast cancer patients carrying the BRCA2999del5 Icelandic founder mutation and sporadic cases, matched with respect to age and year of diagnosis. Tumor and normal adjacent tissue samples from these patients were embedded in paraffin and Quantitative Fluorescence in Situ Hybridization method (Q-FISH) was used to carry out telomere length measurements. Telomeres were visualized using a telomeric sequence-specific fluorescence peptide nucleic acid (PNA) probe. A centromere-specific PNA probe was used as an inner control for hybridization efficiency. The results show a clear difference in telomere length of different cell types in normal breast tissue with inner epithelial cells having shorter telomeres than both myoepithelial cells and fibroblasts. These findings support previously reported data in the literature2. The majority of examined tumors have very short telomeres. In mutation carriers, there are indications that less variable telomere length in normal adjacent tissue is associated with reduced breast cancer specific survival. This seems to be more pronounced in the mutation carrier group compared to the sporadic group. Telomere length in adjacent normal tissue appears to be shorter in cases with luminal type than with basal-like tumors.

Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis.

Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGHs) harbouring the HBV pre-S2 mutant large surface protein (LHBS) have been recognized as a morphologically distinct hallmark of HCC in the advanced stages of chronic HBV infection. Considering its preneoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin α1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin α/β-associated nuclear pore complex (NPC). By interacting with importin α1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transport of an essential DNA repair and recombination factor, Nijmegen breakage syndrome 1 (NBS1), upon DNA damage, thereby delaying the formation of nuclear foci at the sites of DNA double-strand breaks (DSBs). Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability, indicated by increased global gene copy number variations (CNVs), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA DSBs representing significant genomic instability. In conclusion, type II GGHs harbouring HBV pre-S2 mutant oncoprotein represent a high-risk marker for the loss of genome integrity in chronic HBV carriers and explain the complex chromosome changes in HCCs. Mouse array CGH raw data: GEO Accession No. GSE61378 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61378).

A myeloma cell line established from a patient refractory to thalidomide therapy revealed high-risk cytogenetic abnormalities and produced vascular endothelial growth factor

A myeloma cell line established from a patient refractory to thalidomide therapy revealed high-risk cytogenetic abnormalities and produced vascular endothelial growth factor

MYC Gene FISH Testing in Aggressive B-Cell Lymphomas: Atypical Rearrangements May Result in Underreporting of Positive Cases

AbstractAbstract 1552 Background:Multifunctional MYC oncogene overexpression resulting from genomic rearrangement plays a critical role in lymphomagenesis and lymphoma manifestation, particularly in aggressive B-cell lymphomas such as Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL including double hit or triple hit lymphomas (DHL/THL). Accurate detection of MYC gene rearrangement, its presence or absence, has become increasingly important with its diagnostic and consequent therapeutic implications. In practice, FISH is the best test for MYC alterations. However, this is technically challenging, requiring knowledgeable and skillful cytogeneticists to design probe panels, correctly analyze and interpret atypical as well as typical signal patterns. For instance, up to 10% of patients with otherwise typical BL can be MYC rearrangement negative by FISH and, gene amplification and miRNA deregulation have been reported as possible reasons in occasional cases, although some of the FISH negative patients could be detected by better application of probes and interpretation of FISH findings. Here we retrospectively analyze FISH results from 879 consecutive MYC positive cases and share our experience with other FISH labs and physicians who are involved in diagnosis, differential diagnosis and treatment of aggressive lymphomas. Results:A MYC/IGH dual fusion translocation probe and a MYC break apart (ba) probe (Vysis) were applied to paraffin embedded tissue, lymph node biopsy, bone marrow and peripheral blood specimens. Of the 879 cases, MYC ba was positive in 258, MYC ba and MYC-IGH both positive in 331, MYC ba positive/MYC-IGH negative in 276, and MYC-IGH positive/MYC ba negative in 14 cases. Two subsets of cases with significant atypical signal patterns were observed. Firstly, in 14 cases with both probes tested, MYC ba was negative but MYC/IGH positive, including 6 BLs, 3 DLBCLs, 1 MCL, 1 Burkitt leukemia transformed from BL, 1 subtype unknown lymphoma and 2 DHLs with history of Hodgkin lymphoma and Burkitt-like lymphoma respectively and with concomitant BCL6 gene rearrangement in both. Eleven of these cases showed a single fusion signal pattern suggesting an insertion of IGH sequences into MYC or vise versa. This single insertion fusion pattern can also be resulted from complex chromosome changes as seen cytogenetically in 2 of the 11 cases. Secondly, 26 cases with various diagnoses including 4 DHLs and 2 THLs showed MYC rearrangement with a concomitant partial deletion of the MYC ba probe with 17 cases for 3' and 9 for 5' deletion. Three of these cases were also positive for concomitant probe deletion and amplification, two with 3' deletion and 5' amplification and one with 5' deletion and 3' amplification. Conclusion and discussion:MYC gene rearrangement detection by FISH in aggressive B-cell lymphomas is widely used. However, FISH labs should realize the technical limitations of each probe/panel based on underlying mechanisms involving MYC rearrangement and minimize false negative results. From our experience and previous studies by other groups, it is recommended that MYC ba probe should not be tested alone but together with a MYC/IGH t(8;14) translocation probe to detect cryptic insertions and variant translocations which often present as MYC ba negative yet MYC/IGH positive with a single fusion signal pattern inserting IGH promoter/enhancer elements into MYC or part or all of the MYC probe into IGH locus. Another observation that can also be misinterpreted is that the MYC ba probe often shows a deletion of either 5’ or 3’ flanking sequence. This is not because of tissue sectioning but indicative of MYC rearrangement with a concomitant loss of the DNA sequences adjacent to the breakpoints. A flanking sequence deletion revealed at translocation breakpoints using a FISH ba probe is a common finding. As well documented, BCR/ABL translocation results in an ASS deletion upstream of ABL in ∼15% of CMLs, and a 5’ probe deletion in ∼30% ALK rearrangement positive NSCLC cases. Three of 26 such cases in our study also showed simultaneous retained MYC probe amplification making FISH interpretation even more difficult. As a diagnostic, prognostic and predictive biomarker, MYC gene plays a pivotal role in aggressive B-cell lymphomas, and its accurate detection will help improve disease risk stratification and therapy selection. Disclosures:No relevant conflicts of interest to declare.

The molecular and cellular heterogeneity of pancreatic ductal adenocarcinoma

Current standard therapies for pancreatic ductal adenocarcinoma have failed to attenuate the aggressiveness of this disease or confer notable improvements in survival. Previous molecular research into pancreatic cancers, along with advances in sequencing technologies, have identified many altered genes in patients with pancreatic cancer and revealed the marked genetic heterogeneity of individual tumors. Thus, the lack of success of conventional empiric therapy can be partly attributed to the underlying heterogeneity of pancreatic tumors. The genetic alterations that have been detected in pancreatic cancer range from simple mutations at the level of base pairs to complex chromosomal structural changes and rearrangements. The identification of molecular changes that are unique to an individual patient's tumors, and the subsequent development of strategies to target the tumors in a personalized approach to therapeutics, is a necessary advance to improve therapy for patients with this disease.

A rare case of chronic myeloid leukemia with secondary chromosomal changes including partial trisomy 17q21 to 17qter and partial monosomy of 16p13.3

BackgroundThe so-called Philadelphia (Ph) chromosome is present in almost all cases with chronic myeloid leukemia (CML). Around 5-10% of these patients show complex translocations involving other chromosomes in addition to and/or besides chromosomes 9 and 22. As nowadays most CML cases are treated with Imatinib, variant rearrangements have in general no specific prognostic significance, though events of therapy resistance remain to be studied.ResultsHere we report a Ph chromosome positive patient with hematological typical chronic phase CML. Untypically, an unbalanced complex rearrangement involving chromosomes 16 and 17 leading to a deletion of 16pter and partial trisomy of 17q21 to 17qter, was identified besides a trisomy 8 and an additional Ph chromosome in a part of malignant cells.ConclusionHere a novel and cytogenetically unique case of a Ph chromosome positive CML clinically in chronic phase is reported, having complex secondary chromosomal aberrations. Thus, CML patients with complex chromosomal changes are nonetheless treatable by Imatinib.

Is Acute Erythroid Leukemia a Transitional Stage from MDS to AML?: A Study by Fluorescence in Situ Hybridization and Cytogenetics

Background: Chromosomal abnormalities of acute erythroid leukemia (AEL) are reported to be complex and nonspecific. We compared the cytogenetic abnormalities of AEL to other subtypes of acute myeloid leukemia (AML) by G- band karyotyping and fluorescence in situ hybridization (FISH) techinique using 18 probes.Methods: 384 patients diagnosed with AML between January 2000 and December 2007 were classified morphologically by French-American-British classification. G-band karyotyping and FISH for 4 recurrent chromosomal abnormalities in AML [AML/ETO rearrangement, PML/RARa rearrangement, MLL rearrangement, inv(16)] were performed. To verify whether common cytogenetic abnormalities of myelodysplastic syndrome (MDS) were present in AEL, FISH for 5q deletion, 7q deletion, trisomy 8 and gain of 1q were performed on bone marrow nucleated cells of 25 patients with AEL. To evaluate the numerical chromosomal changes, ten additional FISH tests were done. Chromosomal enumeration probes (CEP) were primarily used and in case where there were no CEP available, probes for chromosomal rearrangement were surrogately used.Results: Incidence of recurrent genetic changes of AML [AML/ETO, PML/RARA, MLL, inv(16)] was 0% in AEL, while the incidence of AML/ETO, PML/RARA, MLL and inv(16) rearrangement in AML excluding AEL was 12.8%, 12.7%, 5.0%, 4.6%, respectively. Frequencies of numerical chromosomal changes were 11.8% in AML excluding AEL and 44% in AEL, showing significantly higher incidence of numerical changes in AEL. Frequencies of cytogenetic changes, which are common in MDS, were 20% for 5q deletion, 32% for trisomy 8, 16% for 1q gain among AEL patients. In total, 40% of the AEL patients showed similar chromosomal changes to MDS. By G-banding, 32% of the AEL patients showed numerical change of chromosome 8 and 20% for chromosome 5. However, numerical chromosomal changes by G-banding were not statistically different between AEL and other AML, while >3 complex chromosomal changes were significantly higher in AEL. (P=0.001) Out of 25 AEL patients, 4 patients (16%) were transformed from MDS and 1 patient (4%) transformed to other subtype of AML during treatment.Discussions: Numerical chromosomal change was the most predominant genetic change of AEL, while recurrent genetic changes of AML were not found in AEL. Instead, AEL patients showed similar chromosomal changes to MDS. This implies that AEL subtype of AML is rather a separate disease entity from the other types of AML, more within the spectrum of MDS. We infer that AEL is a transitional stage from MDS to AML.Table 1. General aspects and the summary of the results of AML and AEL*CharacteristicsAMLAELGenderfemale16141.9%622.2%male22358.1%2177.8%total384100.0%27100.0%FAB classificationM0164.2%M14311.2%M212432.3%M34311.2%M48121.2%M5174.4%M6277.0%M771.8%undetermined266.8%AML excluding AELAELAge, years (median)15–77 (51)17–84 (60)<6024869.5%1140.7%°Ã6010930.5%1659.3%Recurrent genetic abnormalities ¢”PML/RARA43/33812.7%0/270.0%AML/ETO42/32712.8%0/270.0%inv(16)12/2594.6%0/270.0%MLL16/3195.0%0/270.0%Abnormal karyotype85/35623.9%13/2748.1%Ploidy change by FISH ¢Ô41/34811.8%14/2556.0%Abbreviations: AML, Acute myeloid leukemia; FAB, French-American-Britishclassification; AEL, acute erythroid leukemia* AEL diagnosed by WHO criteria¢” FISH was done on available specimen only¢Ô Ploidy changes were determined by 18 kinds of probes in AEL, and 4 kinds of Probes in AML subtypes excluding AEL

A Biologic Definition of Burkitt's Lymphoma from Transcriptional and Genomic Profiling

The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is unclear. We used transcriptional and genomic profiling to define Burkitt's lymphoma more precisely and to distinguish subgroups in other types of mature aggressive B-cell lymphomas. We performed gene-expression profiling using Affymetrix U133A GeneChips with RNA from 220 mature aggressive B-cell lymphomas, including a core group of 8 Burkitt's lymphomas that met all World Health Organization (WHO) criteria. A molecular signature for Burkitt's lymphoma was generated, and chromosomal abnormalities were detected with interphase fluorescence in situ hybridization and array-based comparative genomic hybridization. We used the molecular signature for Burkitt's lymphoma to identify 44 cases: 11 had the morphologic features of diffuse large-B-cell lymphomas, 4 were unclassifiable mature aggressive B-cell lymphomas, and 29 had a classic or atypical Burkitt's morphologic appearance. Also, five did not have a detectable IG-myc Burkitt's translocation, whereas the others contained an IG-myc fusion, mostly in simple karyotypes. Of the 176 lymphomas without the molecular signature for Burkitt's lymphoma, 155 were diffuse large-B-cell lymphomas. Of these 155 cases, 21 percent had a chromosomal breakpoint at the myc locus associated with complex chromosomal changes and an unfavorable clinical course. Our molecular definition of Burkitt's lymphoma clarifies and extends the spectrum of the WHO criteria for Burkitt's lymphoma. In mature aggressive B-cell lymphomas without a gene signature for Burkitt's lymphoma, chromosomal breakpoints at the myc locus were associated with an adverse clinical outcome.

A der(14)t(1;14)(q12;p11) in chronic myelomonocytic leukemia

Duplication of the long arm of chromosome 1 (1q) is widely reported in human neoplasia, including the myelodysplastic syndromes (MDS). So far, it has not been described as a single aberration in the chronic myelomonocytic leukemia (CMML), a subtype of MDS. Rather, trisomy 1q was always a part of complex chromosome changes affecting the subtypes of MDS other than CMML. We report on a patient with CMML with an unbalanced translocation of the entire 1q onto the short arm of chromosome 14 as a sole cytogenetic abnormality. Fluorescence in situ hybridization (FISH) analysis with an α-satellite probe for the paracentric region of the long arm of chromosome 1 confirmed the presence of trisomy 1q in a derivative chromosome, der(14)t(1;14)(q12;p11). The discrepant results between the metaphase cytogenetics (100% abnormal) and interphase cytogenetic (71% nuclei with 3 signals) suggest that trisomy 1q, even in the absence of additional cytogenetic changes, has a sufficient leukemogenic potential to confer a proliferative advantage on hematopoietic cells committed to monocyte stemline both in vitro and in vivo. The literature data on partial and complete trisomy 1q in CMML is reviewed.

Chromosomal Abnormalities in Non-Small Cell Lung Carcinomas and in Bronchial Epithelia of High-Risk Smokers Detected by Multi-Target Interphase Fluorescence in Situ Hybridization

Human lung carcinogenesis is accompanied by complex chromosomal changes that may be detected in interphase cells by fluorescence in situ hybridization (FISH) assay using recently developed multitarget DNA probes. Touch preparations of 20 non-small cell lung carcinomas, sputum specimens from 3 patients with lung cancer and from 11 ex-smokers without lung cancer, and cultured benign bronchial epithelium of 42 high-risk smokers, 9 of whom had concurrent invasive carcinoma, were tested using a four-color FISH probe (LAVysion) targeting centromere 6, 5p15.2, 7p12 (EGFR), and 8q24 (MYC). Significantly high frequencies of abnormal cells were found in each of the 20 NSCLC (100%) and in the 3 sputum specimens from lung cancer patients. None of the cytologically normal sputa contained FISH abnormalities. Cultured bronchial epithelial cells from 11 of 42 patients (26%) were abnormal for at least one probe. Abnormal FISH patterns had no association with gender, presence of tumor or histology. Multicolor FISH can readily detect chromosomal abnormalities in imprints and sputa from lung carcinomas. Chromosomal aneusomy is also frequent in bronchial epithelial cells from long-term smokers. The prognostic significance of the multicolor LAVysion FISH probe set should be validated in a controlled clinical trial.

Multitude multicolor chromosome banding (mMCB) – a comprehensive one-step multicolor FISH banding method

Multicolor chromosome banding (MCB) using one single chromosome-specific MCB probe set per experiment was previously reported as powerful tool in molecular cytogenetics for the characterization of all kinds of human marker chromosomes. However, a quick analysis of karyotypes with highly complex chromosomal changes was hampered by the problem that up to 24 MCB experiments were necessary for a comprehensive karyotype description. To overcome that limitation the 138 available region-specific microdissection-derived libraries for all human chromosomes were combined to one single probe set, called multitude MCB (mMCB). A typical fluorescence banding pattern along the human karyotype is produced, which can be evaluated either by transforming these profiles into chromosome region-specific pseudo-colors or more reliably by studying the fluorescence profiles. The mMCB probe set has been applied on chromosomes of normal male and female probands, two primary myelodysplastic syndromes and two solid tumor cell lines. Additionally, a cell line of Gorilla gorilla (GGO) studied previously by single chromosome-specific MCB was reevaluated by the mMCB method. All results were in concordance with those obtained in parallel or by other cytogenetic and molecular cytogenetic approaches indicating that mMCB is a powerful multicolor FISH banding tool for fast characterization of complex karyotypes.

Simultaneous mapping of human papillomavirus integration sites and molecular karyotyping in short-term cultures of cervical carcinomas by using 49-color combined binary ratio labeling fluorescence in situ hybridization

Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes' shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21∼23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures.

Re-evaluation of refractory anemia with excess blasts in transformation

The category ‘refractory anemia with excess blasts in transformation (RAEBt)’ consists of two sub-sets; one group is categorized based on the percentage of blasts in the marrow (≥20%) and other is based on the percentage of blasts in the peripheral blood (≥5%). We separated RAEBt patients based on these two criteria and compared hematologic and clinical relevance to assess the reasonable basis for the new classification. All RAEBt patients showing peripheral blood (PB) blasts of ≥5% were re-classified as RAEB by the WHO classification. This subset of RAEBt patients had lower percentages of bone marrow (BM) blasts, and notably they showed frequent complex cytogenetic abnormalities, including −5/5q- and/or −7/7q-. Moreover, the RAEBt patients of this group had shorter survivals compared to RAEBt patients with BM blasts between 20 and 30%. We next assessed hematologic and clinical relevance between refractory anemia with excess blasts (RAEB) and RAEBt patients with PB blasts of ≥5%. Except for the percentage of blasts in the PB ( P=0.0037) and BM ( P=0.0073), there was no significant difference in hematologic or clinical features between RAEB patients with BM blasts of ≥11% and RAEBt patients with PB blasts of ≥5%. When MDS patients with PB blasts of ≥5% (RAEBt by the FAB classification) were included as RAEB-II based on the “MDS 2000 classification’, there was a high frequency of patients with complex chromosome changes, involving 5q and 7q, with significant poorer outcome compared to those with RAEB-I. Although it is still controversial whether MDS patients with BM blasts 20% or more should be considered as acute leukemia, the utilization of the ‘MDS 2000 classification’ might be useful to designate MDS patients diagnosed based on the percentage of blasts in the peripheral blood.

Spectral karyotyping and chromosome banding studies of primary breast carcinomas and their lymph node metastases.

Three primary breast tumors and their lymph node metastases were characterized by G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). In each case, the karyotypic abnormalities detected were similar in the primary tumor and its matched metastasis. Two of the pairs had near-diploid karyotypes with three to four chromosomal aberrations, whereas the third pair had a near-pentaploid chromosome content and many marker chromosomes in the primary tumor and a near-tetraploid chromosome number with almost the same marker chromosomes in the metastasis. SKY and FISH confirmed the karyotypic similarities between the primary tumors and their metastases and, in addition, improved the identification and characterization of marker chromosomes. One of the tumor pairs with near-diploid karyotypes had gain of 8q, 16q, and 17q, whereas the other had gain of 1q and chromosome 8 material in the form of ring chromosomes. The third pair had more complex chromosomal translocations and numerical changes resulting in net gain of material from chromosomes X, 1, 2, 6, 7, 14, 16, 19, and 20, and chromosome arms 8q and 11q, as well as net loss of material from chromosomes 3, 13, 18, 21, and 22. The present study underscores the need to combine conventional chromosome banding and molecular cytogenetic techniques in the cytogenetic analysis of solid tumors.

Cytogenetics of Hodgkin’s disease

Cytogenetic studies over the past 35 years have made a major contribution towards the understanding of the nature of Hodgkin’s disease by demonstrating unequivocally the consistent presence of a clonal population of cells that have the cardinal features of malignancy e.g. more or less gross aneuploidy, frequently with complex chromosomal changes and showing considerable variation from case to case, thus comparable to the findings in carcinomas and other solid cancers. The mode is frequently in the triploid-tetraploid region, as we found in 17 of 27 cases studied in this laboratory by Feulgen microspectrophotometry, compared to only 10 cases with neardiploid modes. It is disappointing that no specific change, such as a translocation that could give a clue to the chromosomal location of a gene or genes involved in the etiology of Hodgkin’s disease, has yet been found. Nevertheless it is clear that a number of nonrandom changes, including several that are also common in other malignancies including the non-Hodgkin’s lymphomas, are frequently present, e.g., deletions of 1p, 6q, and 7q. Interestingly, deletions of 4q, with loss of 4q25→q27, that have also been reported may show some specificity for Hodgkin’s disease.

Chromosome 1p aberrations are frequent in human primary hepatocellular carcinoma

To investigate the chromosomal changes in hepatocellular carcinoma in humans, metaphase chromosomes obtained directly from short-term culture of resected human primary hepato-cellular carcinoma tissues were studied in seven patients. Among the five successfully karyotyped cases, cytogenetic analysis showed highly different and complex chromosomal changes in the cancer tissue in four patients. Structural aberrations of chromosome 1 and deletion of 1p with breakpoints at p22 or p32 were the most common abnormalities, and were found in the four successfully analyzed cases. Loss of 1p may be important in hepatocarcinogenesis, and warrants further characterization by molecular genetic analysis.

FISH studies of urinary cells of patients with bladder cancer

Generally, bladder cancers are characterized by complex and numerous chromosome changes that vary from tumor to tumor. Nevertheless, certain chromosome changes recur with a consistency, (eg, +7, +8, −9, and −Y). In applying fluorescence in situ hybridization (FISH) studies to urinary cells in bladder cancer we chose probes for these chromosomes as well as those for chromosomes 10 and 11. A probe for the X chromosome was used for female patients in place of the Y. In the present study, we show that FISH of urine samples can detect the presence of cancer cells in transitional cell carcinoma (TCC) of the bladder of any grade and stage, including carcinomas in situ (CIS). We analyzed 27 samples from 25 patients (three were from the same patient); 24 samples were recurrent or newly diagnosed TCC and 3 were CIS. Our results show that FISH of urine samples is a reliable test for the detection of bladder cancer cells, regardless of the grade and stage of the tumor, and that a correlation appeared to exist between invasiveness of the tumor and the number of abnormalities in such tumor.