FISH panels for hematologic malignancies.

Cytogenetic analysis of hematological malignancies has played a crucial role in the diagnosis and clinical management of patients, as well as in providing fundamental insights into the genetic basis of the pathogenesis of these diseases. Leukemias and lymphomas have lent themselves readily to karyotypic analysis and undoubtedly represent the greatest successes of cytogenetics in human cancer. Several cytogenetic changes have been shown to have considerable prognostic significance also and are being used as measurable targets for response to therapy. Molecular characterization of the recurrent cytogenetic abnormalities has identified genes involved in leukemogenesis and formed a basis for specific treatment strategies. Fluorescence in situ hybridization (FISH) analysis, since its introduction, has revolutionized the field and enabled a more precise determination of the presence and frequency of genetic abnormalities. It is particularly indispensable where metaphase cytogenetics may be difficult in the largely quiescent cells of some hematological malignancies, particularly the lymphoid disorders. FISH probes have been used extensively to detect nonrandom abnormalities in interphase nuclei and the true incidence of chromosome abnormalities has been proven to be much higher than that detected by conventional chromosomal analysis. The avail- ability of a comprehensive line of commercial probes for rapid identification of critical genetic aberrations has contributed to the widespread use of this technique. It has also led to the current practice in most laboratories to test for genetic aberrations by using FISH panels that have been designed to detect genetic changes important not only in the diagnosis of leukemias and lymphomas, but also because of their association with prognosis, to identify high-risk populations in specific hematological cancers, so they can be targeted for aggressive therapy.

Secondary acute myelogenous leukemia and myelodysplasia without abnormalities of chromosome 11q23 following treatment of acute leukemia with topoisomerase II-based chemotherapy.

Therapy-related MDS and AML are complications of intensive chemotherapy regimens. Traditionally, patients exposed to topoisomerase II inhibitors are reported to develop secondary AML with abnormalities of chromosome 11q23. We evaluated the long-term hematologic toxicity of topoisomerase II-intensive high-dose mitoxantrone-based chemotherapy in 163 newly diagnosed acute leukemia patients treated over an 8 year period. Nine (5.5%) patients developed new cytogenetic abnormalities. Four patients developed MDS with progression to AML, three patients developed new abnormalities at the time of relapse, and three patients (including one of the former patients) had changes that were not associated with hematologic disease. The abnormalities most frequently involved chromosomes 7q, 20q, 1q, and 13q. Despite the use of topoisomerase II-intensive treatment, no patient developed an abnormality involving chromosome 11q23. Spontaneous resolution of some changes and prolonged persistence of others in the absence of hematologic disease indicates that some cytogenetic changes are not sufficient to promote leukemogenesis.

Cytogenetic and molecular characterization of a malignant mixed müllerian tumor of the uterus with a t(8;22)(q24.1;q12).

We report the cytogenetic and molecular findings in a malignant mixed Müllerian tumor of the uterus in a 65-year-old woman. Karyotypic analysis revealed a t(8;22)(q24.1;q12) as the sole abnormality in all 20 cells analyzed. Southern blot analysis of two positional candidate genes, MYC at 8q24, and EWS at 22q12, showed no genomic rearrangement. The finding of the t(8;22) as the only abnormality may be of etiologic significance.

Characterization of a complex chromosomal rearrangement in a patient with a typical catlike cry and no other clinical findings of cri-du-chat syndrome.

We report on the clinical, cytogenetic, and molecular cytogenetic findings in a 4-year-old girl who was evaluated for developmental delay and a catlike cry from birth. No other findings of cri-du-chat syndrome were present. Karyotype analysis demonstrated a de novo deletion and inverted duplication of the 5p region. The abnormality was confirmed and further defined by detailed FISH analysis using cosmid and lambda phage clones previously mapped to distinct regions of 5p. The analyses documented deletion of 5p15.3-->pter and an inverted duplication of 5p14-->5p15.3. The deleted segment on 5p contains the region implicated in the isolated catlike cry feature of the cri-du-chat syndrome, confirming that the genes involved in the catlike cry map to the distal end of 5p. Except for the catlike cry and possibly the developmental delay that may be due to the deletion of 5p, the duplication of 5p14-->5p15.3 in this patient did not present with additional anomalies. This study further demonstrates the usefulness of the molecular cytogenetic approach for characterizing complex chromosome rearrangements. Such analyses of patients with an isolated catlike cry can avoid an incorrect diagnosis of the cri-du-chat syndrome, which is associated with a more severe prognosis.

Biphenotypic (mixed myeloid/T-cell) extramedullary myeloid cell tumor.

A 32-year-old male with a 4-year history of chronic myelogenous leukemia (CML) in chronic phase for 4 years, then myeloid blast crisis for 7 months, developed diffuse bulky lymphadenopathy in association with a white blood count (WBC) of 17,100/mm3 with 70% blasts. Biopsy of a cervical lymph node revealed a blastic extramedullary myeloid cell tumor, which showed a biphenotypic (mixed myeloid/T-cell) immunophenotype. Chromosomal analysis revealed karyotypic features of both myeloid and lymphoid lineages. Although extramedullary myeloid cell tumor (EMT, granulocytic sarcoma, chloroma) is well known to occur in chronic myelogenous leukemia (CML), to our knowledge this is the first description of evolution of CML into a biphenotypic EMT.

The cytogenetic and molecular characterization of benign and malignant soft tissue tumors.

Cytogenetic analyses of benign and malignant soft tissue tumors have led to the description of recurrent, specific, and even pathognomonic chromosomal translocations and/or other rearrangements in most types of soft tissue tumors. The consistent karyotypic rearrangements have provided critical diagnostic information in this group of neoplasms that often presents significant diagnostic challenges to the clinician and the pathologist. These findings have also been instrumental in the characterization of the abnormalities at the molecular level. Novel genes have been isolated from the translocation junctions and the mechanisms of their deregulation identified. This has increased our understanding of the histogenesis of these tumors, paved the way for the molecular diagnosis of many sarcomas, aided in directing therapy, and also provided important prognostic information.

Isochromosomes 7q and 17q in Wilms tumor.

We report the cytogenetic findings in a Wilms tumor from a 4-year-old boy. Karyotypic analysis revealed isochromosomes of 7q and 17q as coexisting clonal aberrations. The finding is notable in view of recent reports of i(7q) as a nonrandom event in Wilms tumor and the emerging evidence for genetic heterogeneity in this tumor.

Congenital pulmonary myofibroblastic tumor: a case report with cytogenetic analysis and review of the literature.

We report a case of congenital pulmonary myofibroblastic tumor, and review prior reports of this rare neoplasm to demonstrate its clinically benign behavior despite histologic features previously interpreted as sarcoma. The patient, a female neonate, presented with severe respiratory distress after cesarean section delivery. A large radio-opaque mass was detected in the right hemithorax and resected by right bilobectomy. The tumor mass, confined to the lung, was composed of interlacing fascicles of plump spindle cells showing myofibroblastic differentiation and complex cytogenetic abnormalities. Though sarcomatous in appearance, with highly cellular areas and numerous mitoses, there has been neither tumor recurrence nor metastases. The patient remains alive and well 1 year after surgery. Review of the few other reported cases confirms the uniformly benign behavior of this tumor.

Cytogenetic analysis of 11 squamous cell carcinomas of the head and neck.

We report clonal chromosomal abnormalities in short-term cultures of 11 squamous cell carcinomas of the head and neck. Recurrent deletions were seen at 1p13 (four cases), 6q15q26 (three cases), 6q21q25 (two cases), 12p11.2 (three cases), and 3p13-p23 (three cases). Structural aberrations affecting chromosome 11 with different breakpoints were seen in 7 of 11 tumors. Cytogenetic evidence for gene amplification in the form of homogeneously staining region (hsr) was seen in three tumors (two at 11q13). The results of this study shows that the 1p13, 3p13-p23, 6q15-q26, 6q21-q25, and 12p11.2 were frequently deleted in squamous cell carcinomas of the head and neck.

Consistent chromosomal losses in head and neck squamous cell carcinoma cell lines.

Clonal chromosomal abnormalities were characterized in nine cell lines established from squamous cell carcinomas of the head and neck. Aneuploidy was a common feature; one cell line was near-diploid, three were near-triploid, four were near-tetraploid, and one cell line showed extensive variation in chromosome numbers. Consistent numerical abnormalities included loss of the sex chromosomes in six cell lines, losses of chromosomes 2 and 21 in six and five cell lines, respectively, and gain of chromosome 20 in five cell lines. Recurrent structural rearrangements included del(10)(q22-q26) (seven cell lines), i(5)(p10) (six cell lines), i(8)(q10) (six cell lines), add(19)(q13) (six cell lines), del(4)(q21-q31.3) (five cell lines), i(3)(q10) (four cell lines), del(12)(p11.1-p12) (four cell lines), and add (18)(q21-q23) (four cell lines). Other changes were noted in lower frequencies. Loss of specific regions on chromosomes 2, 3p, 4q, 5q, 8p, 10q, 12p, 18q, 19q, and 21 suggests that they may represent sites of putative tumor suppressor genes, loss of which may play a role in the pathogenesis of squamous cell carcinomas of the head and neck. Alternatively, gain of chromosomal region 3q, 5p, and 8q due to isochromosome formation suggests that more than one mechanism is involved in malignant transformation. Cytogenetic evidence of gene amplification was found in two cell lines; as an hsr(11)(q13) in one and as dmins in the other. The clonal karyotypes of four cell lines were compared with those of their respective primary tumors. In all cell lines, clonal evolution had occurred, with loss of some rearrangements present in the primary tumors or the gain of additional abnormalities.

Genetic changes in epithelial solid neoplasia.

Although chromosomal analysis of solid epithelial neoplasms has lagged behind that of hematopoietic, mesenchymal, and germ cell tumors, gradual accumulation of data over the past 5 years enables development of a general view. Thus, these tumors appear to be characterized by a set of nonrandom deletions the incidence of which varies in tumors of different histological types. Most tumors were studied at advanced stages; therefore, essentially no data are available on the cytogenetic characteristics of the earliest stages of tumorigenesis. In contrast to the status of cytogenetic data, a large body of information on deletions at the molecular level assayed by the loss of heterozygosity analysis has accumulated over the same period. These data have been less complete than the cytogenetic data, although in cases such as colorectal carcinoma, genetic changes from the earliest to the most advanced stages have been studied in detail providing a genetic view of progression. Equally important is the fact that deletion mapping studies by the loss of heterozygosity assay directly lead to isolation of a number of tumor suppressor genes. A comparison of the pattern of deletions identified by chromosomal and loss of heterozygosity analysis revealed, as expected, a concordance. Comparison of the patterns of chromosomal (and the underlying molecular) changes in tumors between major embryological cell types demonstrates fundamental differences in genetic mechanisms which lead to tumorigenesis.

Chromosomal aberrations in soft tissue tumors. Relevance to diagnosis, classification, and molecular mechanisms.

In recent years, significant progress has been made in identifying characteristic chromosomal rearrangements associated with several solid tumor types, notably sarcomas, a relatively rare subset of human cancer. Most sarcomas analyzed have been found to be characterized by recurrent chromosome translocations that are specific to histological types. We have reviewed published reports of chromosomal aberrations in benign and malignant soft tissue tumors and found an incidence of specific translocations in these neoplasms that ranged from 20% to 93% within histological tumor types. Identification of recurrent chromosomal abnormalities in benign tumors has resulted in a reappraisal of the general concept that benign tumors have a normal (diploid) chromosome constitution. The variety of recurrent changes present in the different tumor types attests to the cytogenetic diversity inherent in these tumors. The chromosomal rearrangements in each of the tumor types were unique and did not correspond to cancer-associated aberrations known from other solid or hematopoietic malignancies. Cytogenetics thus provides an essential adjunct to diagnostic surgical pathology in the case of malignant soft tissue tumors, which often present substantial diagnostic challenges. In addition, it represents another approach to determine the histogenetic origin of some tumors and identifies sites of gene deregulation for molecular analysis. Indeed, recent molecular analyses of several sarcoma-associated translocations have identified novel genes and novel mechanisms of their dysregulation.

A recurring translocation, t(11;22)(p13;q11.2), characterizes intra-abdominal desmoplastic small round-cell tumors.

We report the cytogenetic analysis of two cases of intra-abdominal desmoplastic small round-cell tumor, one of which had a t(11;22)(p13;q11.2) translocation. The same translocation has previously been reported in two other cases analyzed cytogenetically, indicating that it could be a consistent abnormality characteristic of this rare tumor entity.

Chromosomal abnormalities in leiomyosarcomas.

Thirty-eight tumors from 30 patients diagnosed as leiomyosarcoma were cytogenetically assessed after short term culture. The specimens were obtained from the retroperitoneum, gastrointestinal tract, and extremities. Chromosomal abnormalities were present in 18 tumors from 13 patients; 15 tumors had clonal changes, whereas 3 tumors had numerous nonclonal changes. Ten tumors from 10 patients had normal karyotypes and no results were obtained in 10 other tumors from 7 patients. Of the tumors with clonal chromosomal aberrations, 4 had near-diploid (3 hypo- and one hyperdiploid) modes, 8 were polyploid, and 3 were bimodal. No specific karyotypic change appeared to characterize the leiomyosarcomas, although involvement of some chromosomes appeared more frequent than others. A comparison of our findings with those reported in the literature revealed certain consistent structural rearrangements involving chromosomes 1, 7, 10, 13, and 14 at bands 1p36, 1p32, 1p13, 1q32, 7p11.1-p21, 7q32, 10q22, 13q14, and 14p11, respectively. Other bands less frequently rearranged were 3p13-p22, 3q21, 4q13-q23, 6q15-q21, 7q11.2-q22, 12q13-q14, 17q12-q25, 19q13.3-q13.4, and 20q12-q13.1. Numerical changes included recurrent loss of chromosomes 4, 9, 14, 15, 16, 18, 21, and 22. Identification of the abnormalities of these chromosomes is important in that it may predict the existence of oncogenes, tumor suppressor genes, and/or growth factor genes at these sites. Subsequent molecular analysis might then lead to the identification of the genes involved and ultimately to a better understanding of the pathogenesis of leiomyosarcomas.

t(12;22)(q13;q13) and trisomy 8 are nonrandom aberrations in clear-cell sarcoma.

We report a case of clear-cell sarcoma with a t(12;22)(q13;q13) and multiple copies of chromosome 8 in addition to other abnormalities. An identical or similar translocation has previously been reported in this type of tumor, suggesting that the t(12;22) is a primary cytogenetic change in the pathogenesis of a subset of clear-cell sarcomas. In addition, the presence of extra copies of chromosome 8, commonly noted in our case and others, suggests that it represents a nonrandom secondary change in these tumors.

Cytogenetic characterisation of small round cell tumours using fine needle aspiration.

Cytogenetic analysis of short term cultures of fine needle aspirates from two tumours, characterised cytologically as small round cell neoplasms, showed specific clonal chromosomal abnormalities. In both cases the cytogenetic finding of a t(11; 22) (q24; q12) helped determine their diagnosis as peripheral neuroectodermal tumours of the thoraco-pulmonary region (Askin's tumour). These findings suggest that cytogenetics can reliably distinguish neoplasms which present as undifferentiated small round cell tumours.

Cytogenetic findings in liposarcoma correlate with histopathologic subtypes.

The cytogenetic findings in 31 liposarcomas from 26 patients are reported. Four other tumors did not grow. Three histologic types are represented in this analysis. The well-differentiated liposarcomas were characterized by telomeric associations, large marker chromosomes and ring chromosomes, and in some cases, double minutes. The pleomorphic liposarcomas contained very high clonal chromosomal numbers with near-tetraploid modes and numerous variable, often unidentifiable, chromosomal abnormalities. The myxoid liposarcomas were characterized primarily by a t(12;16)(q13;p11) as the sole abnormality or additional changes. These results indicate that cytogenetic findings may provide a new criterion, not only for establishing the diagnosis of liposarcoma, but also for differentiating confusing histologic types of liposarcoma and these lesions from other types of sarcomas.

Complex karyotypic aberrations, including i(12p), in malignant mixed mullerian tumor of uterus.

We report the cytogenetic findings in a malignant mixed mullerian tumor of the uterus in a 59-year-old woman. Karyotypic analysis of short-term cultures revealed two abnormal clones of cells characterized by extensive structural and numerical rearrangements. An i(12p) maker chromosome was present in addition to other changes in both clones. This marker, characteristically associated with testicular germ cell tumors in males, has recently been reported in ovarian germ cell tumors, a mediastinal germ cell tumor and in a mixed mullerian tumor of the ovary.

Herpes simplex virus and human papillomavirus sites correlate with chromosomal breakpoints in human cervical carcinoma.

The distribution of 1,912 breakpoints observed in a series of 148 cervical cancers was analyzed. Fifty bands were shown to be nonrandomly involved in chromosome structural rearrangements. One hundred thirty-three breaks were noted in bands known to contain a human papillomavirus integration site, and 454 breaks were noted in bands containing a herpes simplex virus breakage site. We suggest that herpes simplex viruses and, possibly, papillomaviruses play an important role in the carcinogenesis and/or development of cytogenetic abnormalities in cervical cancers.

Cytogenetic findings in a malignant fibrous histiocytoma of the gallbladder.

We report the cytogenetic findings in a rare tumor, a malignant fibrous histiocytoma of the gallbladder. Four related clones, two near-diploid and two near-tetraploid, which appeared to have been formed by a doubling of the near-diploid clones, were present. Numerous structural and numerical abnormalities characterized the tumor. Structural rearrangements included reciprocal translocations, translocations of unidentified material onto chromosomes, and deletions. Chromosomes involved in the rearrangements included 1, 3, 10, 12, 14, 16, and 19. Numerical changes included trisomy of chromosomes 2, 8, 10, and 20. Double minute chromatin bodies ranging in number from 5 to several were present in over a third of the cells.