Hon Fong Louie Mark

Fluorescent in situ hybridization as an adjunct to conventional cytogenetics

Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that exploits the availability of recombinant deoxyribonucleic acid (DNA) technology. In metaphase FISH, a specific nucleic acid sequence (probe) is bound to the homologous segment on a metaphase chromosome in a fixed preparation on a glass slide. The presence of a region-specific DNA sequence in a nondividing cell can be detected using interphase FISH. Interphase cytogenetics via FISH can be performed on fixed cells harvested during a routine culture, on tissue sections and on many cytologic specimens. Specific examples of clinical and research applications are discussed to illustrate the utility of FISH in the detection of constitutional and acquired chromosomal abnormalities.Publisher Summary This chapter discusses the mechanism of fluorescent in situ hybridization (FISH) as an adjunct to conventional cytogenetics. Cytogenetics is a subspecialty of medical genetics. Conventional cytogenetic analysis by banding is a powerful, established technique that can provide a picture of the human genome at a glance. The advent of FISH has been made possible by the explosion in recombinant DNA technology and the Human Genome Project. It offers an unprecedented opportunity for the analysis of a large number of non-dividing cells in the interphase stage. Thus, FISH is sometimes called “interphase cytogenetics.” FISH is the most direct method for visualizing a specific DNA sequence on the metaphase chromosome or an interphase cell. It allows for both the enumeration and localization of a DNA sequence underlying a cytogenetic abnormality. FISH requires no additional acquisitions of new samples. It can be performed on unstained or previously Giemsa-stained slides and also on unstained and previously Wright-stained smears of both peripheral blood and bone marrow. It can also be performed on many other tissues and archival materials.

Fluorescent in situ hybridization assessment of chromosome 8 copy number in breast cancer.

SummaryConventional cytogenetics of breast and other solid tumors has been hampered by a number of factors. An analysis of breast tumor tissues was therefore undertaken using fluorescentin situ hybridization (FISH). A total of 34 specimens were analyzed using a chromosome 8-specific α-satellite probe. Various approaches were tested and compared. Among 30 informative samples, 11 infiltrating ductal carcinomas, not otherwise specified (NOS), 5 ductal carcinomasin situ, 5 lobular carcinomas, 3 papillary carcinomas, and 6 benign lesions were studied. Of the 11 cases of infiltrating ductal carcinomas (NOS) analyzed, four cases showed 3 signals, one case showed 4 signals, and the rest showed 2 signals. Of the 5 cases of ductal carcinomain situ samples, 1 showed 3 signals and the other 4 cases showed 2 signals. All cases of lobular carcinomas, papillary carcinomas, and benign lesions showed 2 signals. We inferred from these data that 36% of the infiltrating ductal carcinomas (NOS) were trisomic and 9% were tetrasomic, whereas 20% of the ductal carcinomasin situ were trisomic. All samples from lobular carcinomas, papillary carcinomas, and the benign lesions were disomic. From our preliminary data, it can further be concluded that a subset of breast cancer is characterized by chromosome 8 trisomy. These data are consistent with an ever-increasing database on the association of chromosomal 8 trisomy with other cancers such as leukemia, lymphoma, prostate cancer, ovarian carcinoma, salivary gland tumor, malignant melanoma, desmoid tumors, and recently gestational trophoblastic disease. It is also noted that the ability to analyze formalin-fixed, paraffin-embedded archival material will enable a more comprehensive cytogenetic study of breast cancer than is currently available.

Cytogenetic analysis of salivary gland type tumors

Fourteen salivary gland type tumors were analyzed with a combination of conventional cytogenetics via GTG-banding, molecular cytogenetics via fluorescent in situ hybridization, and chromosome morphometry. Nine tumors were benign (eight pleomorphic adenomas and one Warthin tumor) five tumors were malignant (one carcinoma ex pleomorphic adenoma, two adenoid cystic carcinomas including one from the breast, a basal cell adenocarcinoma, and an acinic cell carcinoma). Thirteen specimens grew in tissue culture; the basal cell adenocarcinoma did not grow. The Warthin tumor had a normal karyotype, one pleomorphic adenoma was normal, one had a clone with a missing Y chromosome, and the other pleomorphic adenomas had structural chromosomal abnormalities including the following: translocations between chromosomes 3 and 8, chromosomes 6 and 16, chromosomes 8 and 9, chromosomes 8 and 12, chromosomes 8 and 14, and chromosomes 8 and 21. Of the four malignant tumors with karyotypes, the acinic cell carcinoma and one adenoid cystic carcinoma were normal, the second adenoid cystic carcinoma showed a normal polymorphic variant, whereas the carcinoma ex pleomorphic adenoma demonstrated the following karyotype: 46,XX,dir ins(8;5)(q12;q12q35), add(12)(p13)/46,XX. In conclusion, 66% of the benign tumors and 25% of the malignant tumors demonstrated abnormal karyotypes.

Discordant Detection of Monosomy 7 by GTG-banding and FISH in a Patient with Shwachman-Diamond Syndrome without Evidence of Myelodysplastic Syndrome or Acute Myelogenous Leukemia

The myelodysplastic syndromes (MDS) are a group of hematologic disorders commonly affecting elderly persons and often leading to acute myelogenous leukemia (AML). Although rare in children, when MDS does occur, it is frequently part of a congenital disorder such as Shwachman-Diamond syndrome (SDS). Monosomy 7 and/or deletion of part or all of 7q are poor prognostic signs in MDS and AML, although the pathophysiologic relationship between this finding and MDS or AML is unclear. Shwachman-Diamond syndrome is an inherited illness characterized by exocrine pancreatic insufficiency and by congenital neutropenia. Patients with SDS are at increased risk of developing myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Because monosomy 7 is a poor prognostic sign in MDS and AML, establishing its presence is important. However, different methods of detection of monosomy 7 may lead to different results in some patients. We present the case of a 10-year-old girl known to have SDS, who had a bone marrow aspiration and biopsy done to rule out MDS and AML. By light microscopy, the patients bone marrow was unremarkable. GTG-banding showed the following karyotype: 45,XX,-C[3]/47,XX,+C[1]/46,XX[45]. Fluorescence in situ hybridization (FISH) was performed with a chromosome 7-specific alpha-satellite probe (D7Z1). Almost all (373 of 376) cells exhibited only one chromosome 7 signal. A second marrow aspiration done 6 months later showed an essentially normal karyotype by GTG-banding. Fluorescence in situ hybridization with the same chromosome 7 probe showed 230 of 250 cells to be monosomic for chromosome 7. A whole chromosome 7 painting probe demonstrated disomy for chromosome 7 in 90 of 90 cells; however, subtle heteromorphism in the centromeric regions of the 2 copies of chromosome 7 was noted in some cells. This case demonstrates that FISH and GTG-banding can give discordant results, that the two should be viewed as complementary technologies, and that both have a place in a full karyotypic analysis. Furthermore, this case demonstrates for the first time that heteromorphism and/or subtle structural abnormalities of chromosome 7, previously associated with MDS and AML, can exist without clinical or morphologic signs of these illnesses. It will be of interest to further study the relationship, if any, between SDS and various structural abnormalities of chromosome 7 in MDS and AML, and to elucidate the molecular mechanisms of pathogenesis, physiology, and treatment of these disorders.

Trisomy 8 in Embryonal Rhabdomyosarcoma Detected by Fluorescence In Situ Hybridization

Embryonal rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood. Cytogenetic studies of this tumor are rare. In one study trisomy 8 was found to be a primary cytogenetic abnormality. In view of the findings of trisomy 8 in a multitude of cancers, we conducted a pilot study to test the hypothesis that a subset of rhabdomyosarcoma also exists with trisomy 8. Accordingly, archival tissues of 12 cases of rhabdomyosarcoma were retrieved and fluorescence in situ hybridization (FISH) using a chromosome 8-specific, alpha-satellite probe was undertaken on formalin-fixed paraffin-embedded tissue sections using the protocol optimized in the Cytogenetics Laboratory at Rhode Island Hospital. The results obtained demonstrated that 6 of 12 tumors showed chromosome 8 trisomy, when a 15% threshold is adopted. In addition, one case was borderline, with 11% of the cells found positive for three fluorescent signals. Future experiments utilizing additional specimens from our centers as well as from other laboratories are needed to confirm and extend the findings of the present study.

Fluorescence in situ hybridization assessment of chromosome 8 copy number in stage I and stage II infiltrating ductal carcinoma of the breast

A total of 34 cases of infiltrating ductal carcinoma of the breast, not otherwise specified (NOS), were selected, based on the clinical stage of the disease (17 cases stage I and 17 cases stage II). The histologic grade and the DNA content of each tumor were evaluated. Each specimen was analyzed and blinded cytogenetically for the frequency of chromosome 8 copy number using fluorescence in situ hybridization (FISH). Among the informative samples, 16 cases were disomic (47%) and 18 cases (53%) were trisomic. Of the 16 disomic tumors, 13 cases (81%) were classified clinically as stage I disease and 3 cases (19%) were stage II disease. Of the 18 trisomic tumors, 4 cases (22%) were stage I, and 14 cases (78%) were stage II. Microscopically, all trisomic tumors were of high histologic grade and aneuploid when analyzed by flow cytometry. We inferred from these data that a subset of infiltrating ductal carcinomas (NOS) is characterized by chromosome 8 trisomy. This chromosomal abnormality correlates well with other markers that predicate aggressive biological behavior of the tumor. While this observation needs to be further extended, the data suggest that chromosome 8 copy number may be used as a possible marker to identify a subgroup of patients with infiltrating ductal carcinoma associated with a poor prognosis.

Abnormal chromosome 8 copy number in stage I to stage IV breast cancer studied by fluorescence in situ hybridization

To test the hypothesis that the frequency of abnormal chromosome 8 copy number increases with the severity of the disease as defined by an increase in clinical stage, we conducted a fluorescence in situ hybridization (FISH) study of a sample of 42 breast cancer specimens utilizing a protocol that was optimized by our laboratory. Cytogenetic results, obtained from blinded analyses of archival specimens, demonstrated that the higher clinical stages (i.e., stages III and IV) yield higher frequencies of abnormal chromosome 8 copy number. Specifically, 45.45% and 50% of the stage I and stage II cases, respectively, were abnormal, whereas 63.64% and 60% of the stage III and stage IV cases, respectively, were abnormal for chromosome 8 copy number. The overall frequency of abnormal chromosome 8 copy number was 54.76% (23 of 42 tumors studied). When the results of a control probe were taken into account, 34.78% (8 of 23) of the abnormal cases were trisomic, whereas the remaining cases were likely triploid. Thus, the present data not only established that chromosome 8 trisomy is a recurrent finding in breast cancer, but also confirmed a higher frequency of occurrence of abnormal chromosome 8 copy number with the higher clinical stages. Future experiments utilizing additional specimens in this laboratory and from other laboratories are necessary to confirm and extend the findings of the present study.

A novel, convenient, and inexpensive approach for deriving ISCN (1985) relative lengths: validation by a morphometric study of 100 karyotyped metaphase cells

The present study stemmed from a need for a rapid means of deriving reproducible chromosome measurements. An internal set of standards can serve as the basis for routine, easy, and reliable morphometric comparisons. In this study, a total of 100 karyotyped metaphases were analyzed using the Nestler Run-Mate, a computerized curvilinear measuring tool. The null hypothesis tested was that there are no significant differences between chromosomal relative-length values obtained via this previously untested approach and those cited in ISCN (1985). The results indicate that this new method is not only feasible and adequate but has advantage over the conventional approach, which requires the use of a projector and screen to measure chromosomes in unkaryotyped metaphase spreads; further, it is less expensive and easier than using computerized digitizing tablets, a conclusion supported by time-and-effort measurements. Immediately obvious applications include routine use in clinical cytogenetics laboratories, as well as for fractional length estimations in fluorescent in situ hybridization studies performed in research laboratories that do not have access to expensive automated instrumentation.

A Multimodal Approach in the Diagnosis of Patients with Hematopoietic Disorders

Myelodysplastic syndromes (MDS) are a group of relatively ill-defined hematopoietic disorders in which both qualitative and quantitative defects of the hematopoietic cells cause bone marrow dysfunction. With an incidence estimated to be approximately 1 per 100,000 persons per year, MDS mainly affects the elderly. Myelodysplastic syndromes share many features with acute nonlymphocytic leukemia; in fact, a proportion of patients with MDS eventually develop acute myeloid leukemia. To illustrate a multimodal approach in the diagnosis of patients with hematopoietic disorders, we describe a 66-year-old patient with a question of myelodysplastic syndrome, leukemia, and two translocations involving chromosome 10:t(5;10) and t(7;10). These structural rearrangements effectively gave rise to monosomy for part of the long arm of chromosome 5 and for the long arm of chromosome 7. Findings of del(5q) and del(7) in MDS have been reported in the literature. The results of chromosome morphometry, which was conducted to compare the lengths of all relevant chromosome segments, are consistent with the hypothesized chromosomal abnormalities. The investigational technique of fluorescence in situ hybridization (FISH), using both painting and alpha-satellite probes, was used as an adjunct to conventional cytogenetics to further delineate the nature of the chromosome abnormalities observed in the GTG-banded studies. Confirmatory studies utilizing the new technique of spectral karyotyping (SKY) were also carried out. Thus, the multimodal approach of hematopathology, GTG-banding, chromosome morphometry, FISH, and SKY can be very useful for delineating complex cytogenetic cases.

A Combined Cytogenetic and Molecular Approach for Diagnosing Delayed Puberty

A phenotypic female aged 15 4/12 years was referred because of delayed puberty and short stature. Chromosomal analysis of peripheral blood leukocytes revealed two subpopulations of cells. The modal cell line was hypodiploid with a missing X chromosome while the other cell line was diploid with one X chromosome and a G-sized chromosome that resembled a Y chromosome in morphology. Subsequent fluorescent in situ hybridization yielded results consistent with the above conventional cytogenetic studies. To provide unequivocal evidence of the Y-chromosome material, molecular analyses using the polymerase chain reaction and various primers were carried out which identified an intact short arm and centromere of the Y chromosome and structurally altered long arms. A laparoscopic bilateral gonadectomy, performed because of the risk of neoplasia, also yielded cells with both 45,X and 46,XY karyotypes. The present report thus illustrates the usefulness of a combined approach for diagnosing delayed puberty.