Damir Babić

Tumor cytosol dipeptidyl peptidase III activity is increased with histological aggressiveness of ovarian primary carcinomas

OBJECTIVE Proteolytic enzymes have been implicated in the progression of various human malignancies, including ovarian cancer. The enhanced expression of dipeptidyl peptidase III (DPP III) was found in endometrial carcinomas of various histological types and grade. The aim of this study was to assess activity of DPP III in ovarian tissue specimens and to correlate it with clinico-pathological data. METHODS DPP III hydrolytic activity toward Arg-Arg-2-naphthylamide was determined in 108 ovarian tissue cytosol specimens of 79 patients. The data obtained for 41 ovarian primary carcinoma specimens were stratified according to clinical stage, histological grade and type, and age of the patients. RESULTS Median DPP III activity expressed as milliunits per milligram protein was 6 in normal ovarian tissues (n = 29), 6.5 in benign ovarian tumors (n = 19), 19.5 in primary ovarian carcinomas (n = 41), 12.5 in nonepithelial primary ovarian tumors (n = 7), and 22.1 in metastatic ovarian malignancies (n = 12). A significant rise in median DPP III specific activity was observed in malignant ovarian tumors (of epithelial, nonepithelial, and metastatic origin), but not in benign ovarian tumors, compared to the activity in normal tissue. A significant difference of DPP III expression was found between the group of normal tissues and tumors of clinical stage I and II, of grade 2 and 3, of serous and mucinous histologic type. CONCLUSIONS DPP III activity of benign ovarian tumors equaled that in normal ovarian tissue. In malignant neoplasms of the ovary it increased with growing histologic grade.

Dipeptidyl peptidase III in malignant and non-malignant gynaecological tissue.

Exopeptidases, in contrast to endopeptidases (proteinases) have been much less studied in relation to cancer. The aim of this study was to investigate one such enzyme, dipeptidyl peptidase III (DPP III), in gynaecological tissues, by measuring both the enzyme activity and enzyme content. DPP III activity was assessed in normal (n = 65), benign (n = 9) and malignant (n = 51) gynaecological tissues. A statistically significant higher DPP III activity was observed in endometrial (n = 40, P = 4.6 x 10(-7)) and ovarian (n = 11, P = 8.1 x 10(-4)) malignant tumours, whereas no significant difference was detected for leiomyomas (n = 8), if compared to the activity in normal tissue. A matched pair analysis of normal and cancerous endometrial tissue confirmed the significance of the DPP III activity increase in the transformed tissue (n = 7, P = 0.022). Western blot analysis revealed a significantly (P = 0.014) increased level of DPP III in endometrial cancer. Further, regression analysis showed a positive correlation between the activity and the content of DPP III in normal tissue (r = 0.637, P = 0.047) and in endometrial cancer (r = 0.574, P < 0.007). The increase of the DPP III activity was observed in the endometrial carcinomas of various histological types, grade or the depth of myometrial invasion. The easy-to-perform determination of this exopeptidase activity may serve as a potential indicator of endometrial and ovarian malignancies.

A low-grade ovarian carcinoma case with coincident LOH of PTCH1 and BRCA1, and a mutation in BRCA1.

We report a case of a 53-year-old woman with Grade 1 serous cystadenocarcinoma on her left ovary and metastatic serous adenocarcinoma on her right ovary. Serous carcinoma is the most common type of ovarian cancer, representing approximately half of all cases. Because of positive family history, the patient was referred for BRCA1/2 screening. Germline BRCA1 mutation c.676delT (p.C226VfsX8) was found, and in tumor tissue the normal allele was lost. Tumor tissue also had loss of heterozygosity in the PTCH1 gene, one of the major members of the Hedgehog-Gli (Hh-Gli) pathway. Gene expression analysis showed upregulation of the Hh-Gli pathway in both ovaries compared with healthy ovarian tissue. Primary cell culture was developed from the patient’s tissue and showed downregulation of gene expression in response to cyclopamine, a Hh-Gli pathway inhibitor. The Hh-Gli signaling pathway may play a role in malignant transformation and metastasis of ovarian cancer.

Cathepsin D content in malignant tumours of corpus uteri

recently, 5 cases of paclitaxel-associated radiation-recall reactions [5-71 and a single case of radiation-recall mucositis associated with docetaxel have been reported [8]. The precise mechanism is unknown. One hypothesis suggests that cytotoxic treatment after radiotherapy causes a ‘remembered’ reaction in the remaining surviving cells within the previously irradiated field. An alternative proposition suggests that radiation induces heritable mutations within surviving cells which then produce a subgroup of defective stem cells that are unable to tolerate the second insult of chemotherapy [9]. Paclitaxel [lo] has been found to be a radiosensitiser, but it is unclear how this might correlate with an ability to reactivate latent radiation effects in normal tissues. It is interesting to note that the skin reaction occurred in only two of the four regions of irradiated skin, i.e. anteriorly where the skin doses were 18.7 Gy and 21.5 Gy over the spine and pelvic areas, respectively, but not posteriorly where the corresponding skin doses were 8.7 Gy and 16.8 Gy, respectively. The existence of four levels of skin doses suggests a ‘threshold’ radiation dose for the recall reaction of between 16.8 and 18.7 Gy in this case. It would be interesting to look for a similar ‘threshold’ dose from the data in other cases, but unfortunately, in most cases, only the tumour dose, rather than the skin dose, are reported. To our knowledge, our patient represents the first case of radiation-recall dermatitis with docetaxel. In the few reported cases of recall reactions associated with taxanes, one case of radiation-recall dermatitis has been reported to be recurring with repeated administration of paclitaxel [6]; however, our patient treated with docetaxel and 2 patients reported by Shenkier and associates [7] who were treated with paclitaxel did not have signs of recurrent reactions with repeated therapy using the same drugs. This suggests that there may be a specific time period during which patients are susceptible to recall dermatitis. Alternatively, in our case, there may be a drug dose threshold since the two cycles in which the dermatitis did not occur were associated with a dose reduction.

Extraskeletal myxoid chondrosarcoma of the vulva with PLAG1 gene activation: molecular genetic characterization of 2 cases.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare mesenchymal neoplasm, rarely reported in the genitourinary tract with only 5 cases reported in the vulva. We investigated 2 cases of vulvar sarcomas whose morphologic appearance and immunohistochemical profiles were consistent with EMC using fluorescence in situ hybridization (FISH), reverse-transcription polymerase chain reaction, and a whole genome expression array. FISH and reverse-transcription polymerase chain reaction assays showed no EWSR1 and NR4A3 loci rearrangements. Microarray-based analysis also revealed no changes in NR4A3 and EWSR1 gene transcription levels. Microarray data showed a significant downregulation of the muscle-related genes (eg, myosin heavy chain family, actins, myoglobin, desmin, creatine kinase, troponins) and cytokeratins (KRT6A, 6B, 13, 14, and 78), upregulation of several neuron-specific genes [neural cell adhesion molecule 1 (NCAM-1/CD56), neurofilament (NEFH)], along with some well-characterized tumor biomarkers [carbonic anhydrase IX (CA-9), topoisomerase II&agr; (TOP2A), matrix metalloproteinases (MMP-7, MMP-9), CDKN2 gene (p16-INK4a), checkpoint homolog 2 (CHEK2)]. Notably, both tumors showed upregulation of the pleomorphic adenoma gene 1 (PLAG1), and in 1 case PLAG1 gene rearrangement was detected by break-apart FISH. Some vulvar tumors with morphologic and immunohistochemical characteristics of EMC may represent a molecular genetic entity separate from EMCs arising in other locations. PLAG1 gene activation appears to be involved in the development of these neoplasms.

Cytology of Cervical Intraepithelial Glandular Lesions

Cytological criteria for the identification of glandular intraepithelial lesions (GIL) have not yet been fully described, especially for the precursors of adenocarcinoma in situ (AIS), thus these lesions may frequently remain unrecognized. As most patients diagnosed with AIS or mild to moderate GIL (grades I, II) are free from clinical symptoms, cytology has a very responsible role in the detection of these lesions. The aim of the study was to achieve the most appropriate cytologic diagnosis of intraepithelial lesions of endocervical columnar epithelium, analyzing the cytology findings in patients with histologically verified AIS and GIL (I, II). The value of cytology in the detection and differential diagnosis was assessed in 123 patients with definitive histologic diagnosis of glandular lesions (AIS, n = 13; GIL I, n = 11; and GIL II, n = 7), and glandular lesions associated with squamous component (AIS associated with cervical intraepithelial neoplasia (CIN) or invasive squamous cell carcinoma (SCC), n = 58; GIL I or GIL II associated with CIN, n = 28; and GIL associated with microinvasive squamous carcinoma (MIC), n = 6). In 95.1% of patients, lesions were detected by cytologic analysis that indicated additional diagnostic procedure. In terms of differential diagnosis, cytology showed higher accuracy in predicting lesion severity vs. type of epithelial alteration (75.6% vs. 55.3%) and abnormalities of columnar epithelium (95.7%; vs. 74.2%). The accuracy of cytology was higher in pure (AIS, 61.5% and GIL I, II, 22.2%) than in mixed lesions (25.9% and 20.6%). Continuous improvement in cervical specimens and cytodiagnostic skills, better understanding of intraepithelial adenocarcinoma and precursors, and their inclusion in the classification of cytologic and histologic findings are expected to upgrade the detection of these lesions, and to reduce the invasive cervical adenocarcinoma morbidity and mortality.

Total tissue lactate dehydrogenase activity in endometrial carcinoma

Lactate dehydrogenase (LDH) is essential for continuous glycolysis necessary for accelerated tumor growth. The aim of this study was to reconsider if assay of total tissue activity of this enzyme could be useful as marker for endometrial carcinoma (EC). Activity of LDH was measured spectrophotometrically in homogenate supernatants of uterine tissue samples of 40 patients (10 normal endometria, 27 normal myometria, and 33 EC), including 30 matched pairs. Data obtained were analyzed in relation to clinical and histopathologic findings and compared with our previously published results on the tissue levels of the same enzyme in ovarian cancer and on the proteolytic activity of dipeptidyl peptidase III (DPP III) in EC (suggested biochemical indicator of this malignancy). Significantly increased (1.8–3.0 times; P< 1 × 10−4) LDH activity was observed in EC samples if compared with normal uterine tissues. This rise was not related to the clinicopathologic findings, however. In contrast to previous results on LDH in ovarian carcinomas, a significant rise in LDH activity was found already in grade 1 EC. Using the cutoff value of 1.06 U/mg, diagnostic sensitivity of 82%, specificity of 100%, and accuracy of 91% for total tissue LDH assay have been calculated. A correlation of tissues LDH and DPP III activities was found, and their combined assay for EC showed increased diagnostic sensitivity (94%) and accuracy (96%).

Correlation of the HPV detection, protein expression and DNA content in cutaneous pre-invasive and invasive carcinoma among Croatian patients

Development of cutaneous carcinomas has been associated with HPV infection. There have been various reports on p16, p53 and pRb expression in cutaneous carcinomas and on its linkage to HPV status. Association of protein expression and HPV infection with DNA content is not clear. The aim of this study was to determine a possible correlation between HPV type, protein expression and DNA content in both pre-invasive and invasive squamous cell carcinoma, as well as differences between studied groups in these parameters. Sections of formalin fixed paraffin-embedded tumor tissue from 54 cases of Morbus Bowen (preinvasive cutaneous carcinoma) and 41 cases of invasive squamous cell carcinoma of the skin were subjected to HPV genotyping using Lipa (Line imuno probe assay), immunohistochemical staining for p16(INK4A), p53, pRb and prepared for flow cytometry DNA content analysis. Obtained data were analyzed in SPSS using Chi square test. Only p16 expression showed statistically significant differences in studied groups. Statistically significant correlations were found only in MB between parameters HPV-p53, p53-pRb and p53-p16. Our results suggest different virus-induced pathobiology pathways for different cutaneous carcinoma groups.

The Value of HPV-HR DNA Testing During the Follow-Up After Treatment of CIN3/AIS

Up to 30 % of women treated for a preinvasive cervical disease are diagnosed with a residual/recurrent disease including an invasive carcinoma during follow-up. Studies have shown a higher sensitivity in detection of residual/recurrent disease with the use of combined testing (cytology and HPV. One hundred and thirty five patients treated with cold-knife conization (CKC) or large loop excision of the transformation zone (LLETZ) for a preinvasive cervical disease, were enrolled in the study. The follow-up consisted of cytology, colposcopy with biopsy if needed and HPV testing. Only patients with at least two cytology smears and one HPV test during the first two post-treatment years remained in the study. The HPV test was performed by EIA PCR and by Line immuno Probe Assay. The statistic analysis was performed using the χ2 test. Results of our study suggest that in our setting the best approach is to have a first control cytology smear 6 months after conization. HPV test should be done in patients with a positive smear any time during follow-up as the point of decision for a second treatment. With this approach we could considerably decrease the number of reoperated patients and co-morbidities without decreasing the quality of follow-up.