No Viral Association Found in a Set of Differentiated Vulvar Intraepithelial Neoplasia Cases by Human Papillomavirus and Pan-Viral Microarray Testing

Vulvar Intraepithelial Neoplasia (VIN) is the precursor lesion of Vulvar Squamous Cell Carcinoma (VSCC), and the differentiated type (dVIN) is more frequently observed in relation to VSCC. In contrast to usual-type VIN (uVIN), which is related to infection by human papillomavirus (HPV), a germline mutation in the p53 gene is thought to be associated with ~90% of dVIN cases. To date, no infectious agent has been identified in association with dVIN, and studies investigating this possibility have been hindered by the difficulty in accurately diagnosing dVIN from small biopsies. Here, we used immunostaining for p16^ink4a, a biomarker for HPV infection, to study 14 uVIN high-grade VIN and 14 dVIN cases, and to select 10 dVIN cases to broadly screen for all known viruses using a pan-viral microarray platform (ViroChip). All of the uVIN tissue samples, including 8 warty and 6 basaloid cases, showed positivity with the p16^ink4a immunostain. The staining pattern was full-thickness for all except two cases in which positive staining was localized in the lower 1/3 of the epidermis. In contrast, immunostaining for p16^ink4a was negative in all dVIN cases. ViroChip analysis of 10 pure dVIN samples confirmed the absence of human papillomavirus subtypes or any other virus with the exception of a single sample that showed a weak microarray signature to a porcine herpesvirus. Follow-up PCR testing of the sample was negative for herpesvirus, and in-depth metagenomic next-generation sequencing revealed only sequences corresponding to non-pathogenic viral flora and bacterial contamination. In this study, we demonstrated lack of a virus association in 10 dVIN cases. Alternative pathways for carcinogenesis such as the p53 mutation should be considered for investigation of potential treatment options in dVIN.     

Expression of vascular endothelial growth factors VEGF- C and D, VEGFR-3, and comparison of lymphatic vessels density labeled with D2-40 antibodies as a prognostic factors in vulvar intraepithelial neoplasia (VIN) and invasive vulvar cancer

OBJECTIVE: The aim of this study was to compare the immunohistochemical expression of vascular endothelial growth factors VEGF-C and D, as well as the expression of VEGFR-3 in VIN and vulvar invasive cancer and to compare the density of lymphatic marker D2-40 antibody in both groups, and to compare them with different clinicopathologic features. Materials & Methods: The study was performed using tissue material and clinical data from 100 women diagnosed with VIN and 100 women diagnosed with invasive vulvar cancer. Results: No significant differences were found in the expression of VEGF-C and -D or VEGFR-3 between those patients with VIN and those with invasive vulvar cancers. Weak expression of VEGF-C was confirmed only in two cases of the analyzed series; in all cases, expression of VEGF-D and VEGFR-3 was observed. The strongest expression of VEGF-D and VEGFR-3 was observed in the group of invasive cancers. The highest density of lymphatic vessels per 2 mm was observed in VIN. In the cancer group, small lymphatic vessels with a narrow oval lumen were observed. Moreover, in two cases of vulvar cancer, the presence of intratumoral lymphatic vessels was observed. Conclusions: These results suggest that lymphangiogenesis begins at the preinvasive stage of vulvar carcinogenesis and suggests the important role of VEGF-C, VEGF-D, VEGFR-3 and LV (D2-40) as prognostic factors in the process of carcinogenesis in the vulvar area.     

Gain in 1q is a common abnormality in phyllodes tumours of the breast.

We studied DNA copy number changes by CGH and allelic imbalance (AI) on 3p by LOH analysis on 22 phyllodes tumours (PT) of the breast in order to gain insight into the genetic basis of tumour progression in PT. Copy number changes were observed in 14 cases (63%). Gain in 1q with 1q21-23 as the minimal overlapping area was seen in 12 cases (55%). The gain was observed both in benign and malignant tumours. Our study did not reveal any DNA copy number changes or allelic loss on 3p. The results suggest that DNA copy number changes are not associated with the histological grade or clinical behaviour of PT and the chromosomal changes on 3p appear to be rare. Colour figure can be viewed on http://www.esacp.org/acp/2003/25-2/jee.htm     

New aspects of vulvar cytology

Smears were taken from vulvar skin in 203 cases (83 normal-appearing, 23 benign, 12 precancerous and 85 malignant conditions). Three cases were analyzed by electron microscopy. The appearance of dyskeratotic cells was regarded as a sign of malignancy; this was supported by ultrastructural study. Smears from normal vulvar skin revealed a high correlation between age and presence of nucleated keratinized squames, which occurred most often in the third and rarely in the eighth decade. Benign conditions showed cellular features like those of the normal vulva. In precancerous lesions, 58% of the smears showed dyskeratotic cells. Malignant conditions showed dyskeratotic cells in 66% of the smears and true cancer cells exclusively in an additional 14%. Our observations indicate that vulvar cytology can be a major diagnostic aid in all suspicious cases, especially in the elderly. Vulvar cytology cannot replace an exact histologic examination, but it can provide a reason for taking a biopsy earlier than might otherwise be the case.     

Mitochondrial genotype in vulvar carcinoma - cuckoo in the nest

Vulvar squamous cell carcinoma (VSCC) is a rare female genital neoplasm. Although numerous molecular changes have been reported in VSCC, biomarkers of clinical relevance are still lacking. On the other hand, there is emerging evidence on the use of mtDNA as a diagnostic tool in oncology. In order to investigate mtDNA status in VSCC patients, haplogroup distribution analysis and D-loop sequencing were performed. The results were compared with available data for the general Polish population, cancer free-centenarians as well as patients with endometrial and head and neck cancer. The obtained data were also compared with the current status of mitochondrial databases. Significant differences in haplogroup distribution between VSCC cohort, general Polish population and cancer-free centenarians cohort were found. Moreover, a correlation between the VSCC patients haplogroup and HPV status was observed. Finally, a specific pattern of mtDNA polymorphisms was found in VSCC. Our results suggest that the mitochondrial genetic background may influence the risk of VSCC occurrence as well as susceptibility to HPV infection.     

Quantitative RT-PCR assay for mRNA of VEGF and histone H4 in the determination of proliferative and angiogenic activity in vulvar pathology

Proliferative and angiogenic activity of tissue specimens taken from women with various vulvar pathologies were evaluated by determining the number of mRNA VEGF molecules and H4 histone mRNA molecules, by means of the QRT-PCR (TaqMan) technique. Following a cluster analysis the results, where normalised. Euclidean distances were used, all the cases were classified into three groups of pathologies. Group I included low degree vulvar pathologies, group II included high degree vulvar pathologies and group III included vulvar pathologies with high proliferative and angiogenic activity. Significant differences were found in the proliferative and angiogenic activity between groups I and III, and between groups II and III, while no statistically significant differences were found between groups I and II.     

Tumor suppressor gene RBSP3 in cervical carcinoma: copy number and transcriptional level

In this work for the first time copy number and expression changes of the tumor suppressor gene RBSP3 (3p21.3) were investigated. The study was performed on HPV-positive squamous cervical carcinoma (SCC) using real-time PCR. Deletions were detected in 42% of cases (19 of 45 studied biopsies). Frequency of deletions was significantly higher in SCC samples with metastases (64%) than in tumors without metastases (32%, P < 0.05). In a few cases amplification of RBSP3 was also found. Altogether copy number changes of RBSP3 were detected in 51% of cases (23 of 45). Expression of RBSP3 was decreased in 64% of SCC samples (21 of 33). Again decreased expression of RBSP3 was detected significantly more frequently (83%) in tumors with metastases compared with SCC without metastases (52%, P < 0.05). In several cases however increased expression was observed. Altogether changes in expression of RBSP3 were detected in 79% (26 of 33) of SCC biopsies. Comparison of copy number and expression changes showed that in 23% of SCC cases decreased expression of RBSP3 was detected in samples with deletions and in 36% cases such decrease was not associated with copy number changes. Rarely more complicated SCC cases were found. For example in some tumors increased expression of RBSP3 was detected in samples with deletions or without changes in copy number. Results of the study suggested that RBSP3 is involved in the progression of SCC and complex mechanisms for inactivation of RBSP3. We also hypothesize that these data indicate that RBSP3 in addition to dephosphorylation of pRb has other functions important for malignant transformation because pRb is almost absent in HPV-positive SCC.     

Cervical carcinoma progression-associated genetic alterations on chromosome 6

To identify the loci associated with progression of cervical carcinoma, chromosome 6 regions were tested for loss of heterozygosity. Detailed analysis with 28 microsatellite markers revealed a high frequency of allelic deletions for several loci of the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16-21, 6q23-24, 6q25, 6q27) arms of chromosome 6. Examination of 37 microdissected carcinoma and 22 cervical dysplasia specimens revealed allelic deletions from the HLA class I-III genes (6p22-21.3) and subtelomeric locus 6p25 were found in more than 40% dysplasia specimens. With multiple microdissection of cryosections, genetic heterogeneity of squamous cervical carcinoma was analyzed, and clonal and subclonal allelic deletions from chromosome 6 were identified. Half of the tumors had clonal allelic deletion of D6S273 (6p21.3), which is in a Ly6G6D (MEGT1) intron in the HLA class III gene locus. The frequency of allelic deletions from the chromosome 6 long arm was no more than 20% in dysplasias. Allelic deletions from two loci, 6q14 and 6q16-21, were for the first time associated with invasion and metastasis in cervical carcinoma.     

Genomic imbalances in 61 renal cancers from the proximal tubulus detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to characterize 61 primary renal cell carcinomas derived histogenetically from the proximal tubulus. The tumor samples comprised 46 clear-cell renal cell carcinomas (ccRCCs) and 15 papillary renal cell carcinomas (pRCCs). Changes in the copy number of entire chromosomes or subregions were detected in 56 tumors (92%). In ccRCCs, losses of chromosome 3 or 3p (63%); 14q (30%); 9 (26%); 1 and 6 or 6q (17% each); 4 and 8 or 8p (15% each); 22 (11%); 2 or 2q and 19 (9% each); 7q, 10, 16, 17p, 18, and Y (7% each); and 5, 11, 13, 15, and 21 (4% each) were detected. Most frequent genomic gains in ccRCC were found on chromosome 5 (63%); 7 (35%); 1 or 1q (33%); 2q (24%); 8 or 8q, 12, and 20 (20% each); 3q (17%); 16 (15%); 19 (13%); 6 and 17 or 17q (11% each); and 4, 10, 11, 21, and Y (9% each). In pRCCs, gains in the copy number of chromosomes 7 and 17 (7/15, each) and 16 and 20 (6/15, each) were frequent. One pRCC showed amplification of subchromosome regions 2q22-->q33, 16q, 17q and the entire X chromosome. In pRCC, losses were less frequently seen than gains. Losses of chromosomes 1, 14, 15, and Y (3/15 each) and 2, 4, 6, and 13 (2/15 each) were observed. In ccRCCs, statistical evaluation revealed significant correlations of chromosomal imbalances with tumor stage and grade, i.e., a gain in copy number of chromosome 5 correlated positively with low tumor grade, whereas a gain of chromosomes 10 and 17 correlated positively with high tumor grade. Furthermore, loss of chromosome 4 correlated positively with high tumor stage.     

Anti-HPV16 E2 protein T-cell responses and viral control in women with usual vulvar intraepithelial neoplasia and their healthy partners

T-cell responses (proliferation, intracellular cytokine synthesis and IFNγ ELISPOT) against human papillomavirus 16 (HPV16) E2 peptides were tested during 18 months in a longitudinal study in eight women presenting with HPV16-related usual vulvar intraepithelial neoplasia (VIN) and their healthy male partners. In six women, anti-E2 proliferative responses and cytokine production (single IFNγ and/or dual IFNγ/IL2 and/or single IL2) by CD4+ T lymphocytes became detectable after treating and healing of the usual VIN. In the women presenting with persistent lesions despite therapy, no proliferation was observed. Anti-E2 proliferative responses were also observed with dual IFNγ/IL2 production by CD4+ T-cells in six male partners who did not exhibit any genital HPV-related diseases. Ex vivo IFNγ ELISPOT showed numerous effector T-cells producing IFNγ after stimulation by a dominant E2 peptide in all men and women. Since the E2 protein is absent from the viral particles but is required for viral DNA replication, these results suggest a recent infection with replicative HPV16 in male partners. The presence of polyfunctional anti-E2 T-cell responses in the blood of asymptomatic men unambiguously establishes HPV infection even without detectable lesions. These results, despite the small size of the studied group, provide an argument in favor of prophylactic HPV vaccination of young men in order to prevent HPV16 infection and viral transmission from men to women.     

Detection of p53 protein and Ki-67 proliferation antigen in human papillomavirus (HPV)-positive and HPV-negative cervical lesions by immunohistochemical double-staining

In HPV-associated genital lesions, low or absent expression of p53 has been attributed to the rapid degradation of p53 through its binding with HPV E6 protein. In this study, we examined p53 protein expression with two antibodies (CM1 polyclonal and PAb 1801 monoclonal antibodies), and Ki-67 proliferation antigen (monoclonal antibody) using an immunohistochemical (IHC) double-staining technique in 77 HPV-positive cervical lesions (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33) and in 15 HPV-negative cases. p53 protein expression was detected in 36/92 (39.1%) of the specimens. of the p53-positive cases, 80.6% (29/36) were HPV-positive samples, including 10/23 (43.5%) of HPV16- and 3/10 (30%) of HPV18-positive biopsies. In 52.8% of the p53-positive samples, the expression was found in less than 5% of the basal cells which were also positive for Ki-67.
Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV16. p53 was more abundant in progressive or persistent lesions, but no differences were found between HPV-positive and HPV-negative samples. the positive IHC double-staining of both p53 and Ki-67 proliferation antigen in the same basal (and parabasal) cells indicates that these two normal cell-cycle proteins are being expressed while the cells are entering from the G₁ to the S phase of the cell cycle. Since the latter property is only attributed to the wild-type p53 (but not to mutated p53), the p53 protein detected in HPV lesions by IHC is likely to be the wild-type p53 rather than mutated p53, and the result was also confirmed by using p53 mutant specific antibody PAb 240. Accordingly, the concept of HPV inactivating the wild-type p53 protein should be re-examined, and other mechanisms for HPV-mediated carcinogenesis should be considered.     

Absence of differentiation-related expression of keratin 10 in earlystages of vulvar squamous carcinoma

Using specific monoclonal antibodies (DE-K10 and DE-SCK respectively), the expression of some differentiation-related epidermal keratins was studied in 38 human vulvar squamous carcinomas. In the epidermis, expression of keratin 10 (K10) strictly paralleled the extent of differentiation; it was absent in the basal layer, appeared in the first suprabasal layers and increased in concentration towards the granular layer. However, K10 was rarely detected (1 case out of 12) in early stages of vulvar squamous carcinomas (tumours less than 2 cm, clinical stage I) regardless of the tumour grade. In larger and more advanced tumours (greater than 2 cm, clinical stages II and III), K10 was detected in 21 out of 26 cases. Its expression appeared to be related to maturation of malignant keratinocytes, being preferentially detected in more-differentiated parts. Occasionally however, cells that did not show histological signs of keratinisation were also K10-positive. Modified stratum corneum keratins (recognized specifically by monoclonal antibody DE-SCK) were detected in the most keratinized areas (horn pearls and their close vicinity) of some K10-positive tumours, i.e., in a pattern close to their normal expression in terminally differentiated epidermal cells. These data suggest differences in the regulation of K10 expression during the differentiation processes in the normal keratinising squamous epithelium and in squamous carcinomas. While the normal pattern of vulvar epithelial differentiation is accompanied by an increasing expression of K10, malignant keratinocytes, also when these are histologically moderately or well differentiated, cease expressing this keratin in the early stages of tumour development.     

Molecular study of the rat liver NADH: Cytochrome c oxidoreductase complex during development and aging

The mechanisms involved in ageing are yet to be fully understood but it is thought that changes produced in energy transfer pathways occurring in the mitochondria may be responsible for the lack of energy typical of the later stages of life. The aim of the present investigation was to determine the enzymatic activity of the liver NADH cytochrome c oxidorectuctase complex (Complex I-III) in mitochondria isolated from the liver of rats of 3 different age groups: lactating, animals (15-17 days), adult females (3-5 months) and old animals (26-30 months). The activities of the unbound Complexes I and III were also determined.An increase in Complex I-III activity was detected during development (142 ± 10 vs. 447 ± 23 mol cyt. c/mg/min, p < 0.001) ang ageing (447 ± 23 vs. 713 ± 45 mol cyt. c/mg//min, p < 0.001). However, unbound Complex I showed a reduction in activity during the ageing period whilst Complex III activity moderately increased. Immunological studies indicated only a moderate increase in the amount of Complex I-III and studies on the purified complex suggested that the increase in activity was due to effects other than an increase in enzyme quantity. The analysis of protein bands and the quantification of prosthetic groups showed particular reductions in the relative concentrations of Complex I subunits including the 51 kDa unit, which binds FMN, confirmed by a similar reduction in levels of the nucleotide. In contrast, 4 of the 5 subunits which increased during the lifetime of the animals corresponded to those of Complex III. These subunits are responsible for the binding of catalytic groups. The results suggest that, in addition to the increase in the amount of enzyme, binding factors between Complexes I and III may also play an important role in the observed increase in Complex I-III activity.     

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.     

Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally

We have determined three opioidmimetics (compounds I-III) in the rat brain dialysates after intraperitoneal (i.p.) administration of compounds I-III using a liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). The dialysate samples with methanol were directly analyzed by online column-switching liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 421 of m/z 657 for compound I, m/z 421 of m/z 643 for compound II, and m/z 407 of m/z 629 for compound III) on LC-MS/MS with electrospray ionization (ESI), opioidmimetics in rat brain dialysates were determined. Calibration curves of the method showed a good linearity in the range of 10-100 ng/ml for each compound. The limit of determination was estimated to be ca. 1 ng/ml for compounds II and III, and ca. 5 ng/ml for compound I, respectively. The precision of analysis showed coefficients of variation ranging from 4.7 to 10.4% at compound III concentration (10-100 ng/ml) in Ringer's solution. As a result, the procedure proved to be very suitable for routine analysis. The method was applied to the analysis of three opioidmimetics in the brain dialysate samples from rats treated with these compounds.     

Genetic Alterations at Chromosome 6 Associated with Cervical Cancer Progression

Loss of heterozygosity (LOH) analysis on chromosome 6 was performed to define the genetic changes that occur in the development of squamous cell cervical cancer (SCC). Detailed analysis with 28 microsatellite markers revealed several loci with high frequency of deletions at the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16–q21, 6q23–q24, 6q25, 6q27) arms of chromosome 6. Examination of microdissected 37 SCC and 22 cervical intraepithelial neoplasias (CIN) revealed allelic deletions in the HLA class I–III region (6p22–p21.3) and at subtelomeric locus 6p25-ter in more than 40% of CIN. By a combination of LOH and microdissection of multiple samples from the same tumor sections, we studied the intratumoral genetic heterogeneity of SCC, and identified clonal and subclonal allelic deletions. Half of SCC had clonal allelic deletion at D6S273, which is localized in intron of Ly6G6D (MEGT1) gene mapped in the HLA class III region. The LOH frequency at 6q in CIN cases did not exceed 20%. Allelic deletions at two loci, 6q14 and 6q16–q21, were for the first time associated with invasion and metastasis in SCC.     

Chromosome imbalances in oligodendroglial tumors detected by comparative genomic hybridization

Seven well-differentiated oligodendrogliomas, 16 anaplastic oligodendrogliomas and two cases of oligoastrocytomas were investigated by comparative genomic hybridization (CGH) on frozen tissue samples. The most frequent losses found involved 1p and 19q in 32% of cases. Loss of 9p was observed during malignant progression in 25% of anaplastic oligodendrogliomas. In two anaplastic oligodendrogliomas gain of 1q was found. The frequent losses of chromosome 16 and 22 have not been reported previously. These results underscore that CGH is a powerful tool for the classification of gliomas complementing the traditional histopathological approach.     

A comprehensive characterization of genome-wide copy number aberrations in colorectal cancer reveals novel oncogenes and patterns of alterations

To develop a comprehensive overview of copy number aberrations (CNAs) in stage-II/III colorectal cancer (CRC), we characterized 302 tumors from the PETACC-3 clinical trial. Microsatellite-stable (MSS) samples (n = 269) had 66 minimal common CNA regions, with frequent gains on 20 q (72.5%), 7 (41.8%), 8 q (33.1%) and 13 q (51.0%) and losses on 18 (58.6%), 4 q (26%) and 21 q (21.6%). MSS tumors have significantly more CNAs than microsatellite-instable (MSI) tumors: within the MSI tumors a novel deletion of the tumor suppressor WWOX at 16 q23.1 was identified (p<0.01). Focal aberrations identified by the GISTIC method confirmed amplifications of oncogenes including EGFR, ERBB2, CCND1, MET, and MYC, and deletions of tumor suppressors including TP53, APC, and SMAD4, and gene expression was highly concordant with copy number aberration for these genes. Novel amplicons included putative oncogenes such as WNK1 and HNF4A, which also showed high concordance between copy number and expression. Survival analysis associated a specific patient segment featured by chromosome 20 q gains to an improved overall survival, which might be due to higher expression of genes such as EEF1B2 and PTK6. The CNA clustering also grouped tumors characterized by a poor prognosis BRAF-mutant-like signature derived from mRNA data from this cohort. We further revealed non-random correlation between CNAs among unlinked loci, including positive correlation between 20 q gain and 8 q gain, and 20 q gain and chromosome 18 loss, consistent with co-selection of these CNAs. These results reinforce the non-random nature of somatic CNAs in stage-II/III CRC and highlight loci and genes that may play an important role in driving the development and outcome of this disease.