Charles P. Harris

A Juvenile Polyposis Tumor Suppressor Locus at 10q22 Is Deleted From Nonepithelial Cells in the Lamina Propria

BACKGROUND & AIMS Juvenile polyps are characterized by an abundant lamina propria that lacks smooth muscle and may contain cystically dilated glands, with epithelium that seems normal and is nondysplastic. Rarely, an autosomal dominant inheritance pattern occurs. The aim of this study was to test the hypothesis that the genetic defect in both sporadic juvenile polyps and hereditary juvenile polyposis involves loss of function for a tumor suppressor gene. METHODS Allelic losses were detected by comparing normal DNA with tumor DNA from a series of 47 juvenile polyps from 16 patients using polymerase chain reaction amplification of microsatellite markers and fluorescent in situ hybridization (FISH). RESULTS Somatic deletions at 10q22 were detected in 39 of 47 juvenile polyps (83%) from 16 unrelated patients with either hereditary or sporadic juvenile polyps, and the minimum overlap localized juvenile polyposis coli to the 3-cM interval D10S219-D10S1696. Fluorescent in situ hybridization shows that the cells affected by deletion mutation reside exclusively in the lamina propria, not in the epithelium. CONCLUSIONS The location of a novel tumor suppressor gene on chromosome 10 that is affected by deletion mutation in the majority of juvenile polyps was mapped. Unlike adenomas and carcinomas of the colonic epithelium, juvenile polyps originate in the lamina propria.

Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma

BackgroundCarcinoma of uterine cervix is the second most common cancers among women worldwide. Combined radiation and chemotherapy is the choice of treatment for advanced stages of the disease. The prognosis is poor, with a five-year survival rate ranging from about 20–65%, depending on stage of the disease. Therefore, genetic characterization is essential for understanding the biology and clinical heterogeneity in cervical cancer (CC).MethodsWe used a genome-wide screening method – comparative genomic hybridization (CGH) to identify DNA copy number changes in 77 patients with cervical cancer. We applied categorical and survival analyses to analyze whether chromosomal changes were related to clinico-pathologic characteristics and patients survival.ResultsThe CGH analysis revealed a loss of 2q33-q37 (57.1%), gain of 3q (54.5%) and chromosomal amplifications (20.77%) as frequent genetic changes. A total of 15 amplified chromosomal sites were detected in 16 cases that include 1p31, 2q32, 7q22, 8q21.2-q24, 9p22, 10q21, 10q24, 11q13, 11q21, 12q15, 14q12, 17p11.2, 17q22, 18p11.2, and 19q13.1. Recurrent amplified sites were noted at 11q13, 11q21, and 19q13.1. The genomic alterations were further evaluated for prognostic significance in CC patients, and we did not find any correlation with a number of clinical or histological parameters. The tumors harboring HPV18 exhibited higher genomic instability compared to tumors with HPV 16.ConclusionsThis study demonstrated that 2q33-q37 deletions, 3q gains and chromosomal amplifications as characteristic changes in invasive CC. These genetic alterations will aid in the identification of novel tumor suppressor gene(s) at 2q33-q37 and oncogenes at amplified chromosomal sites. Molecular characterization of these chromosomal changes utilizing the current genomic technologies will provide new insights into the biology and clinical behavior of CC.

Deletion of 1p32-p36 Is the Most Frequent Genetic Change and Poor Prognostic Marker in Adenoid Cystic Carcinoma of the Salivary Glands

Purpose: Adenoid cystic carcinoma (ACC) is a relatively uncommon salivary gland malignancy known for its protean phenotypic features and pernicious clinical behavior. Currently, no effective therapy is available for patients with advanced nonresectable, recurrent, and/or metastatic disease. The purpose of this study is to identify prognostic factors other than tumor stage that can be used to predict the outcome of the patients with ACC. Experimental Design: We used comparative genomic hybridization (CGH) to identify copy number aberrations in 53 primary ACCs. Array CGH and fluorescence in situ hybridization analysis was used to validate CGH results on selected cases. We correlated these copy number aberrations with clinicopathologic factors using Pearsons χ2 or by the two-tailed Fisher exact test. The disease-specific survival and disease-free intervals were generated by the Kaplan-Meier product limit method. Results: Chromosomal losses (n = 134) were more frequent than gains (n = 74). The most frequent genetic change was the loss of 1p32-p36 in 44% of the cases followed by 6q23-q27, and 12q12-q14. The most frequently gained chromosomal regions were 8 and 18. Of the chromosomal aberrations, loss of 1p32-p36 was the only abnormality significantly associated with patients outcome. Conclusions: This study, for the first time, identifies loss of 1p32-p36 as a significant aberration in ACC. Molecular characterization of 1p32-36 region using the available genomic technologies may lead to the identification of new genes critical to the development of novel therapeutic targets for this disease copy number aberration.

Heteropagus conjoined twins due to fusion of two embryos: Report and review

We report on a case of conjoined twinning (CT) consistent with fusion of two embryos followed by resorption of the cranial half of one of them, resulting in a normal male baby with the lower half of a male parasitic twin fused to his chest. Fluorescent in situ hybridization (FISH) studies suggested that the parasitic twin was male, and DNA typing studies demonstrated dizygosity. Although incomplete fission is the usual explanation for conjoined twins, the unusual perpendicular orientation of the parasite to the autosite supports a mechanism observed in mares in which early fusion of two embryos is followed by resorption due to compromised embryonic polarity.

FISH detection of HER-2/neu oncogene amplification in early onset breast cancer

HER-2/neu (c-erbB-2) gene amplification based on Southern blotting or immunohistochemistry has been shown to be predictive of poor outcome in breast cancer occurring in women over 40, but there is little data on the role of HER-2/neu in young women with breast cancer, many of whom may have inherited BRCA1 or other predisposing genes. The present study used fluorescent in situ hybridization (FISH) on archival specimens of breast cancer from 37 women under the age of 40 to evaluate the role of HER-2/neu amplification in this cohort, and to also evaluate the efficacy of FISH for quantifying amplification. The frequency of primary tumors with a greater than fourfold increase in gene copy number was found to be 38%, which is similar to the frequency of amplification reported in Southern blot studies in older women. However, the greater sensitivity of FISH enabled detection of low level amplification (more than 2 but less than 8 gene copies), which was found in an additional 30% of the tumors. Patients with low level amplification demonstrated a 54% recurrence rate, compared to 86% in those with high amplification and 17% in those with no amplification. HER-2/neu amplification appeared to be more prognostic of recurrence than nodal status, with 45% of node negative tumors recurring compared to 62% of those which were node positive, nor was tumor size predictive of recurrence in this cohort since tumors of 2 cm or less recurred in 44% of cases compared to 57% of those larger than 2 cm. Thus, this study demonstrates that FISH is a reproducible and sensitive technique for detecting HER-2/neu amplification, and that amplification of the oncogene is the strongest independent indicator of recurrence of breast cancer in young women.

Comparison of chromosome aberrations in leiomyoma and leiomyosarcoma using FISH on archival tissues

Fluorescence in situ hybridization (FISH) with chromosome-specific probes was used to study cytogenetic changes in five cases of leiomyosarcoma (LMS) and nine cases of uterine leiomyoma (LM). Biotinylated DNA probes for the centromeric regions of chromosomes 1, 6, 8, 9, 17, and 18, painting probes for chromosomes 1 and 22, and the cosmid probe for chromosome region 21q22.3 were used on nuclei isolated from paraffin blocks. Four of five LMS cases revealed major chromosomal aberrations, while the only case with minor clonal aberrations was subsequently found not to be a typical LMS. The most common numerical aberrations found in the LMS cases were extra copies of chromosome 8 (three of five cases), loss of chromosome 1 (three of five cases), and loss of chromosome 6 (two of five cases). One of two LMS cases studied with a chromosome 1 painting probe demonstrated translocations of chromosome 1. In contrast to LMS, only five of nine uterine LM cases had abnormal clones, and these were smaller than those in LMS. Two LM cases showed 9% tetrasomy 8 with 17 or 20% monosomy 6, and three other cases had monosomy 6 clones in 18-34% of cells. These results indicate that typical LMS is characterized by multiple chromosomal aberrations affecting most of the cells, whereas borderline LMS and LM have fewer affected chromosomes and less clonal involvement.

Detection of aneuploidy and possible deletion in paraffin-embedded rhabdomyosarcoma cells with FISH

Conventional cytogenetic studies of solid tumors are limited by the difficulty of culturing tumor cells, while in situ hybridization using paraffin sections of interphase cells results in too many truncated cells. To solve these problems, fluorescent in situ hybridization (FISH) technique was used on free nuclei isolated from formalin-fixed paraffin-embedded embryonal rhabdomyosarcoma (RMS) tissue using our modification of Hedleys method for isolation of nuclei. Biotinylated DNA probes for the centromeric regions of chromosomes 6, 8, 11, 12, 17, and 18, painting probes for chromosomes 8 and 11, and a cosmid probe for the HER-2/neu oncogene, were used. The centromeric probes worked well, demonstrating two copies of chromosomes 6, 17, and 18, but three copies of chromosome 11 in 52.9% of nuclei. Four copies of chromosome 8 were observed in 57.1% of nuclei and five or more in 17.1%. Chromosome 12 demonstrated 21.8% trisomy and 62.2% tetrasomy. Painting probes for chromosome 11 also worked well and matched the results of the centromeric probes, with no suggestion of structural aberration. However, the results of the painting probe for chromosome 8 yielded fluorescent areas of different sizes, suggesting that some of the extra chromosomes 8 could be deleted. The cosmid probe for the HER-2/neu oncogene also worked well, and revealed two signals in each nucleus without evidence of amplification. This study illustrates the successful use of a new technique for studying chromosomal aberration in paraffin-embedded solid tumors. The importance of this technique is that it has not been previously possible to use painting probes or cosmid probes on paraffin tissue sections. Use of this procedure will broaden the type of retrospective studies that can be performed to include detection of deletions or translocations.

Identical Cytogenetic Clones and Clonal Evolution in Pediatric Monozygotic Twins With Acute Myeloid Leukemia: Presymptomatic Disease Detection by Interphase Fluorescence In Situ Hybridization and Review of the Literature

Purpose Observation of identical acquired genetic changes in infant monozygotic (MZG) twins with acute leukemia has provided strong evidence for in utero twin-twin transfusion as the cause of concordance. Documentation of similar phenomenon in older MZG twins offers insight into the latency period for leukemia and may provide the opportunity for presymptomatic disease detection in one twin. Design The literature describing leukemia in MZG twins is reviewed and the results of classical and molecular cytogenetic studies of one pair of MZG twins at 3 and 4 years with acute non-lymphocytic leukemia-FAB type Ml are reported. Results The twins studied had cytogenetically identical neo-plastic clones with identical clonal evolution. Retrospective fluorescence in situ hybridization studies demonstrated the presence of the abnormal clone in the asymptomatic twin at the time of bone marrow transplant of the first twin. Conclusions These observations support in utero twin-twin transfer as the origin of leukemic clones in pediatric and infant leukemia, demonstrate that clonal evolution of a leukemic clone may occur years before onset of overt disease, and indicate that knowledge of acquired genetic change(s) in one twin may provide markers to assess disease in the asymptomatic twin.

Use of Fluorescent in Situ Hybridization to Detect Trisomy 13 in Archival Tissues for Cytogenetic Diagnosis

The present report describes the use of molecular probes to investigate the chromosomal constitution of interphase nuclei of formalin-fixed, paraffin-embedded tissue from three infants with multiple congenital malformations and a provisional diagnosis of trisomy 13 in two. Fluorescent in situ hybridization with the probe for the 13 and 21 centromeric regions revealed five nuclear signals in two of the cases, indicating the presence of an extra chromosome, and only four nuclear signals in the other case. Only the two positive cases had phenotypic features consistent with trisomy 13. Routine cytogenetic analysis was performed on one child and confirmed an additional chromosome 13. The child without an extra chromosome had features consistent with Ivemark syndrome. This study demonstrates the utility of fluorescent DNA probes for the retrospective diagnosis of aneuploidies in archival material.

Granulocytic sarcoma presenting as pneumonia in a child with t(8;21) acute myelogenous leukemia: diagnosis by fluorescent in situ hybridization.

Granulocytic sarcoma is a soft tissue collection of leukemic cells. The authors describe a 4-year-old boy with M2 acute myelogenous leukemia (AML) who presented with fever, mild nonproductive cough, and hematemesis. Although he was initially diagnosed with nodular pneumonia, rapid resolution of a pulmonary infiltrate following induction chemotherapy was suggestive of a pulmonary granulocytic sarcoma. Interphase fluorescent in situ hybridization (FISH) of the lung biopsy specimen for the t(8;21)(q22;q22) translocation confirmed the retrospective diagnosis of a well-differentiated pulmonary granulocytic sarcoma. Pulmonary granulocytic sarcomas may be underrecognized in children with AML; this may delay anti-leukemic therapy and may lead to ineffective therapy if misdiagnosed as pneumonia.