Integrated morphologic and molecular analysis of Trichomonas vaginalis, Mycoplasma hominis, and human papillomavirus using cytologic smear preparations.

Pathogenic microbes may colonize the female genital tract via sexual transmission and cause health issues like inflammation or malignancy, summarized as sexually transmitted disease (STD). A major representative of such pathogens is Trichomonas vaginalis (T.v.), whose role in the etiology of cervical cancer remains elusive. Traditional morphologic screening of cervical smears is able to detect T.v., although its identification may be complicated by look-alikes such as degenerated granulocytes and basal cells. In addition, the parasite's endosymbiont Mycoplasma hominis (M.h.) cannot be detected in the Pap test. This investigation was aimed at designing a PCR-based method to detect specific pathogenic germs by using cervical cytology slides to overcome morphologic uncertainty and increase diagnostic accuracy. To test our molecular screening method on T.v., M.h., and HPV in archival smears, we elaborated a multiplex PCR approach based on microdissection. This assay was applied to a minute quantity of starting material which harbored or was suspected to harbor T.v.; the resulting isolated DNA was used for subsequent molecular analyses of T.v., M.h., and HPV. We clarified the diagnosis of genital T.v. infection in 88 and 1.8% of morphologically suspicious and T.v.-negative cases, respectively. We also revealed a tendency of M.h. co-infection in high-risk HPV cases. In conclusion, a microdissection-based approach to detect pathogenic microbes such as T.v., HPV, and M.h. is a molecular tool easy to implement and may help to better understand the interactivity of these germs with respect to pathogenesis.

Specific lentiviral shRNA-mediated knockdown of cyclin D1 in mantle cell lymphoma has minimal effects on cell survival and reveals a regulatory circuit with cyclin D2.

Cyclin D1 overexpression is the hallmark of mantle cell lymphoma (MCL). However, the importance of cyclin D1 in the maintenance and progression of the disease remains to be defined. The aim of this study was to elucidate the role of cyclin D1 overexpression using an efficient cyclin D1-shRNA and a lentiviral system in well-characterized MCL cell lines. Surprisingly, the knockdown of cyclin D1 led to a moderate retardation in growth, without induction of apoptosis. The cyclin D1-shRNA-transduced MCL cells showed a 15% shift from S phase to G(1) phase of the cell cycle, a weak induction of p27(Kip1), decreased Rb (Ser807/811) phosphorylation, and a consistent upregulation of cyclin D2 mRNA and protein expression. However, double knockdown of cyclins D1 and D2 did not intensify the effects observed with cyclin D1 knockdown alone. These data suggest that the moderate effects of cyclin D1 downregulation on survival and proliferation are likely the result of compensatory cyclin-independent mechanisms governing proliferation or alternatively, secondary genetic events that make cyclin D1 dispensable. These findings have important implications for MCL therapy, as strategies targeting only cyclin D1 function might be hampered by compensatory regulatory mechanisms, resulting in a low probability of treatment response.

Chromosomal imbalances in Barrett's adenocarcinoma and the metaplasia-dysplasia-carcinoma sequence.

To characterize cytogenetic alterations found in Barrett's adenocarcinoma (BA) and, more importantly, its premalignant stages, we studied chromosomal imbalances in various lesions in the histologically proposed metaplasia-dysplasia-carcinoma sequence using comparative genomic hybridization (CGH). Using 30 esophageal adenocarcinoma resection specimens, we were able to study 30 areas of Barrett's adenocarcinoma and 8 lymph node metastases (LN). In addition, we investigated 25 premalignant lesions adjacent to BA derived from a subset of 14 resection specimens including 11 areas of high grade dysplasia (HGD), 8 areas of low grade dysplasia (LGD), and 6 areas of intestinal metaplasia (IM), which were laser-microdissected and studied with CGH. To validate the CGH findings, fluorescence in situ hybridization analysis on 13 BA with probes specific for HER-2/neu and 20q13.2 were performed. The chromosomal alterations most often identified in BA were: gains on 8q (80%), 20q (60%), 2p, 7p and 10q (47% each), 6p (37%), 15q (33%) and 17q (30%). Losses were observed predominantly on the Y-chromosome (76%), 4q (50%), 5q and 9p (43% each), 18q (40%), 7q (33%) and 14q (30%). High-level amplifications were observed on 8q23-qter, 8p12-pter, 7p11-p14, 7q21-31, 17q11-q23. Recurrent chromosomal changes were also identified in metaplastic (gains on 8q, 6p, 10q, losses on 13q, Y, 9p) and dysplastic epithelium (gains on 8q, 20q, 2p, 10q, 15q, losses on Y, 5q, 9p, 13q, 18q). Novel amplified chromosomal regions on chromosomes 2p and 10q were detected in both Barrett's adenocarcinoma and premalignant lesions. An increase of the average number of detected chromosomal imbalances from IM (7.0 +/- 1.7), to LGD (10.8 +/- 2.2), HGD (13.4 +/- 1.1), BA (13.3 +/- 1.4), and LN (22 +/- 1.2) was seen. Although the detection of common chromosomal alterations in premalignant lesions and adjacent carcinomas suggest a process of clonal expansion, the occurrence of several chromosomal changes in an apparently random order relative to one another is striking evidence that clonal evolution is more complex than would be predicted by linear models. This is probably a reflection of the existence of many divergent neoplastic subpopulations and highlights one of the main problems associated with surveillance of Barrett's patients, namely sampling error.