Numerous colonic adenomas in an individual with Bloom's syndrome.

Bloom's syndrome (BS) is a rare recessive disorder caused by germline mutation of the BLM gene. Individuals with BS manifest growth retardation, immunodeficiency, and a predisposition to cancer. In this report, we describe an individual with BS and multiple colonic adenomas reminiscent of familial adenomatous polyposis coli (FAP). Molecular studies revealed APC mutations in 4 of 6 adenomas, including 2 adenomas with the identical APC mutation and microsatellite instability in 1 of 6 adenomas. These results demonstrate similar pathways to colorectal neoplasia in BS as in the normal population and suggest that individuals with BS may be particularly susceptible to colorectal neoplasia.

Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells.

Rearrangements involving the RET gene are common in radiation-associated papillary thyroid cancer (PTC). The RET/PTC1 type of rearrangement is an inversion of chromosome 10 mediated by illegitimate recombination between the RET and the H4 genes, which are 30 megabases apart. Here we ask whether despite the great linear distance between them, RET and H4 recombination might be promoted by their proximity in the nucleus. We used two-color fluorescence in situ hybridization and three-dimensional microscopy to map the positions of the RET and H4 loci within interphase nuclei. At least one pair of RET and H4 was juxtaposed in 35% of normal human thyroid cells and in 21% of peripheral blood lymphocytes, but only in 6% of normal mammary epithelial cells. Spatial contiguity of RET and H4 may provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double-strand break in each gene at the same site in the nucleus.

Phase II trial of menogaril in patients with previously treated multiple myeloma or chronic lymphocytic leukemia.

Menogaril is a semisynthetic anthracycline with relative lack of cardiotoxicity. Ten patients with multiple myeloma (MM), seven patients with chronic lymphocytic leukemia (CLL), and one patient with diffuse well-differentiated lymphocytic lymphoma (DWDL) were treated with menogaril, 160 mg/m2 (for MM) or 200 mg/m2 (for CLL/DWDL), given as a 2-hour intravenous infusion, repeated every 28 days. All patients except one with CLL had been previously treated with one chemotherapy regimen and had either not responded or had relapsed after a response to prior treatment. There were no objective responses to treatment. Among the six evaluable patients with MM, two had stable disease with subjective improvement in symptoms for five to 25 cycles, and among the eight patients with CLL/DWDL, five patients remained stable for two to eight cycles on treatment. The remainder of the patients had progressive disease after one to two cycles of chemotherapy. Five grade 4 hematologic toxicities were observed. There was one fatal neutropenic sepsis. Menogaril, as administered in this study, does not appear to have significant activity in patients with previously treated MM or CLL.

Hyalinizing spindle cell tumor with giant rosettes--a soft tissue tumor with mesenchymal and neuroendocrine features. An immunohistochemical, ultrastructural, and cytogenetic analysis.

CONTEXT: Hyalinizing spindle cell tumor with giant rosettes is a recently described biphasic neoplasm of soft tissues that shares mesenchymal and neuroendocrine features. Its morphologic structure is distinctive, with the presence of hyalinized paucicellular foci that are termed rosettes. The cells around the latter display positive immunoreactivity for neuroendocrine markers. The small number of cases described to date indicates that they tend to be localized in the extremities. OBJECTIVE: To describe the clinicopathologic features of 2 unusual cases of hyalinizing spindle cell tumor with giant rosettes. METHODS AND RESULTS: One tumor was located in the prestyloid parapharyngeal space and the second in the left thigh. Both tumors were well circumscribed and surrounded by a thin capsule-like fibrous band without infiltrating projections. The rosettes were embedded in a spindle cell proliferation. Immunohistochemical stains showed positive results for S100 protein, synaptophysin, CD57, protein gene product 9.5, and neuron-specific enolase exclusively in the cells palisading the rosettes. These markers were negative in the spindle cell portions of the tumor. The latter were immunoreactive for factor XIIIa, vimentin, HAM56, collagen IV, and CD68. Vimentin was the only marker shared by the rosette-forming cells and the spindle cells. Ultrastructurally, the rosette-forming cells contained neurosecretory granules. This study describes the first cytogenetic analysis in this type of tumor revealing 2 cell lines, both containing a balanced translocation between chromosomes 7 and 16. Follow-up of the patients at 16 and 8 months did not disclose evidence of recurrence. CONCLUSIONS: These 2 new cases increase the awareness of hyalinizing spindle cell tumor with giant rosettes and demonstrate that it is a spindle cell neoplasm of unique cytogenetic rearrangements composed of dendritic, histiocytic, and fibroblastic cells admixed with cells that have neuroendocrine differentiation.

Proliferation-associated SNF2-like gene (PASG): a SNF2 family member altered in leukemia.

To identify genes involved in cell growth and/or apoptosis in leukemia, differential display was used to identify mRNAs that showed altered expression levels after cytokine withdrawal from the cytokine-dependent MO7e cell line. Sequence analysis of one transcript that showed a profound decrease in expression after cytokine withdrawal revealed it to be a member of the SNF2 family of chromatin remodeling ATPases. This cDNA had a 2514-nucleotide (838-amino acid) open reading frame and encoded an additional 230 amino acids at the NH2 terminus compared with the murine homologue, lsh, and the human counterpart, Hells. This gene locus has been designated SMARCA6 (SWI/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 6). The highest levels of mRNA expression in humans are observed in proliferative tissues such as the thymus, testis, and bone marrow. Whereas cytokine withdrawal in MO7e cells leads to apoptosis and decreased mRNA expression, growth arrest without the induction of apoptosis of MO7e cells also leads to down-regulation of mRNA expression, suggesting an association with cell proliferation and not suppression of apoptosis. Nuclear localization of this SNF2-like putative helicase is dependent on a nuclear localization sequence located in the NH2-terminal region. Based on sequence homology to other SNF2-like helicases, the pattern of tissue expression, and the association of expression with cell proliferation, we refer to the protein product as proliferation-associated SNF2-like gene product [PASG (D. W. Lee et al., Blood, 94: 594a, 1999)]. Examination of acute myelogenous leukemia and acute lymphoblastic leukemia samples revealed a high frequency of a PASG transcript containing an in-frame 75-nucleotide deletion, which codes for a conserved motif known to be critical for the transactivation activity of a related yeast SWI/SNF polypeptide. These results extend our knowledge of this SNF2-like family member and suggest a role for PASG in leukemogenesis.

Diagnostic analysis of the Rubinstein-Taybi syndrome: five cosmids should be used for microdeletion detection and low number of protein truncating mutations.

Rubinstein-Taybi syndrome (RTS) is a malformation syndrome characterised by facial abnormalities, broad thumbs, broad big toes, and mental retardation. In a subset of RTS patients, microdeletions, translocations, and inversions involving chromosome band 16p13.3 can be detected. We have previously shown that disruption of the human CREB binding protein (CREBBP or CBP) gene, either by these gross chromosomal rearrangements or by point mutations, leads to RTS. CBP is a large nuclear protein involved in transcription regulation, chromatin remodelling, and the integration of several different signal transduction pathways. Here we report diagnostic analysis of CBP in 194 RTS patients, divided into several subsets. In one case the mother is also suspect of having RTS. Analyses of the entire CBP gene by the protein truncation test showed 4/37 truncating mutations. Two point mutations, one 11 bp deletion, and one mutation affecting the splicing of the second exon were detected by subsequent sequencing. Screening the CBP gene for larger deletions, by using different cosmid probes in FISH, showed 14/171 microdeletions. Using five cosmid probes that contain the entire gene, we found 8/89 microdeletions of which 4/8 were 5' or interstitial. This last subset of microdeletions would not have been detected using the commonly used 3' probe RT1, showing the necessity of using all five probes.

A multicenter investigation with D-FISH BCR/ABL1 probes.

Twenty-eight laboratories evaluated a new fluorescence in situ hybridization (FISH) strategy for chronic myeloid leukemia. In a three-part study, bcr/abl1 D-FISH probes were used to study bone marrow specimens. First, laboratories familiarized themselves with the strategy by applying it to known normal and abnormal specimens. Then, collectively the laboratories studied 20 normal and 20 abnormal specimens blindly and measured workload. Finally, each laboratory and two experts studied six serial dilutions with 98-0% abnormal nuclei. Using the reported normal cutoff of < 1% abnormal nuclei, participants reported no false-negative cases and 15 false-positive cases (1-6.6% abnormal nuclei). Results provided by participants for serial dilutions approximated the expected percentages of abnormal nuclei, but those from the experts exhibited greater precision. The clinical sensitivity, precision, nomenclature, workload, recommendations for training, and quality assurance in methods using D-FISH in clinical practice are discussed.

Variation in microdeletions of the cyclic AMP-responsive element-binding protein gene at chromosome band 16p13.3 in the Rubinstein-Taybi syndrome.

Most reported microdeletions of the CREB-binding protein (CBP) gene in the Rubinstein-Taybi syndrome (RTS) were detected by fluorescence in situ hybridization (FISH) with a single cosmid probe specific to the 3' region of the gene. In order to test the hypothesis that the rate of microdeletion-positive cases would be greater if the entire gene was evaluated, we performed FISH on 66 patients with an established diagnosis of RTS, using a panel of five cosmids that span the CBP gene. Five of 66 patients had deletions by FISH (9%), consistent with those rates reported in various series that ranged between 3-25%. Among our cases, different deletions were observed; one was deleted for the 5' but not the 3' region of the CBP gene (case 055). Other deletions included a total CBP deletion extending from the 5' through the 3' region (case 017), a deletion of all but the 5' region (cases 006 and 060), and an interstitial deletion in the 3' region (case 028). Fine breakpoint mapping with additional cosmid and yeast artificial chromosome (YAC) constructs was performed on these patients. The findings of a partial 5' deletion and of interstitial deletions of the CBP gene add to the known spectrum of mutations of this gene in RTS and demonstrate the need for evaluation of the entire CBP gene region for deletions rather than only the 3' region in RTS patients. These results further suggest that the true rate of microdeletion across the CBP gene detectable by FISH has yet to be established firmly. No phenotypic differences between partial deletion, complete deletion, and nondeletion patients were observed, supporting a haploinsufficiency model for RSTS.

45,X/46,X,r(Y) karyotype transmitted by father to son after intracytoplasmic sperm injection for oligospermia. A case report.

BACKGROUND: The advent of assisted reproductive techniques, such as intracytoplasmic sperm injection (ICSI), has permitted conception and successful pregnancy for an increasing population of infertile men. Approximately 13.7% of infertile men with aspermia and 4.6% with oligospermia have a coexistent chromosome abnormality. Although the ICSI procedure appears safe thus far, early studies are in progress to evaluate outcomes of such pregnancies. For men whose infertility is linked to genetic conditions, it is an unprecedented challenge to predict the potential effects on their offspring. CASE: At 18 weeks' gestation, a 45,X/46,X,r(Y) karyotype was found on genetic amniocentesis performed for advanced maternal age. The pregnancy was achieved by ICSI using sperm from the husband, who was infertile due to severe oligospermia. Subsequently the same karyotype was found in the father. To our knowledge, this is the first reported case of familial transmission of ring Y chromosome. CONCLUSION: It is strongly recommended that ICSI and other new assisted reproductive techniques be preceded by genetic screening for male infertility as well as other indications warranted by the family history since traditional risk assessment may require revision and outcomes may be uncertain in some cases.

Cytogenetic analyses of 85 testicular germ cell tumors: comparison of postchemotherapy and untreated tumors.

Cytogenetic analyses of 85 testicular germ cell tumors, of which 54 were karyotypically abnormal, showed recurrent breakpoints at chromosome bands 1p36, 1p13-1qh, 11q23, 19q13, and the pericentromeric regions of the acrocentric chromosomes. Postchemotherapy tumors had significantly more rearrangements of bands 3p25-p26, 6q16-q21, 8p22-p23 when compared with untreated tumors, while untreated tumors had more rearrangements of 9p22-p24 when compared with postchemotherapy tumors. Frequent breakpoints also were identified at 15q15 and 9qh in untreated tumors. Tumors of different histopathology, clinical stage, and treatment status showed no significant differences in the frequencies of i(12p)-positive and i(12p)-negative tumors.

Third report of t(19q)(13.4) in mesenchymal hamartoma of liver with comments on link to embryonal sarcoma.

We report the third known case of mesenchymal hamartoma of the liver (MHL) with a balanced translocation involving a common breakpoint, 19q13.4. A common clonal chromosome abnormality appears to characterize an important subset of MHL, some of which may be low-grade neoplasms. We found no consistent karyotype abnormality in a post-treatment sample of embryonal sarcoma of the liver (ESL). Reports of coexistent MHL and ESL in two patients and detection of 19q abnormalities in two ESLs appear to support Stocker's hypothesis of a histogenetic link between these two rare liver lesions. More data are needed to clarify this relationship. It is possible that MHLs are etiologically heterogenous and may be developmental disorders, disruptions, or neoplasms.

Characterization of multiple 12p rearrangements in testicular germ cell tumor cell line 833K and its subclone 64CP by chromosome microdissection.

Rearrangements of chromosome arm 12p are known to be common in germ cell tumors (GCT). Previous studies, using fluorescence in situ hybridization (FISH) with a whole chromosome 12 painting probe, showed unusual distributions of chromosome 12-derived chromatin in GCT cell line 833K and its cisplatin-resistant subclone, 64CP, located next to AgNOR (silver staining nucleolus organizer regions), some of which were ectopic. In this study, the ectopic stalk regions were shown by FISH to be composed of 18s and 28s rDNA, but were flanked by beta-satellite DNA, which may form a barrier around the rDNA. In order to determine the specific origins of the rearranged chromosome 12 segments, three different derived chromosome 12 regions were isolated from 64CP, using chromosomal microdissection. The microdissected fragments were labeled and hybridized by FISH to normal human chromosomes. All three segments localized to distal 12p; 12p12-->12pter, but with apparently different breakpoints for each segment. Furthermore, three-color FISH experiments with 12p band-specific probes demonstrated that the derivative chromosome 12 regions in 833K also originate from distal 12p (12p12-->p13). These sequences now can be evaluated for degree of overlap or common breakpoints which may be of significance in the development or progression of GCT.

An assessment of two training interventions designed to increase the knowledge of obstetrical nurses and nurse-midwives about the maternal serum triple screen.

The purpose of this study was to assess the effects of two brief training interventions designed to improve nurses' and nurse-midwives' knowledge about the maternal serum triple screen. The low intervention consisted of written information on the triple screen; the high intervention consisted of written information plus a one hour oral presentation. Knowledge was assessed at baseline, immediately following the oral presentation (high intervention only), and one month following the interventions. Forty-seven nurses, nurse-midwives and nursing assistants participated. Sixteen respondents (34 per cent) who routinely talk to patients about the triple screen obtained a score of less than 70 per cent on the knowledge questionnaire at baseline assessment. Respondents' knowledge about the maternal serum triple screen included areas that needed to be improved in order for them to be able to provide patients with accurate and complete information. Both interventions assessed in this study resulted in an increase in participants' knowledge about the maternal serum triple screen, however the high intervention was more effective. This study presents evidence that improvements in health care professionals' knowledge can be made with brief educational interventions.

Interphase chromosome painting of paraffin-embedded tissue in the differential diagnosis of possible germ cell tumors.

Germ cell tumors (GCTs) are the most frequent cancer in men aged 15 to 34 years. These tumors are highly responsive to therapy with platinum-containing regimens, and 80% of cases so treated can be considered cured. Cytogenetically, 80% of GCTs have an i(12p) regardless of tumor site or histopathology, and those that are i(12p) negative have other manifestations of 12p amplification. GCTs occasionally arise extragonadally, and such cases can be especially difficult to distinguish from poorly differentiated somatic carcinomas, a situation that poses a diagnostic and treatment dilemma We developed a technique for two-color fluorescence in situ hybridization chromosome painting on nuclei released from paraffin-embedded sections. In four tumors for which GCT was a differential diagnosis, we examined the 12p and 12q chromosome arm distributions by this technique. By use of 12p and 12q painting probes developed by microdissection, 12p and 12q were distinguished and their relative distributions evaluated. In each of the four cases, 12p regions seemed to be rearranged and over-represented relative to 12q regions. In three of the cases, an apparent i(12p) could be identified. These results support a diagnosis of GCT or GCT origin in these four cases. In tumors for which specific cytogenetic abnormalities are known, chromosome painting by fluorescence in situ hybridization using paraffin-embedded tissue is a useful technique to aid in the diagnosis of tumors that are difficult to differentiate. The patients can then be placed on treatment regimens appropriate for their specific tumor type.

Genomic structure and chromosome location of the murine PDE1B phosphodiesterase gene.

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby participating in regulation of the intracellular concentrations of these second messengers. The PDE1 family is defined by regulation of activity by calcium and calmodulin. We have cloned and characterized the mouse PDE1B gene, which encodes the 63-kDa calcium/calmodulin-dependent PDE (CaM-PDE), an isozyme that is expressed in the CNS in the olfactory tract, dentate gyrus, and striatum and may participate in learning, memory, and regulation of phosphorylation of DARPP-32 in dopaminergic neurons. We screened an I-129/SvJ mouse genomic library and identified exons 2-13 of the PDE1B gene that span 8.4 kb of genomic DNA. Exons range from 67 to 205 nucleotides and introns from 91 to 2250 nucleotides in length. Exon 1 was not present in the 3 kb of genomic DNA 5' to exon 2 in our clones. The mouse PDE1B gene shares many similar or identical exon boundaries as well as considerable sequence identity with the rat PDE4B and PDE4D genes and the Drosophila dunce cAMP-specific PDE gene dnc, suggesting that these genes all arose from a common ancestor. Using fluorescence in situ hybridization, we localized the PDE1B gene to the distal tip of mouse Chromosome (Chr) 15.

Partial trisomy 1q with growth hormone deficiency and normal intelligence.

We present two sibs with partial trisomy 1 (q31.1-q32.1) due to a familial insertion. Patient 1 is a girl who presented at age 9 months with minor anomalies, short stature, and normal psychomotor development. Karyotype was 46,XX,der(4)ins(4;1) (p14;q31.1q32.1)pat. The father had a balanced inverted insertion of 1q into 4p, with karyotype 46,XY,ins(4;1)(p14;q31.1q32.1). At age 5 years, patient 1 was found to have short stature with documented growth hormone deficiency and ectopic pituitary. Her growth velocity responded well to treatment with growth hormone. Cognitive testing at 5 9/12 years showed normal intelligence with an IQ of 90. Patient 2, the brother of patient 1, presented with intrauterine growth retardation. He has the same chromosomal insertion as his sister, with partial trisomy 1q. We suggest that there is a recognizable phenotype of trisomy 1(q31.1-q32.1) which includes prenatal and postnatal growth retardation, narrow palpebral fissures, microphthalmia, microstomia, pituitary abnormalities, and normal intelligence in some individuals.

A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes.

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98-99% XY with no XX cells, and 97-98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96-97% XX with 0.03% XY cells, and 94-96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service.

Fluorescence in situ hybridization on nuclei from paraffin-embedded tissue in low stage pure embryonal carcinoma of the testis.

Approximately 30% of patients who present with clinical stage A nonseminomatous testis cancer are in fact pathologic stage B. In previous studies an increasing volume of embryonal carcinoma in the orchiectomy specimen was associated with a higher likelihood of being pathologic stage B. However, not all patients with pure embryonal carcinoma in the primary tumor were pathologic stage B. In an effort to discriminate patients with pure embryonal carcinoma in the testicular specimen relative to pathologic stage, archival specimens from patients presenting with clinical stage A pure embryonal carcinoma were examined by fluorescence in situ hybridization (FISH) with newly developed probes for chromosome arms 12p and 12q. Whole nuclei from archival material from 14 patients (six pathologic stage A, seven pathologic stage B and one stage C) with 100% embryonal carcinoma in the orchiectomy specimen were studied using bicolor FISH with chromosome arm 12p- and 12q-specific painting probes developed by chromosome microdissection. In all cases a blinded analysis showed distinct regions of 12p and 12q probe hybridization simultaneously and allowed identification of probable normal chromosomes 12, as well as regions of amplification of 12p sequences, including possible i(12p). In 5/14 specimens, a distinct and peculiar pattern of 12p hybridization was observed which resembled 12p "disarray" or "multifocal 12p". Of the five specimens demonstrating multifocal 12p, four were pathologic stage B, while one was pathologic stage A. Whether the trend toward multifocal 12p predicts metastatic potential in primary testicular embryonal carcinoma will need to be assessed using a larger series of patients.