Glycolysis gene expression analysis and selective metabolic advantage in the clinical progression of colorectal cancer.

Production of lactate even in the presence of sufficient levels of oxygen (aerobic glycolysis) seems the prevalent energy metabolism pathway in cancer cells. The analysis of altered expression of effectors causing redirection of glucose metabolism would help to characterize this phenomenon with possible therapeutic implications. We analyzed mRNA expression of the key enzymes involved in aerobic glycolysis in normal mucosa (NM), primary tumor (PT) and liver metastasis (LM) of colorectal cancer (CRC) patients (pts) who underwent primary tumor surgery and liver metastasectomy. Tissues of 48 CRC pts were analyzed by RT-qPCR for mRNA expression of the following genes: hexokinase-1 (HK-1) and 2 (HK-2), embryonic pyruvate kinase (PKM-2), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (GLUT-1), voltage-dependent anion-selective channel protein-1 (VDAC-1). Differences in the expression of the candidate genes between tissues and associations with clinical/pathologic features were studied. GLUT-1, LDH-A, HK-1, PKM-2 and VDAC-1 mRNA expression levels were significantly higher in PT/LM tissues compared with NM. There was a trend for higher expression of these genes in LM compared with PT tissues, but differences were statistically significant for LDH-A expression only. RAS mutation-positive disease was associated with high GLUT-1 mRNA expression levels only. Right-sided colon tumors showed significantly higher GLUT-1, PKM-2 and LDH-A mRNA expression levels. High glycolytic profile was significantly associated with poor prognosis in 20 metastatic, RAS-mutated pts treated with first-line chemotherapy plus Bevacizumab. Altered expression of effectors associated with upregulated glucose uptake and aerobic glycolysis occurs in CRC tissues. Additional analyses are warranted for addressing the role of these changes in anti-angiogenic resistance and for developing novel therapeutics.

Clinical impact of the HGF/MET pathway activation in patients with advanced gastric cancer treated with palliative chemotherapy.

In gastric cancer, available clinical studies focusing on the activated hepatocyte growth factor (HGF)/MET pathway are limited to surgical and often heterogeneous series. MET copy number gain (CNG) and an activating truncation in the HGF promoter (deoxyadenosine tract element, DATE+) were studied in tumors of 95 patients with advanced gastric cancer treated with palliative chemotherapy. Associations with overall survival (OS) and the pattern of metastatic disease were studied. Median OS was 9.7 months in 80 MET CNG <5 copies cases (MET-), and 6.4 months in 15 MET CNG was ⩾5 copies cases (MET+) (P=0.001). MET+ status confirmed the adverse prognostic effect in the multivariate model. A significantly different distribution of MET+/DATE+ and MET-/DATE- cases was observed between patients with and without peritoneal carcinomatosis (PC). MET+ status confirms its adverse prognostic role in advanced gastric cancer patients. The activated MET/HGF axis seems to be associated with PC. These findings are relevant to the development of anti-MET/HGF compounds.

Breast cancer and primary systemic therapy. Results of the Consensus Meeting on the recommendations for pathological examination and histological report of breast cancer specimens in the Marche Region.

Primary systemic therapy (PST) adds some practical problems to the pathologic examination of neoplastic breast tissue obtained from patients before and after chemotherapy. Pathologists, oncologists, breast surgeons, radiotherapists and radiologists in the Marche Region held a Consensus Meeting in Ancona on May 13, 2010, in which 15 statements dealing with neoadjuvant chemotherapy were approved by all participants. The first two statements are related to the pre-PST phase and concern the technical procedures and the histological report of the core biopsy. The other statements deal with similar issues of the post-PST surgical specimen.

Loss of hMLH1 expression correlates with improved survival in stage III-IV ovarian cancer patients.

Pre-clinical data suggest a relationship between DNA MisMatch Repair (MMR) system failure, particularly the inactivation of genes hMLH1 and hMSH2, and resistance to drugs like cisplatin and carboplatin. We studied the correlation between loss of hMLH1 expression in tumour cells and clinical outcome in 38 patients with ovarian cancer, who underwent cisplatin-based chemotherapy. 19 patients (56%) showed loss of hMLH1 expression (Group A) while 15 patients (44%) showed normal hMLH1 expression (Group B). 4 patients were not evaluable for hMLH1 expression. The 2 groups of patients were similar for clinical characteristics, response to chemotherapy and time to progression. Group A patients showed a median survival of 55 months whereas Group B patients had a median survival of 12 months (P=0.014). Loss of hMLH1 expression was the only independent predictor of survival in the multivariate analysis. Our observations suggest a relationship between loss of hMLH1 and improved survival in advanced ovarian cancer.

PPARgamma expression in normal human placenta, hydatidiform mole and choriocarcinoma.

Peroxisome proliferator-activated receptor (PPAR) gamma belongs to a subclass of nuclear hormone receptors that execute their transcriptional functions as heterodimers with the retinoid X receptors (RXR). PPARgamma plays a pivotal role in cellular differentiation. This study investigated PPARgamma protein expression in normal human placentas, hydatidiform moles and choriocarcinoma, using immunohistochemical and Western blot analyses. In first trimester normal placenta, PPARgamma was mainly localized in the nuclei of the villous cytotrophoblastic cells, whereas at term it was mainly localized in the nuclei of the syncytiotrophoblast. Extravillous cytotrophoblast of cell islands and cell columns also showed nuclear PPARgamma immunostaining. A striking result was the altered expression patterns of PPARgamma in pathological tissues; PPARgamma showed a reduced immunostaining in the trophoblastic diseases. In hydatidiform moles, PPARgamma was mainly localized in the nuclei of the trophoblastic collections of the pathological villi and in the extravillous trophoblastic cells, whereas in the choriocarcinoma, only a few trophoblastic cells showed weak PPARgamma nuclear immunostaining. These findings suggest an involvement of PPARgamma in trophoblast differentiation during normal placental development. The down-regulation of PPARgamma expression in the gestational trophoblastic diseases analysed in this study provides a new insight into the progression of these pathologies.

Expression of ZO-1 and occludin in normal human placenta and in hydatidiform moles.

Zonula occludens-1 (ZO-1) and occludin are key molecules in cell-cell contacts. They are tight junction constituents and therefore play a pivotal role in tissue differentiation and organogenesis. In the present report we have investigated the expression of ZO-1 and occludin in normal human placentae and in hydatidiform moles using immunohistochemical and Western blot analyses. In normal placentae, ZO-1 and occludin were mainly localized in the apical part of the syncytium, in cell-cell contacts between syncytium and villous cytotrophoblastic cells as well as between the latter. Extravillous cytotrophoblast of cell islands and cell columns was positive for ZO-1 and occludin in the cell layers proximally located to the villous stroma whereas the cytotrophoblastic cells, distally located from the villous stroma, were totally negative. Furthermore, fetal vessels showed a positive staining pattern for ZO-1 throughout gestation, whereas a positive reaction for occludin was produced mainly at term. A striking result was the altered expression of ZO-1 and occludin in partial and complete moles. In 11 moles, these two molecules were not expressed at all, while in the other nine, their expression was only cytoplasmic in syncytium and villous cytotrophoblastic cells. These findings suggest that ZO-1 and occludin participate in normal placental development, maintaining the organization and functions of different tissue components. The down-regulation and/or dysregulation of these two molecules may be related to phenotypic changes associated with epithelial cell transformation of the chorionic villi in partial and complete moles.

Cisplatin, epirubicin and cyclophosphamide (PEC) in the treatment of advanced ovarian cancer.

We report the long-term results of a series of patients affected by advanced epithelial ovarian cancer treated with the PEC combination (cisplatin 60 mg/m2, epirubicin 60 mg/m2 and cyclophosphamide 750 mg/m2, all at day 1, every 21 days). Response was evaluated after three cycles, and treatment continued in responsive patients. A total of 80 patients with a median follow-up of 55 months were studied. Fifty-eight patients with stage III ovarian cancer and 22 patients with stage IV received PEC as primary treatment (41 patients), or for residual disease after surgery (37 patients), or for relapsed disease after primary surgery (2 patients). The overall response rate was 67.5% (20.0% complete response, 47.5% partial response), with 22.5% stable disease and 3.7% progressive disease. Median progression free survival was 13.0 months, and median survival was 25 months. Grade III-IV toxicity was moderate: leukopenia 20.0% of patients, thrombocytopenia 5.0%, anemia 16.2%. No cardiac toxicity was observed. In conclusion, the PEC combination, an anthracycline-containing platinum-based regimen, proved to be effective in advanced ovarian cancer, in terms of response rate and overall survival. The regimen was devoid of significant toxicity and in particular of cardiac toxicity.

Combined malignant hemangiopericytoma and deep venous thrombosis. A case report.

Malignancies, antiproliferative drug treatment, cancer-related conditions like immobilization, perioperative status and radiotherapy are risk factors for hypercoagulability. Setting aside mass or invasion-related venous thrombosis, the differential diagnosis regarding the etiopathogenesis (paraneoplastic syndrome or antiproliferative treatment) is usually problematic. The authors report a case of combined malignant hemangiopericytoma and recurrent deep venous thrombosis in the right inferior limb. Through a literature review, the following issues are discussed: 1) the criteria for cyto-histopathologic assessment; 2) the involvement of pericytes both in coagulation and platelet aggregation; 3) the importance of discriminating true paraneoplastic syndromes from other tumor-related clinical manifestations; 4) the response to external radiotherapy of malignant hemangiopericytoma as limited disease; 5) the poor results of doxorubicin-ifosfamide polychemotherapy and dacarbazine monochemotherapy in metastatic disease. Although doxorubicin-ifosfamide treatment was in progress in the reported case, the authors conclude that the recurrent deep venous thrombosis is likely to be paraneoplastic, even if such a diagnosis has not been previously reported in the literature.

Vascular endothelial growth factor expression as a prognostic index in serous ovarian cystoadenocarcinomas: relationship with MIB1 immunostaining.

OBJECTIVE: The aim of our study was to investigate the expression of vascular endothelial growth factor (VEGF) by neoplastic cells in serous ovarian cystoadenocarcinomas; the correlation between this marker of angiogenesis, histopathologic parameters, disease-free survival, and MIB1 immunostaining was also evaluated. MATERIALS AND METHODS: Thirty-two patients with serous ovarian cystoadenocarcinoma, treated at the Institute of Gynecology and Obstetrics, Ancona University (Italy), were used as study population; 10 women with serous cystoadenoma were also analyzed. The expression of VEGF was immunohistochemically evaluated by polyclonal antibody anti-VEGF (Santa Cruz, CA, dilution 1:100) on formalin-fixed paraffin-embedded tissue. RESULTS: Compared to cystoadenomas, the tissutal VEGF immunostaining was significantly higher in cystoadenocarcinomas, with the highest values in architectural grade 3 neoplasms (P < 0. 001). A direct relationship was observed between VEGF immunostaining and MIB1 index (r = 0.44, P = 0.013). A relationship was defined between VEGF expression and disease-free survival, evaluated by Cox hazards analysis (P < 0.001). CONCLUSIONS: Angiogenesis, evaluated by VEGF immunostaining, seems to be an interesting prognostic indicator in serous ovarian cystoadenocarcinoma, involved in neoplastic proliferation.

Expression of vascular endothelial growth factor related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors.

BACKGROUND: The aim of the current study was to investigate the expression of vascular endothelial growth factor (VEGF) by neoplastic cells in patients with serous ovarian tumors. The correlation between neoangiogenesis and 72-kilodalton metalloproteinase (MMP2) immunostaining also was evaluated. METHODS: Fifty-eight patients with serous ovarian tumors who were treated at the Institute of Gynecology and Obstetrics of Ancona University (Ancona, Italy) were used as the study population; 10 women had serous cystoadenoma, 16 women had a serous borderline tumor, and 32 women had invasive cystoadenocarcinoma. Expression of VEGF and MMP2 was evaluated immunohistochemically by polyclonal antibody anti-VEGF (dilution, 1:100) and affinity purified, rabbit anti-MMP2, formalin fixed, paraffin embedded tissue. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells. RESULTS: Compared with cystoadenomas and borderline tumors, the tessutal VEGF immunostaining was significantly higher in cystoadenocarcinomas, with the highest values detected in architectural International Federation of Gynecology and Obstetrics Grade 3 neoplasms (P2 < 0.001). A direct relation was observed between VEGF and MMP2 immunostaining (correlation coefficient, 0.44; P2 = 0.013). A relation was found between VEGF expression and disease free survival as evaluated by Cox hazards analysis (P2 = 0.03). CONCLUSIONS: Neoangiogenesis detected by VEGF immunostaining appears to be a promising indicator of aggressiveness in serous ovarian tumors. In cystoadenocarcinomas, VEGF expression appears to be related to MMP2 index.

The role of human papillomavirus DNAs in cervical carcinoma and risk of lymph node metastasis: association with 72-kilodalton metalloproteinase immunostaining.

BACKGROUND: The role of human papillomavirus (HPV) as a prognostic factor in cervical carcinoma is not understood completely and little is known regarding the intrinsic mechanisms involved in the metastatic process of HPV positive carcinoma. The authors evaluated HPV status with respect to clinical features in early stage cervical carcinoma, with special emphasis on lymph node spread. The authors also analyzed the relation between HPV, lymph node involvement, and 72-kilodalton (kDa) metalloproteinase immunostaining, an enzyme that cleaves Type IV collagen and may play a role in tumor metastasis. METHODS: Thirty-two patients with International Federation of Gynecology and Obstetrics Stage I and IIA squamous cell cervical carcinoma treated by primary radical surgery were reviewed. Histologic grade of differentiation, tumor size, fractional depth of invasion, and lymph node spread were evaluated with respect to HPV status and 72-kDa metalloproteinase immunostaining. HPV DNA was detected by polymerase chain reaction and the primers potentially recognized at least the following HPV subtypes: 6, 11, 16, 18, 31, 33, 34, 35, 42, 51, 56, and 58. Immunohistochemical staining was performed using the avidin-biotin complex technique. Affinity-purified rabbit anti-72-kDa metalloproteinase antibody was used. RESULTS: HPV DNA was detected in a total of 69% of cases, and HPV-16 was the most frequent type detected. HPV positive carcinomas showed a significantly higher rate of lymph node metastases than HPV negative carcinomas (45% vs. 10%; P = 0.03); similarly, 72-kDa metalloproteinase index was significantly higher (P = 0.001). CONCLUSIONS: These findings suggest a relation between HPV and risk of lymph node metastasis, which may be mediated by an increased production of 72-kDa metalloproteinase.

MIB 1 immunostaining in cervical carcinoma of young patients.

OBJECTIVE: In the present study, we assessed whether biologic characteristics of tumors in young patients differ from those observed in older patients with the same clinical and histologic characteristics, but ranging in age from 50 to 70 years. The hypothesis to be verified was whether cervical carcinoma in young patients presented an increased proliferative activity which might explain more aggressive behavior. MATERIALS AND METHODS: Locally advanced cervical carcinoma tumor samples were obtained from our series of patients, maximum age 40 years, and immunohistochemically evaluated by monoclonal MIB 1 antibody (Immunotech, Marseille Cedex, France) on microwave-oven-processed Formalin-fixed paraffin-embedded tissue. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells for each case. For each young patient, a control was selected among patients aged >/=50 years (range 50-70) matched for stage, tumor size, histologic type and grading, and lymphvascular invasion. RESULTS: Fourteen of 73 patients (19.2%) with stage I and IIa cervical carcinoma who underwent primary radical surgery at our Institute between 1986 and 1994 were aged

p53 immunostaining and HPV DNA detection by PCR in cervical intraepithelial neoplasia: clinical implications of a combinated evaluation.

We analyzed p53 immunoreactivity and clinical outcome in a series of cervical intraepithelial neoplasias (CIN), with respect to HPV DNA positivity. Cervical biopsy samples were obtained from 86 women who attended our Colposcopic Service from January 1993 to June 1994 due to abnormal pap-smear suspicious for CIN and/or human papillomavirus infection. Forty-one women with histologically confirmed CIN were included in the study. p53 positivity was immunohistochemically detected by monoclonal antibody anti-human p53 (pAb D0-7, Dako Denmark; dilution 1:50), and expressed as the percentage of positive cells. p53 positivity was observed in 78% of CIN lesions. In particular, all the HPV DNA-negative dysplasias expressed p53 protein while only 12 out of 21 (57%) HPV DNA-positive were p53 immunoreactive; (P = .003) the p53 immunostaining was also significantly higher in HPV DNA-negative than in positive CIN (P = .049). By analyzing p53 positivity with respect to clinical-pathologic evolution of the disease, among HPV DNA-negative cases, progressive dysplasia had significantly higher values of p53 immunostaining when compared to persistent and/or regressive lesions (P = .002). These findings imply that p53 immunostaining, when analyzed with respect to HPV DNA status, may help to understand the behavior of dysplastic lesions and define their therapeutic approach. Extensive p53 staining in HPV DNA-negative CIN is probably correlated with a high risk of progression.

Microinvasive cervical carcinoma and cervical intraepithelial neoplasia: biologic significance and clinical implications of 72-kDa metalloproteinase immunostaining.

OBJECTIVE: The immunohistochemical expression of 72-kDa metalloproteinase was evaluated in cervical intraepithelial neoplasia (CIN) and microinvasive carcinoma, with the aim to define a relationship between 72-kDa metalloproteinase expression and neoplastic invasiveness, useful to identify subsets of intraepithelial lesions with higher risk of progression. MATERIALS AND METHODS: Cervical bioptic samples were obtained consecutively from 54 women who attended our Colposcopic Service from January 1993 to July 1993 because of abnormal pap smear, suspicious for cervical dysplasia and/or human papillomavirus infection. After written consent, 29 women with CIN were included in the study. All women with CIN 3 lesion underwent conization; in 21 women with mild or moderate cervical dysplasia, we did not perform any medical or physical treatment but followed them longitudinally at close interval. After 12 months, the clinical evolution was classified as spontaneous remission, persistence, or progression depending on the absence or presence of lesion and/or HPV infection in colposcopy, histology, and polymerase chain reaction findings. In the study we also included surgical specimens from 10 women with microinvasive squamous carcinoma who underwent primary radical surgery. Seventy-two kilodalton metalloproteinase positivity was immunohistochemically stained on serial sections by using the avidin-biotin complex technique (Vector Laboratories, Burlingame, CA) and expressed as percentage of cells per 10(3) counted neoplastic cells. RESULTS: Cytoplasmatic positive 72-kDa metalloproteinase immunostaining was significantly higher in microinvasive cervical carcinomas than in CIN lesion (Student's t test; P < 0.001). Considering only cervical intraepithelial neoplasias, a significant increase in 72-kDa metalloproteinase immunostaining was observed with CIN degree increasing (one-way analysis of variance; P = 0.002). No correlation was found between 72-kDa metalloproteinase immunostaining and HPV infection and lesion size defined by quadrants of the cervix involved with colposcopically evident dysplasia. By analyzing 72-kDa metalloproteinase positivity, regressive dysplasia showed low values of 72-kDa metalloproteinase immunostaining (median 1.2%, range 0.5-1.8%), while persistent (median 2.6%, range 1.9-3.6%) and progressive lesions (median 4.6%, range 2.3-6.9%) presented a significantly higher positivity (one-way analysis of variance; P < 0.001). DISCUSSION: In conclusion, the 72-kDa metalloproteinase expression is related to invasive potential with a significant increase in staining positivity in microinvasive carcinomas; 72-kDa metalloproteinase is detectable in cervical dysplasia, and it is related to the severity of cellular atypia. A clinical implication of 72-kDa metalloproteinase immunostaining seems to be indicated, by analyzing the differences in 72-kDa metalloproteinase positivity rates between regressive and persistent or progressive disease.

Epithelioid haemangioma of the heart.

The authors report a case of epithelioid haemangioma (EH) of the right atrium, the first description of this tumour originating in the heart. The lesion was found incidentally during a cardiac echocardiogram and diagnosed pre-operatively as cardiac myxoma. The tumour must be differentiated from the exceptionally rare epithelioid haemangioendothelioma (EHE) of the heart and from a cardiac myxoma. A correct pathological diagnosis is clinically important since EH is a benign tumour, whereas EHE and cardiac myxoma can recur and metastasize. The uneventful follow-up of this patient confirms the benign nature of EH.

Proliferating cell nuclear antigen in endometrial carcinoma: pretreatment identification of high-risk patients.

OBJECTIVE: The aim of our study was to retrospectively examine the proliferating cell nuclear antigen (PCNA) immunoreactivity of tumor cells in curettage specimens containing endometrioid adenocarcinoma and obtained immediately before definitive surgical staging. This PCNA index was compared with the one subsequently derived from surgical specimens and assessed as a function of histologic grade, depth of myometrial invasion, neoplastic nodal involvement, cervical spread, and progression-free survival in order to determine a new prognostic parameter valuable at the time of diagnosis. MATERIALS AND METHODS: A population of 79 patients with locally advanced (stage I and II) endometrioid carcinoma, who underwent both the preliminary diagnostic curettage and the subsequent definitive surgical management, selected from January 1986 to June 1993 at the Department of Gynecology and Obstetrics, Ancona University, was retrospectively recruited from our series of 99 endometrial carcinomas. The archival paraffin blocks from the curettage and uterine specimens were identified and assessed for histologic reexamination and PCNA immunostaining [PC10 monoclonal antibody (Dako, Denmark)]. RESULTS: After a median follow-up of 47 months, recurrences were detected in 7 cases, and the Kaplan-Meier disease-free survival curve estimated for the entire study group was 91%. The median PCNA index of the curettage specimens presented a good overlap with the PCNA immunostaining in corresponding uterine samples with a correlation coefficient of 0.4 (P=0.02). A PCNA index >/=30% in curettage specimen was predictive of deep myometrial invasion; of 35 patients with PCNA index > or = 30%, 29 (83%) had myometrial invasion > or = 50%. No significant relationship was observed with neoplastic cervical spread, and histologic differentiation. By Cox hazard analysis, the PCNA index evaluated on curettage specimens was significantly related to disease-free survival, with significant disease-free survival advantages for patients with PCNA <30% (P<0.001). CONCLUSION: Our findings suggest that the PCNA immunostaining has proved to be considerably promising for the risk assessment in locally advanced endometrial carcinoma. The PCNA index is an objective and reproducible parameter accruably valuable also before starting the treatment; in presence of a high PCNA index, the patients should be referred to gynecologic oncologists for appropriate management.