Aneuploidy in carcinomas may be initiated by the acquisition of a single trisomy.

Karyotypic studies of eight endometrioid carcinomas of the endometrium in this laboratory, four colorectal polyps (from this laboratory or reported in the literature), and four early carcinomas of the ovary (from the literature), provide evidence that clonal evolution leading to malignant neoplasms at these sites originates when a cell acquires a single additional chromosome. In different tumors, different chromosomes may be involved in this change from euploidy to aneuploidy. Since the resultant clones of trisomic cells occur at an early stage of tumor development, their presence is only likely to be determined when they are at a location that is accessible for study. As aneuploidy is a virtually constant feature of malignancy, the possibility that the concept of a single trisomy as the initial event in the development of all malignant solid neoplasias should be addressed.

Frequency of hyperdiploid chromosome complements in endometrioid tumors of the endometrium whereas similar tumors in the ovary tend to show hypodiploidy: a significant difference that may not be distinguishable by flow cytometry of DNA content.

Recent interest has focused on endometrioid carcinomas of the endometrium in view of the frequent occurrence of microsatellite stability, accompanied by a favorable prognosis, and by near-diploidy when studied by flow cytometry. The latter feature fails to address the question whether (and to what extent) the karyotypes of the tumor cells may or may not be truly diploid, an important feature on which there is virtually no information. A reconsideration of earlier published and unpublished work in this laboratory on near-diploid carcinomas of the endometrium, and comparable studies on near-diploid ovarian carcinomas (a site where endometrioid carcinomas are also commonly found) has therefore been undertaken. In the endometrium, these studies have clearly shown that carcinomas with near-diploid chromosomes are in fact commonly hyperdiploid, often of endometrioid histology, and in many instances show a single additional chromosome, differing in different tumors, as the apparently sole chromosome change. By contrast, similar studies on the ovary (which also included several endometrioid carcinomas) revealed a tendency towards hypodiploidy, with loss of a few chromosomes, as well as the presence of structural chromosome changes and a generally poor prognosis.

Microsatellite instability.

Unlike aneuploidy, considered to be the cardinal feature of malignant tumors ever since the chromosomal analysis of neoplastic cells became technically feasible, a second pathway toward malignancy has emerged over the past decade that is not characterized by gross aneuploidy but, instead, by inactivation of the DNA mismatch repair system, leading to a hypermutable state in which simple repetitive DNA sequences are unstable during DNA replication. Although mutations of many of these microsatellite sequences are presumably innocuous, because they do not occur in the coding or regulatory regions of genes, other such sequences are critically located in the coding regions of genes involved in the regulation of cell growth. First discovered in the rather uncommon hereditary nonpolyposis colorectal cancer syndrome, where there is an inactivating germline mutation in one of the DNA mismatch repair genes and most of the tumors show microsatellite instability, the latter phenomenon has since been implicated in about 15% of sporadic colorectal cancers, as well as in cancers at several other sites, such as the endometrium. Tumors showing microsatellite instability are generally near-diploid, are at a low stage of development, have a favorable prognosis, and, in the colon, are commonly located on the right side. In recent years, epigenetic phenomena, including hypermethylation and loss of imprinting, have come to be recognized as having a significant bearing on the development of these tumors.

Chromosomal doubling: the significance of polyploidization in the development of human tumors: possibly relevant findings on a lymphoma.

Recent molecular evidence points to defects in cell cycle checkpoints as one of the most important events in the transformation of normal to malignant cells. A byproduct of, if not a critical step brought about by, these defects is the occurrence of polyploidization; near-tetraploid and near-octoploid cells are a common feature of cancers, and the neoplastic stemline may itself attain a high (e.g., near-tetraploid) value. This short review cites cases in which polyploidization is frequent, even at an early stage of tumor development, and considers the probability that, once a high stemline has arisen, there is increased instability with the likelihood of further chromosome changes. A possible example of the latter is a lymphoma in which tetraploid and hypotetraploid metaphases were found, the latter, interestingly, showing an apparently preferential loss from tetraploidy of chromosome 10. It appears, therefore, that a stemline was emerging consequent to (a) chromosome doubling resulting in tetraploid cells, and (b) the appearance of a hypotetraploid line in which chromosome 10 was under-represented. Alternately, there might have been a repeated loss of chromosomes from tetraploid cells that preferentially included chromosomes 10.

Significance of chromosome 5 and 17 changes in the development of carcinoma of the cervix uteri.

Two chromosomes that undergo nonrandom changes in carcinoma of the cervix and have been studied for several decades in this laboratory are discussed. The first, chromosome 5, is discussed in view of the frequent appearance of an isochromosome for 5p, often in two or more copies and commonly associated with fewer that the expected number of normal copies of this chromosome. The second is chromosome 17, where a translocation involving another chromosome may result in a 17p+, and the significant change appears to be a loss from 17p that may include the p53 gene (TP53) and/or other tumor-suppressor genes located on this chromosome arm.

A 3-cM commonly deleted region in 6q21 in leukemias and lymphomas delineated by fluorescence in situ hybridization.

Deletions of the long arm of chromosome 6 (6q) are frequent chromosome aberrations in non-Hodgkin lymphomas (NHLs) and acute lymphoblastic leukemias (ALLs). It is presumed that one or more tumor suppressor genes are localized on 6q. By means of fluorescence in situ hybridization (FISH), we attempted to detect and delineate deletions of 6q in leukemias and lymphomas. We performed FISH on 148 cases of lymphoma and acute leukemia using a panel of 36 YAC probes distributed from 6q12 to 6q27 and a centromeric probe of chromosome 6 as internal control. Deletions of 6q that included a 7-cM commonly deleted region in 6q21 were detected in 59 patients who had B- and T-cell low-grade and high-grade NHL and ALL. FISH with two YAC probes flanking this region was performed on an additional 97 cases of NHL and leukemia. Deletions in 6q21 were detected in an additional 21 cases. In five cases of high-grade B- and T-cell NHL and ALL, the deletion breakpoints were located within the commonly deleted region. To define the deletion breakpoints exactly and to narrow this region further, FISH was performed with six additional YAC probes that have been physically localized within this region. A 3-cM (4-5 Mb) commonly deleted region in 6q21 was delineated. Our study suggests that this commonly deleted region harbors a putative tumor suppressor gene involved in the pathogenesis of both low-grade and high-grade NHL and ALL. Genes Chromosomes Cancer 27:52-58, 2000.

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.

Diffuse large cell (Kiel-1) lymphoma with a t(9;11)(p21-22;q13) and a missing Y as the only chromosome changes.

We describe a diffuse large cell (Kiel-1) lymphoma in a 76-year-old man that is noteworthy because, apart from a missing Y, the only chromosome change was a hitherto undescribed reciprocal translocation, t(9;11)(p21-22;q13). It is interesting that the breakpoints lay in the vicinity of genes that encode proteins engaged in cell cycle control: CCND1 situated at 11q13 and p15 and p16 at 9p21.

Cytogenetics of carcinoma of the cervix uteri: a review.

Cytogenetic studies on carcinoma of the cervix have shown the nonrandom involvement in structural changes of a number of chromosomes, particularly chromosomes 1, 3, 5, 11, and 17. Apart from chromosome 5, where a short-arm isochromosome is the commonest derivative, these chromosomes most often undergo short-arm deletions. Notably, chromosome 17 may have undergone structural changes that result in loss of the tumor suppressor gene TP53 on 17p; chromosomal translocations may in some tumors perform the function that in others is provided by human papillomavirus protein complexing with and inactivating this gene. The chromosome 1 changes may sometimes result in the duplication of long-arm material. Although there have been few comparable studies on the preinvasive stages of cancer of the cervix, it is clear from earlier chromosome and quantitative DNA studies that, except perhaps in the "mild dysplasias," there already is clonal development that has resulted in an aneuploid population with a mode that, as in carcinomas, is either in the diploid or (in 50% or more) triploid-tetraploid range; spindle defects are prominent and may result in unequal segregation of the chromosomes into the daughter cells. Further characterization of the chromosomal changes in carcinoma of the cervix, and more particularly its preinvasive stages, using the new molecular DNA techniques is eagerly awaited.

Evidence for somatic pairing of chromosome 7 and 10 homologs in a follicular lymphoma.

Fluorescence in situ hybridization using centromeric probes for chromosomes 7 and 10 in a follicular lymphoma revealed only one signal in about 40% of interphases, whereas two copies of each chromosome were consistently seen in metaphases. In four out of 18 metaphases both copies of chromosome 7 were situated close to one another. In contrast, two signals for chromosome 1 were seen in 94% of interphases, consistent with observations on metaphases. The findings suggest chromosome-specific somatic pairing, the functional significance of which is at present unknown, and reinforce previous evidence suggesting that care should be taken in the interpretation of interphase signal numbers using centromeric probes in neoplastic as well as normal material.

Genomic alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status.

Specific human papilloma virus (HPV) types appear to be necessary etiological factors for most cervical cancers, yet additional genetic alterations seem to be required for their development and progression. The aim of this study is to determine the likely chromosomes location of tumorigenicity suppressor-like genes, the loss of function of which might be important in the origin or progression of cervical carcinomas. PCR with primers for 75 highly polymorphic microsatellite loci located on the major autosome arms were used to estimate the incidence of loss of heterozygosity (LOH) in 38 tumors. The HPV status of the tumors was also determined. LOH was found to involve 19 chromosome arms in 20-43% of the tumors. Chromosome arms 6p, 3p, and 18q are most frequently involved in LOH in 43, 39, and 35% of the informative carcinomas, respectively. The respective regions involved are 6p21.1-23, 3p13-25.3, and 18q12.2-21.2. LOH is generally limited to specific band segments within these regions. Similar high incidences of LOH of the same 3p segments have been reported in cervical carcinomas from different parts of the world. The same 3p and 6p segments are involved in many types of common cancers, whereas 18q changes are less frequent in other cancers. Chromosome arms 1q, 2q, 3q, 4p, 4q, 5p, 5q, 6q, 7q, 8p, 8q, 11q, 13q, 16p, 18p, and 19p are involved in LOH in 20-33% of the cervical tumors. Chromosome 11 alterations are among the most frequently found in many different types of neoplasias. In this study, 11p was involved in 16% of the tumors, and 11q was involved in 22%. Chromosome 17 alterations are found in more cancers than those of any other chromosome, frequently involving the p53 gene on 17p. LOH of 17p was found in 5 (15%) cervical tumors; 2 of these were HPV negative and expressed mutant p53. In such HPV-negative tumors, direct mutation of the wild-type p53 appears to replace the inactivation of the p53 product by oncogenic HPV types. Tumors with LOH at many loci were, on the average, at more advanced stages, as were tumors with mutant p53. The higher overall incidence of LOH in cervical carcinomas as compared to other cancers, and the diversity of LOH patterns found, suggest that different cervical carcinomas probably arise and/or progress, in part, because of the loss of function of different yet finite sets of tumorigenicity suppressor genes and genes that are involved in tumor progression and metastasis. The findings also indicate that certain chromosome segments that are often altered in cervical carcinomas are also frequently altered in several other types of cancers. It remains to be determined whether the same or different genes located within these segments are involved in the different cancer types.

Ectopic nucleolar organizer regions. A common anomaly revealed by Ag-NOR staining of metaphases from nine cancers.

In view of the sparsity of reports on nucleolar organizer regions (NORs) in human tumor metaphase chromosomes, we have applied the silver (Ag-NOR) technique to a previously studied testicular germ-cell tumor that had an abnormal translocation, which involved a 13p, and to nine new sequentially studied tumors. Six of the new tumors, and the germ cell tumor, showed ectopic NORs (e.g., at the end of the long arm of acrocentrics or metacentrics, or interstitially in metacentrics): five carcinomas and a leiomyosarcoma, all of which also revealed numerous structural chromosome changes after G-banding. The three tumors that did not show ectopic NORs were lymphomas with relatively simple karyotypic changes. It seems that the presence of ectopic NORs in the majority of the tumors is a reflection of the multiplicity of structural changes in these tumors and does not signify that there is any particular propensity for acrocentrics to take part in these changes. It was interesting that several of the chromosomes showed large notably a metacentric in a squamous cell carcinoma of the skin in which the Ag-NOR-positive region was seen as an unstained gap in unbanded and G-banded chromosomes.

Mutation of the MXI1 gene in prostate cancer.

The Mxi1 protein negatively regulates Myc oncoprotein activity and thus potentially serves a tumour suppressor function. MXI1 maps to chromosome 10q24-q25, a region that is deleted in some cases of prostate cancer. We have detected mutations in the retained MXI1 alleles in four primary prostate tumours with 10q24-q25 deletions. Two tumours contained inactivating mutations, whereas two others contained the identical missense mutation. Fluorescence in situ hybridization also demonstrated loss of one MXI1 allele in an additional tumour lacking chromosome 10 abnormalities. MXI1 thus displays allelic loss and mutation in some cases of prostate cancer that may contribute to the pathogenesis or neoplastic evolution of this common malignancy.

Chromosome abnormalities and p53 expression in a small cell carcinoma of the bladder.

Chromosome studies on a highly malignant tumor, a small cell carcinoma of the bladder (the first to be studied cytogenetically), showed a hypertriploid mainline and a hypertetraploid minor line. Extensive chromosomal rearrangements were present in both lines, some rearranged chromosomes being seen in only one of the lines, while others, derived from chromosomes 6, 9, 11, 13, and 18, were seen in both. Although different giant chromosomes were present in the two lines, they shared a possibly significant common feature: multiple copies of 2q. DNA flow cytometry confirmed that the tumor had a hypertriploid main mode and showed that dysplastic surface epithelium present in the histologic material also had a hypertriploid DNA index. p53 expression in the tumor was demonstrated by flow cytometry.

Derivative chromosome, der(17;22)(q10;q10), in two carcinomas of the cervix uteri and one of the skin.

In a previous study, we described 17p+ chromosomes in about 40% of carcinomas of the cervix, but it was usually not possible to identify the additional material on the short arm of the chromosome 17. Here we report an apparently identical rearranged chromosome in two squamous cell carcinomas of the cervix and one of the skin, in which the whole of 17p has been replaced by the long arm of a chromosome 22: der(17;22)(q10;q10), suggesting that this rearrangement may represent a significant step in the development of carcinomas of the cervix and other sites.

Use of the mitotic counts for the prognosis and grading of breast cancer. Method evaluation study.

Reproducibility of the volume fraction-corrected mitotic index (M/VV index) was studied in 144 unselected breast cancer specimens. The influence on decision making of variation in determining the index was also analysed. In the complete series of specimens the correlation between two observers, one subjectively estimating the epithelial fraction of tumor epithelium and the other using point-counting (10 x 10 ocular grid), was good (Pearson's r = 0.82, p < 0.001, 95% CI 0.70-0.92). A subset of 30 specimens was used to evaluate the grading efficiency (GE) of the M/VV index method. The mean grading efficiency as estimated from this subset varied between 90% and 93%. The average minimum GE value was 82.8% (SD = 3.4%). The findings suggest that when the M/VV index method is used, the grading is correct on average in 90% or more of the cases, but dependent on the cutoff point. The over-all grading efficiency of the M/VV index method was comparable to that obtained from published S-phase fraction data on breast cancer specimens from three independed laboratories. We conclude that the M/VV index in breast cancer analysis is a sufficiently reproducible method in mitosis counting, and that it can be used with subjective or point count estimation of the area fraction of neoplastic epithelium.