Carleton T. Garrett

Cardiac myocytes and dendritic cells harbor human immunodeficiency virus in infected patients with and without cardiac dysfunction: detection by multiplex, nested, polymerase chain reaction in individually microdissected cells from right ventricular endomyocardial biopsy tissue.

Two hundred fifteen patients infected with human immunodeficiency virus (HIV) participated in a prospective longitudinal study of HIV-related heart disease. Evaluation included signal-averaged electrocardiography and echocardiography. Fifteen patients underwent endomyocardial biopsy, 5 had cardiovascular symptoms and 10 did not. Cardiac myocytes or dendritic cells were prepared by individual cell microdissection to sort them from other cell types such as interstitial cells or circulating blood elements. HIV proviral sequences were amplified in samples of 15 to 20 cells of each type by multiplex, nested, polymerase chain reaction and hybridized to 32P-labeled probes specific for regions within the gag and pol genes of HIV-1. The results showed the presence of HIV sequences in myocytes of 2 of 5 patients with cardiac symptoms and in 6 of 10 without. Thus, symptomatic HIV cardiomyopathy did not appear to be a direct consequence of the virus on myocardial cells. In dendritic cells, HIV sequences were detected in 5 of 5 patients with cardiac symptoms and in 8 of 10 with apparently normal ventricular function. Furthermore, dendritic cells were somewhat more numerous in the myocardium of symptomatic than asymptomatic patients. Our studies are the first to directly detect the HIV genome in purified cardiac myocytes from patients with and without cardiac dysfunction. Our findings do not support a direct role of the virus in myocardial dysfunction. However, the results do suggest that the interstitial dendritic cells may be involved in some manner in the development of cardiac dysfunction observed in HIV-infected patients.

Protooncogene amplification and tumor ploidy in human ovarian neoplasms

DNA from 24 ovarian tumors, including 16 carcinomas, was examined for amplification of the proto-oncogenes c-myc, int-2, and rc-erbB-2. All cases of carcinoma were also examined by flow cytometry for DNA ploidy and cell cycle analysis, and eight cases of carcinoma were examined for estrogen and progesterone receptors. Protooncogene amplification was not detected in the DNA of benign ovarian neoplasms, or of ovarian carcinomas with low malignant potential. Amplification of c-myc was detected in six of 12 cases of invasive carcinoma, int-2 amplification was present in one case, and c-erbB-2 amplification was not detected in any case. Among the seven cases evidencing protooncogene amplification, three cases showed aneuploidy in tumor DNA, while four showed diploidy. Two cases which showed aneuploidy in tumor DNA did not demonstrate any degree of protooncogene amplification. Protooncogene amplification was frequently associated with morphologic nuclear anaplasia and high mitotic count. Six of the seven cases demonstrating c-myc or int-2 were of the serous type or showed some degree of serous differentiation, while none of the four cases of purely mucinous carcinoma had evidence of amplification. While the total number of cases in the study was limited, it would appear from the trend demonstrated by the data that protooncogene amplification (particularly c-myc) may be involved in the pathogenesis of aggressive common epithelial tumors of the ovary.

An analysis of abnormalities of the retinoblastoma gene in human ovarian and endometrial carcinoma

The altered expression of the human retinoblastoma (RB) gene has been demonstrated to play an important role in the pathogenesis of RB and other tumors. To determine whether the RB gene might be involved in the pathogenesis of human ovarian and endometrial cancer, DNA from 24 human ovarian tumors, 3 normal ovaries, 3 endometrial carcinomas, and 1 endometrial hyperplasia was examined with an RB complementary DNA probe. Evidence for homozygous deletion of the RB gene was observed in only one specimen. Interestingly, the specimen was an endometrioid tumor of the ovary of low malignant potential (LMP). This patient experienced rapid progression of the tumor and died 8 months after diagnosis. Abnormalities of the RB gene may be involved in the aggressive biologic behavior of certain forms of ovarian carcinoma, particularly those of LMP.

Amplification of the proto-oncogenes int-2, c-erbB-2 and c-myc in human breast cancer

int-2 is a proto-oncogene that is partially homologous to angiogenesis-inducing fibroblast growth factor and is believed to play a role in mouse mammary carcinogenesis. Recent evidence has suggested that this proto-oncogene may also play a role in human breast cancer. In the present study, we used Southern hybridization analysis to examine DNA from 79 primary and 11 recurrent human breast cancers for evidence of activation of int-2 through either gene rearrangement or amplification. A similar analysis was performed for two other proto-oncogenes, c-erbB-2 and c-myc, also suspected of playing a role in the development of human breast cancer. Proto-oncogene status was correlated with estrogen (ER) and progesterone (PR) receptor status, patient age, and lymph node (LN) status at the time of surgery. Gene rearrangement was not a frequent occurrence with any of the proto-oncogenes. However, amplification of int-2 occurred at a significantly higher frequency in recurrent breast cancers than in primary cancers and in patients with primary cancers who were less than or equal to 50 years of age versus greater than 50 years of age at surgery. Although amplification of all three proto-oncogenes occurred at a greater frequency in primary tumors from patients with lymph node metastases than from those without lymph node metastases, a significant difference was noted only in the case of c-myc amplification. These findings confirm and extend earlier results of studies of int-2, c-erbB-2 and c-myc amplification in human breast cancers and point to a role for int-2 activation in certain cases of recurrent breast malignant neoplasia.

Polymerase chain reaction detection of immunoglobulin gene rearrangement and bcl-2 translocation in archival glass slides of cytologic material.

Cytologic evaluation of lymph node fine-needle aspirates and serous effusions is a rapid and useful means for establishing the diagnosis of a variety of lymphoproliferative disorders. However, in some instances, cytologic findings are not sufficient to establish a diagnosis of lymphoma, thus necessitating the use of ancillary procedures, the most frequent of which is immunophenotyping. In this respect, the usefulness of molecular markers, such as clonal immunoglobulin gene rearrangements or chromosomal translocations, have been less well evaluated. Follicular lymphoma constitutes an interesting disease for such a study because these tumors possess characteristic histopathologic features and contain two potential molecular markers, that is, a clonal immunoglobulin gene rearrangement and a bcl-2 gene translocation [t(14;18)]. In the present study, we evaluated, retrospectively, the cytologic material from four lymph node fine-needle aspirates and one pleural effusion of five patients with biopsy-proven follicular lymphoma. In four of the cases, definitive diagnosis of lymphoma had not been possible solely from cytologic evaluation. DNA was isolated from archival air-dried samples present on glass slides and amplified by the polymerase chain reaction (PCR) for detection of either a clonal immunoglobulin heavy chain gene rearrangement or bcl-2 translocation (major breakpoint region). An immunoglobulin heavy chain gene rearrangement was detected in four of five patients, and two patients had the bcl-2 translocation by PCR. The effusion case was identical by gel electrophoresis with product amplified from a lymph node biopsy of the same patient and DNA extracted directly from fresh pleural effusion cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Serous papillary adenocarcinoma of the endometrium. Analysis of proto-oncogene amplification, flow cytometry, estrogen and progesterone receptors, and immunohistochemistry

Primary and metastatic tumor tissues of serous papillary adenocarcinoma of the endometrium were examined for the following: (1) amplification of int‐2, c‐erbB‐2 and c‐myc proto‐oncogenes by Southern blot hybridization; (2) DNA ploidy by flow cytometric study; (3) and expression of specific proteins, such as estrogen and progesterone receptors, keratin, vimentin, and carcinoembryonic antigen (CEA) using immunohistochemical and biochemical techniques. Amplification of c‐myc was observed in the specimens from the endometrium (ten‐fold) and from omental metastasis (five‐fold). Both int‐2 and c‐erbB‐2 amplification were not observed. The tumor showed aneuploidy, with the specimens from the endometrium and omental metastasis exhibiting multiple populations of aneuploid tumor cells. Estrogen and progesterone receptors could not be detected biochemically; however, immunohistochemically, estrogen receptors were observed in tumor cells forming papillary structures but not in the tumor cells of the solid, more poorly differentiated areas. A similar distribution was observed for both low and high molecular weight keratin. The findings of c‐myc amplification and aneuploidy in the serous papillary adenocarcinoma of the endometrium are consistent with its aggressive behavior observed clinically and emphasize the importance of distinguishing this lesion from other types of endometrial carcinoma.

Optimization for detection of cytomegalovirus by the polymerase chain reaction (PCR) in clinical samples

PCR is 100 times more sensitive than traditional tube culture for detecting cytomegalovirus (CMV) but may require up to 12 reactions per specimen (Sandin et al., 1991). In order to make the assay practical for use in a clinical laboratory the procedure used to detect CMV must be simplified. In this study, the effect of reducing the number of reactions per specimen on sensitivity and specificity of the PCR assay was evaluated. 53 residual samples from specimens processed for CMV by shell vial assay/routine tube tissue culture (SVA/TTC) were analyzed by PCR. The residual samples were separated into a supernatant and pellet fractions, then tested for CMV with primers to the immediate early (IEP) and late protein (LP) genes using a nested procedure. To exclude false negatives due to the presence of inhibitors in the sample fractions, all fractions were tested for the presence of the human myosin heavy chain gene also using a nested procedure. SVA/TTC had a sensitivity and specificity of 52/96% in comparison to PCR when data from all 12 PCR reactions was considered. However, high sensitivity and specificity were retained when only the data of the IEP primers with two samples were considered. The results from examining only the 1:10 dilution of pellet and the undiluted supernatant by PCR provided a 60% increase in sensitivity over SVA/TTC, high specificity and a clinically feasible assay.

Human papilloma virus in uterine cervix: a comparison of detection by morphology and by dot-blot hybridization.

Colposcopically directed cervical biopsies, smears, and swabs obtained from 210 women with a previous abnormal cervical cytology were evaluated for the presence of human papilloma virus (HPV) using morphology and dot-blot hybridization. The diagnosis of HPV infection in biopsies and smears examined morphologically was rendered using established criteria for condyloma/cervical intraepithelial neoplasia (CIN). In hybridization studies. DNA was isolated from cells obtained from cervical swabs and annealed with probes that detected HPV types 6/11, 16/18, and 31/33/35 using a dot-blot procedure. Ninety-five cases demonstrated morphologic evidence of condyloma/CIN; 51 of these (54%) were positive for HPV DNA (five cases 6/11, 21 cases 6/18, 20 cases 31/33/35. and five cases two different probes). HPV DNA was also detected in 6 of the 115 cases (5.29?) that were morphologically negative (three cases 16/18, three cases 31/33/35). The results demonstrated that morphology was more sensitive than dot-blot hybridization for detection of HPV-related lesions. The dot-blot hybridization did detect HPV DNA in a small percentage of the cases that showed no morphologic abnormality and was useful for typing of the HPV. At this juncture, however, the clinical significance of the latter findings is unclear.