Hon Fong L. Mark

Constitutional trisomy 8 mosaicism and gestational trophoblastic disease

Concurrence of congenital trisomy 8 mosaicism and gestational trophoblastic disease in a 42-year-old gravida IV, para IV female is described in the present report. In contrast to other cases described in the literature, our patient had no known additional confounding chromosomal abnormalities other than trisomy 8. The finding of trisomy 8 mosaicism in yet another type of cancer provides further support for the hypothesis of an increased predisposition to cancer in tissues with constitutional genomic imbalance, which can manifest itself as numerical chromosomal abnormalities (e.g., trisomies) or structural chromosomal abnormalities (e.g., translocations). To the best of our knowledge, this is the only report in the English literature of constitutional trisomy 8 mosaicism associated with gestational trophoblastic disease, a rare gynecologic disease entity in itself.

Fluorescent in situ Hybridization Detection of HER-2/neu Gene Amplification in Rhabdomyosarcoma

Embryonal rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood, comprising 45–50% of childhood sarcomas. Cytogenetic studies of this tumor are rare. In view of the paucity of cytogenetic data on this cancer and based on the finding of HER-2/neu gene amplification in a number of cancers that was detected mostly using the traditional technique of immunohistochemistry, we decided to conduct a pilot study to investigate whether HER-2/neu gene amplification in this tumor can be detected using the newer technique of fluorescent in situ hybridization (FISH). Archival tissues of rhabdomyosarcoma were retrieved and FISH using an HER-2/neu probe was undertaken on formalin-fixed paraffin-embedded tissue sections using a protocol optimized for our laboratory at Rhode Island Hospital. Out of 9 cases of rhabdomyosarcoma studied to date, 1 case clearly showed HER-2/neu gene amplification. Thus, FISH is a sensitive technique suitable for the detection of oncogene amplification and the delineation of tumor heterogeneity in this tumor. Future experiments utilizing additional specimens from our centers as well as from other laboratories will be needed to extend the finding in the present pilot study.

Distal 5q trisomy resulting from an X;5 translocation detected by chromosome painting

We describe the case of a 13-year-old girl with an apparently de novo unbalanced translocation resulting in the presence of additional chromosomal material on the short arm of one X chromosome, which was detected by conventional G-banding studies. Fluorescence in situ hybridization (FISH) using the Chromoprobe Multiprobe-M protocol confirmed that the additional chromosomal material originated from chromosome 5. The karyotype of this patient is now established to be 46,X,der(X) t(X;5)(p22.3;q33), with a deletion of Xp22.3-pter and partial trisomy of 5q33-qter. The distal 5q trisomy genotype has been associated with clinical signs that include growth and mental retardation, eczema, craniofacial anomalies, and malformations of heart, lungs, abdomen, limbs, and genitalia. Our patient also has short stature, a prominent nasal bridge, a flat philtrum, a thin upper lip, dental caries, and limb and cardiac malformations, but she appears to be mildly affected compared with previously reported cases. This is the first case of distal 5q trisomy arising from a translocation with the X chromosome. Replication studies on this patient show that the derivative t(X;5) chromosome is late replicating in almost all cells examined, which indicates that this chromosome is preferentially inactivated. However, the translocated segment of chromosome 5 appears to be early replicating, which implies that the trisomic 5q segment is transcriptionally active. We cannot determine from these studies whether all or only some genes in this segment are expressed, but this patients relatively mild clinical signs suggest that the critical region(s) that contribute to the distal 5q trisomy phenotype are at least partly suppressed. A review of other patients with X-chromosome translocations indicates that many but not all of them also have attenuated phenotypes. The mechanism of inactivation of autosomal material attached to the X chromosome is complex, with varying effects on the phenotype of the patients that depend on the nature of the autosomal chromatin. Replication studies are of limited utility in predicting expression of autosomal genes involved in X-chromosome translocations. Am. J. Med. Genet. 94:392–399, 2000.

Characterization of an analphoid supernumerary marker chromosome derived from 15q25→qter using high‐resolution CGH and multiplex FISH analyses

Supernumerary marker chromosomes (SMCs) without detectable alphoid DNA are predicted to have a neocentromere and have been referred to as mitotically stable neocentromere marker chromosomes (NMCs). We report the molecular cytogenetic characterization of a new case with analphoid NMC derived from 15q25→qter using high‐resolution comparative genomic hybridization (HR‐CGH) and multiplex fluorescence in situ hybridization analyses with various α‐satellite DNA probes, all‐human‐centromere probe (AHC), whole chromosome painting probes, and a subtelomere probe. The propositus is a dysmorphic infant who, at age 3 months, showed accelerated growth, partial deafness, and a phenotype similar to that of the eight previously reported cases of distal 15q tetrasomy. Chromosome studies showed that he had a de novo extra SMC in 80% of cells examined. HR‐CGH revealed rev ish enh(15)(q25qter). Molecular cytogenetic analysis and molecular DNA polymorphism study demonstrated that this extra SMC is an NMC containing an inverted duplication of the distal long arm of chromosome 15 (tetrasomy 15q25→qter) which originated paternally, i.e. ish der(15)(qte→q25::q25[neocen]→qter)(AHC–, CEP15–, WCP15+, PCP15q++). This case further elucidates the phenotype related to tetrasomy of this specific chromosome segment and represents a new report of a neocentromere on distal chromosome 15q suggesting that this region appears to be susceptible to the formation of neocentromeres.

Trisomy 10 in leukemia

Previous studies had raised questions about whether the relatively rare finding of trisomy 10 in leukemia is nonrandomly associated with a specific immunophenotype or ethnic origin. To shed light on the above questions and to obtain additional clinical and pathologic information on this unique class of leukemic patients, we conducted a retrospective study of leukemia cases at our laboratory from July 1, 1990, to July 31, 1996. The results not only support the rarity of trisomy 10, but they also reject the hypotheses that all trisomy 10 cases are CD7-positive, or found in Orientals.

Interphase cytogenetics for studying solid tumors.

Conventional cytogenetic analysis is a powerful, established technique that can provide a picture of the human genome at a glance. Most laboratories use G-banding using trypsin and Giemsa stain (GTG-banding) for conventional cytogenetic analysis. In a routine cytogenetic study, a short term culture is either established in the presence of a mitogen—such as phytohemagglutinin (PHA), where it is called a stimulated culture— or grown without such an agent, in which case it is called an unstimulated culture. The former is used for peripheral blood cultures to rule out constitutional abnormalities, whereas the latter is used for the study of neoplastic tissues. Longterm tissue cultures are usually established for solid tumor studies. Harvesting chromosomes for conventional cytogenetics is a rather lengthy and tedious process. Colcemid, a derivative of colchicine, is usually used to block spindle fiber formation and arrest the chromosomes in metaphase. This is followed by a hypotonic treatment to cause the cells to take up water and swell so that the chromosomes will spread well when dropped onto glass slides at a later step. After the hypotonic treatment step, the cell pellet is fixed with a fixative consisting of three parts methanol to one part glacial acetic acid. After repeated rinsings, the cells are then dropped onto glass slides and airdried. Slides are aged for a variable amount of time, then banded and stained according to one of the banding protocols. GTG-banding seems to be the most popular method in the U.S., probably because of its simplicity. Prior to the advent of modern-day imaging systems, photographs were taken of the best metaphase spreads, and the photographs were enlarged and hand-cut to separate the images of the chromosomes for identification. Most cytogenetics laboratories now own computer-assisted karyotyping systems. Karyotyping involves arranging the 46 chromosomes in the human genome according to shape, size, and banding patterns. Thus, conventional cytogenetics is a labor-intensive process requiring highly trained personnel. In addition, conventional cytogenetics depends entirely on the availability of high quality metaphases, thus excluding from analysis the vast majority of cells that are in interphase.

HER-2/neu gene amplification in stages I-IV breast cancer detected by fluorescent in situ hybridization.

Purpose: Approximately 25–30% of breast and ovarian carcinomas have amplification of the HER-2/neu oncogene. The aim of the present study was to focus on HER-2/neu gene amplification in different clinical stages of breast cancer in order to (1) determine if fluorescent in situ hybridization (FISH) can be used to detect HER-2/neu gene amplification in different clinical stages of breast cancer, (2) establish whether HER-2/neu gene amplification characterizes a subset of breast cancer in each of these stages, and (3) determine whether a trend for correlation of amplification with the clinical stage of the disease can be detected using the FISH technology.Methods: A total of 40 specimens of formalin-fixed, paraffin-embedded breast cancer tissues were analyzed cytogenetically, in a blinded fashion, for HER-2/neu gene amplification using FISH and the Vysis LSI HER-2/neu Orange and CEP 17 Green DNA dual color probe. The criterion for “high amplification” was an amplification ratio of >4.0, that for “moderate amplification” a ratio between 2.1 and 4.0, and that for “low amplification” a ratio of 1.5–2.0.Results: Using a cutoff point of ≥1.5, the overall frequency of HER-2/neu gene amplification among stage I tumors was 30% (3 out of 10). Of these, one-third (1 out of 3) showed low amplification, one-third (1 out of 3) were moderately amplified, and one-third (1 out of 3) were highly amplified. The overall frequency of HER-2/neu gene amplification among stage II tumors was 0% (0 out of 10). The overall frequency of HER-2/neu gene amplification among stage III tumors was 10% (1 out of 10). The sole tumor found positive was classified as moderately amplified by our criteria. The overall frequency of HER-2/neu gene amplification among stage IV tumors was 50% (5 out of 10). Four of the 5 tumors found positive were highly amplified. The overall frequency of gene amplification in the 40 cases studied was 22.5% (9 out of 40 tumors studied).Conclusion: Although a linear correlation between HER-2/neu amplification and clinical stage cannot be established at this time, it is interesting to note that when stages I and II, and when stages III and IV are combined, respectively, the latter category has a higher amplification frequency than the former. Furthermore, stage IV has the highest frequency (5 out of 10) of HER-2/neu gene amplification than all three lower stages combined (4 out of 30). This is no doubt due to the high frequency of gene amplification observed in stage IV tumors, which, interestingly, also demonstrate high level amplification of HER-2/neu gene copy numbers. Although the biologic and clinical basis for gene amplification is not clear, given the observation that the most aggressive disease stage is associated with the highest frequency of gene amplification and the most high level amplification, further exploration of HER-2/neu as a prognostic marker of poor outcome using FISH is warranted.

Construction and characterization of radiation hybrids for chromosome 9, and their use in mapping cosmid probes on the chromosome

Radiation hybrids were produced from a monochromosomal microcell hybrid (PK87-9) which contains only human chromosome 9 with an inserted marker on 9p. Doses of radiation ranging from 1000 to 8000 rads were used to produce a series of hybrids with different size fragments of human chromosome 9. The inserted dominant selectable marker was used to select for hybrids that preferentially maintain fragments of 9p. A panel of 53 radiation hybrids were characterized for 17 chromosome 9 markers. In addition, 17 hybrids were analyzed by fluorescent in situ hybridization (FISH). Hybrids were produced with breaks on both 9p and 9q, many of which appear to contain a single fragment of human chromosome 9. These hybrid cell lines were used to regionally localize 31 cosmids isolated from a chromosome 9 cosmid library. Six cosmids were mapped to intervals on 9p, six cosmids mapped to the centromeric region of the chromosome, and 19 mapped to 9q.

Delineation of a supernumerary marker chromosome utilizing a multimodal approach of G-banding, fluorescent in situ hybridization, confirmatory P1 artificial chromosome fluorescent in situ hybridization, and high-resolution comparative genomic hybridization

We describe the structure of a supernumerary marker in a child who presented with a right atretic ear and multiple congenital anomalies. Using G‐banding, fluorescent in situ hybridization (FISH), P1 artificial chromosome FISH and high‐resolution comparative genomic hybridization (CGH), the marker was demonstrated to be a derivative chromosome resulting from malsegregation of a paternal 8;22 translocation: 47,XY, +der(22)t(8;22)(q24.1; q11.2). This case is noteworthy because the marker, while sharing similarities to der(22) in the Cat Eye syndrome (CES), also contains chromosome 8q material. This partial 8q trisomy confounds the diagnosis of CES associated with pure trisomy or pure tetrasomy 22q. The paternal translocation is noted with prolonged infertility and oligospermia, which again highlights the utility and necessity of chromosome analysis in this setting.

Study of Chromosome 12 Copy Number in Breast Cancer Using Fluorescence In Situ Hybridization

Trisomy 12 is the most frequent numerical chromosomal abnormality reported in chronic lymphocytic leukemia (CLL). Its significance in other cancers, however, has not been extensively investigated until recently. Less than 20 cases of polysomy for chromosome 12 have been studied thus far. The most recent data in the literature suggest that gain of chromosome 12 may be a recurrent and sometimes early event in breast carcinogenesis. To test the hypothesis that a subset of breast cancer may be characterized by chromosome 12 trisomy, we conducted a retrospective study of 40 specimens. Of these, ten were stage I, ten were stage II, ten were stage III, and ten were stage IV. Out of the total sample, 12 cases (30%) were found to be presumably trisomic, if a conservative cutoff point of greater than or equal to 15% cells with three signals is adopted. Furthermore, some, but not all, of the 12 cases were found to be likely triploid, when data from a control chromosome 17 probe were taken into account. Thus, our data support the hypothesis that a subset of breast cancer exists which is characterized by an abnormal copy of chromosome 12, in either a diploid or a triploid background.