Adverse prognostic value of peritumoral vascular invasion: is it abrogated by adequate endocrine adjuvant therapy? Results from two International Breast Cancer Study Group randomized trials of chemoendocrine adjuvant therapy for early breast cancer.

BACKGROUND: Peritumoral vascular invasion (PVI) may assist in assigning optimal adjuvant systemic therapy for women with early breast cancer. PATIENTS AND METHODS: Patients participated in two International Breast Cancer Study Group randomized trials testing chemoendocrine adjuvant therapies in premenopausal (trial VIII) or postmenopausal (trial IX) node-negative breast cancer. PVI was assessed by institutional pathologists and/or central review on hematoxylin-eosin-stained slides in 99% of patients (analysis cohort 2754 patients, median follow-up >9 years). RESULTS: PVI, present in 23% of the tumors, was associated with higher grade tumors and larger tumor size (trial IX only). Presence of PVI increased locoregional and distant recurrence and was significantly associated with poorer disease-free survival. The adverse prognostic impact of PVI in trial VIII was limited to premenopausal patients with endocrine-responsive tumors randomized to therapies not containing goserelin, and conversely the beneficial effect of goserelin was limited to patients whose tumors showed PVI. In trial IX, all patients received tamoxifen: the adverse prognostic impact of PVI was limited to patients with receptor-negative tumors regardless of chemotherapy. CONCLUSION: Adequate endocrine adjuvant therapy appears to abrogate the adverse impact of PVI in node-negative disease, while PVI may identify patients who will benefit particularly from adjuvant therapy.

Extracapsular tumor spread and the risk of local, axillary and supraclavicular recurrence in node-positive, premenopausal patients with breast cancer.

BACKGROUND: Extracapsular tumor spread (ECS) has been identified as a possible risk factor for breast cancer recurrence, but controversy exists regarding its role in decision making for regional radiotherapy. This study evaluates ECS as a predictor of local, axillary, and supraclavicular recurrence. PATIENTS AND METHODS: International Breast Cancer Study Group Trial VI accrued 1475 eligible pre- and perimenopausal women with node-positive breast cancer who were randomly assigned to receive three to nine courses of classical combination chemotherapy with cyclophosphamide, methotrexate, and fluorouracil. ECS status was determined retrospectively in 933 patients based on review of pathology reports. Cumulative incidence and hazard ratios (HRs) were estimated using methods for competing risks analysis. Adjustment factors included treatment group and baseline patient and tumor characteristics. The median follow-up was 14 years. RESULTS: In univariable analysis, ECS was significantly associated with supraclavicular recurrence (HR = 1.96; 95% confidence interval 1.23-3.13; P = 0.005). HRs for local and axillary recurrence were 1.38 (P = 0.06) and 1.81 (P = 0.11), respectively. Following adjustment for number of lymph node metastases and other baseline prognostic factors, ECS was not significantly associated with any of the three recurrence types studied. CONCLUSIONS: Our results indicate that the decision for additional regional radiotherapy should not be based solely on the presence of ECS.

Prognostic significance of PU.1 in follicular lymphoma.

Very few prognostic factors are known in follicular lymphoma (FL), a common malignancy of germinal centre (GC) B-cells. The Follicular Lymphoma International Prognostic Index (FLIPI) thus far appears to be the most important predictor of clinical outcome. This study explores the predictive power of the degree of GC differentiation for outcome in FL. Samples from 73 patients with FL were evaluated by immunohistochemistry for expression of GC markers. Strong PU.1, CD20, and CD75 expression were significantly associated with longer progression-free survival (PFS) and overall survival (OS). Results for PFS were independent of the International Prognostic Index or the Italian Lymphoma Intergroup prognostic index for CD75 and PU.1, but only PU.1 expression was independent of FLIPI for PFS and OS. Oct-2 was weakly expressed overall, but more strongly in higher grades of FL; it had a trend for negative linear association with PU.1 and strong positive linear association with CD27, which possibly reflects its role in terminal B-cell differentiation. We show that the level of GC differentiation, as determined by the levels of PU.1, CD75, CD20, Bcl-6, and CD10 expression, has an association with outcome in patients with FL. While this is determined qualitatively in most studies of diffuse large B-cell lymphoma, in FL there is a quantitative positive association between a high level of expression of GC antigens and longer OS and PFS even when data are stratified by the FLIPI score.

Human papillomavirus DNA in oral squamous cell carcinomas and normal oral mucosa.

To elucidate the putative etiologic role of human papillomaviruses (HPV) in oral carcinogenesis, a comparative study was carried out on 62 tissue specimens of oral squamous cell carcinoma (OSCC) and on 62 specimens of histologically normal oral mucosa obtained from the individuals who matched the subjects with OSCC in age, gender, localization of obtained tissue specimens, drinking and smoking habits. Internal control amplification showed that amplifiable DNA was recovered from 59/62 and 61/62 tissue samples of OSCC and normal oral mucosa, respectively. The amplification with two different HPV L1 and one HPV E6 consensus primer sets showed the presence of the HPV DNA genotypes 16, 33, 58 in 5/59 (8.4%) OSCC specimens and HPV genotypes 11, 16, 31, 68 in 4/61 (6.6%) tissue samples of normal oral mucosa tested. In the study in which a comparative examination of the presence of HPV DNA was for the first time performed on the tissue samples of the patients with OSCC and the age- and gender-matched control subjects there was no significant difference in the prevalence of HPV DNA among both study groups. Our results suggest that occasional findings of HPV DNA in OSCC tissue specimens may be the result of an incidental HPV colonization of oral mucosa, rather than of viral infection, and that HPVs play a limited role in the etiopathogenesis of the majority of OSCC.

Added value of blue dye in sentinel node biopsy for breast cancer.

Sentinel node biopsy in breast cancer is a new rapidly advancing minimal invasive procedure which enables nodal staging of clinically node negative breast cancer patients without performing complete axillary dissection. There are still controversies over the added value of Blue Dye when lymphoscintigraphy and gamma probe are used. In our series, 91 consecutive patients with invasive breast carcinoma were operated by a single surgeon, using lymphoscintigraphy, gamma probe and Blue Dye. The sentinel nodes (SLN) were histologically examined by HE and immunohistochemistry. Lymphoscintigraphy was succesful in 81 patients (89%). After the injection of Blue Dye, SLN could be identified in all 91 patients. Metastases in the SLN were present in 35 patients. We retrieved 128 SLN, of these 93 were hot and blue, 19 only hot and 16 only blue. The distribution of metastatic and nonmetastatic SLN between these three labeling groups was not different (P = 0.9361). We could not show any difference in the metastatic involvement of SLN in patients in whom preoperative lymphoscintigraphy could visualise the SLN preoperatively compared to those in whom it could not (P = 0.7315). False negativity calculated in our initial series of 36 patients was 0%. Our study showed added value of Blue Dye in detection of metastatic and nonmetastatic SLN.

The role of P-glycoprotein (MDR1) polymorphisms and mutations in colorectal cancer.

To better understand physiological function of P-glycoprotein (P-gp), encoded by MDR1 gene, and its role in cancer, we analyzed tumor and corresponding normal tissue from 400 patients with previously non treated colorectal cancer for germline and somatic alterations in MDR1 gene and compared the results to histopathological data and microsatellite instability status of tumors. We have identified naturally occurring mutations in the MDRI gene associated with colorectal cancers with high microsatellite instability (MSI-H) suggesting that tumor cells with MDR1 mutations are selected for during development of MSI-H cancers and that MDR1 plays an important role in tumor initiation and progression in at least a proportion of MSI-H cancers. We found that in all MSI-H tumors with MDR1 mutations, both, the coding and promoter regions were mutated. These results and results from others suggest that alterations in MDR1 promoter are important for P-gp function and that screening for naturally occurring mutations in the promoter region of MDR1 is important in some of the human cancers. We have identified also 12 different germline polymorphisms and at least two of them were significantly associated with increased lymphoid infiltration in tumors suggesting physiological function for P-gp in immune response in addition to protection from xenobotics.

Immunochemical localisation of cathepsin S, cathepsin L and MHC class II-associated p41 isoform of invariant chain in human lymph node tissue.

Antigen presentation by MHC class II molecules requires cysteine proteases (CP) for two convergent proteolytic processes: stepwise degradation of the invariant chain (Ii) and generation of immunogenic peptides. Their activity is controlled by intracellular CP inhibitors, including presumably the p41 isoform of invariant chain (p41 Ii), which is in vitro a potent inhibitor of cathepsin L but not of cathepsin S. In order to evaluate the inhibitory potential of p41 Ii in antigen-presenting cells (APC), these three proteins were stained in lymph node tissue using specific monoclonal and polyclonal antibodies. The most abundant labelling was observed in subcapsular (cortical) and trabecular sinuses of the lymph node. In this area the most frequent APC were macrophages, as confirmed by the CD68 cell marker. Using confocal fluorescence microscopy, co-localisation of p41 Ii with cathepsin S, but not with cathepsin L was found in these cells. Our results are consistent with the hypothesis that cathepsin S participates in degradation of the invariant chain, but they do not support the association between cathepsin L and p41 Ii in APC.

Immunohistochemical localization of group II phospholipase A2 in the tumours and mucosa of the colon and rectum.

BACKGROUND: Group II phospholipase A(2)(PLA(2)) is an enzyme important in malignant transformation and in the invasion process of malignant cells. The aim of the present study was to investigate the expression of group II PLA(2)in the cancers of the colorectum, peritumoural mucosa and in the mucosa distant from the tumour. METHODS: Resection specimens from 57 patients with colorectal carcinoma (caecum 10, ascending 10, transverse 10, sigmoid colon nine, and rectum 18) were analysed immunohistochemically. Histological slides from paraffin blocks were stained by the human monoclonal group II PLA(2)antibody ('Upstate Biotechnology', Lake Placid, NY 12946, USA. Antibody Class: IgG1k. Immunogen: HPC purifed human sperm phospholipase A(2)- 14 kDa enzyme) using the standard DAKO peroxidase-labelled streptavidin-biotin method by TechMate 500 stainer. Group II PLA(2)expression was evaluated semi-quantitatively according to the extensivity and intensivity of the positive cells. For statistical evaluation the Kruskal-Wallis one way analysis of variance on ranks and the Mann-Whitney rank sum tests were used. RESULTS: The highest expression of group II PLA(2)was found in the peritumoural mucosa (median 4.00), much lower in the mucosa distal from the tumour (median 0.70) and almost no activity in the tumour itself (median 0.00), all differences were statistically significant (all pairwise multiple comparison procedures - Dunn's Method P<0.05). The expression of group II PLA(2)was higher in the left colon and rectum than in the right colon (Mann-Whitney rank sum test P<0.05). CONCLUSIONS: Our results suggest that there is variation of the group II PLA(2)expression throughout the mucosa and tumours of the colorectum, which might reflect the progression of neoplastic process.

Effect of primary treatment on survival in anaplastic thyroid carcinoma.

AIMS: Anaplastic thyroid carcinoma (ATC) is a fatal disease despite combined treatment consisting of chemotherapy, radiotherapy and surgery. The optimal sequence of treatment modalities is not known. The purpose of our retrospective non-randomized study was to find out whether timing of the treatment modality had any influence on survival, and to find out if primary surgery prolongs survival in comparison to primary chemotherapy and/or radiotherapy. METHODS: From our database of 162 patients with ATC treated at the Institute of Oncology Ljubljana from 1972-98, 79 patients (26 men, 53 women; age: 40-86 years, mean age 65 years) were included in this retrospective study. The 83 patients with distant metastases on admission, with the survival shorter than one month or patients without any treatment were excluded. The 79 patients were classified into (1) primary surgery group (n=26) and (2) primary chemotherapy and/or radiotherapy group (n=53), including the 12 patients in whom surgery was performed after chemotherapy and/or radiotherapy. The survival of both groups was compared by log-rank test and group characteristics by ANOVA and(2 test using SPSS program. RESULTS: In comparison to the primary surgery group, the patients from the primary chemotherapy and/or radiotherapy group were older and had faster growing, and larger tumours, which were not confined to the thyroid, and more frequently had regional metastases. There was no difference in the survival of the two groups (P=0.17). Survival for longer than one year was observed in 25% of patients with primary surgery and in 21% of patients with primary chemotherapy and/or radiotherapy. The best results (50% survival at one year) were obtained in patients in whom the tumour was surgically removed after primary chemotherapy and radiotherapy. CONCLUSION: This study suggests that the timing of the treatment modalities has an impact on survival and that treatment should start with chemotherapy and/or radiotherapy, with surgery to follow if possible.)

Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening.

Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.

Correlation of MTS1/p16 and nm23 mRNA expression with survival in patients with peripheral synovial sarcoma.

BACKGROUND AND OBJECTIVES: Tumor suppressor gene MTS1/p16 (cyclin-dependent kinase-4 inhibitor) and a putative tumor metastasis suppressor gene nm23 (nucleoside diphosphate A kinase) have been identified in a variety of human tumors but have not been well studied in mesenchymal neoplasms. METHODS: Expression of nm23 and MTS1 mRNA was determined by quantitative analysis from paraffin-embedded tumor tissue. The series comprised 31 patients with localized primary synovial sarcoma of soft tissues who were followed for a median of 83 months. RESULTS: Neither MTS1 nor nm23 expression levels correlated with the patient's age or sex, tumor type, depth, size, mitotic rate, or extent of tumor necrosis. In addition, there was no correlation between MTS1 and nm23 levels. Patients' survival was not related to sex, age, tumor type, location, mitotic rate, or MTS1 mRNA level. The only factors that correlated with poor survival in multivariate analysis were the presence of extensive tumor necrosis (> 15%) and higher levels of nm23 mRNA. CONCLUSIONS: Our results suggest that increased expression level of nm23 mRNA may be implicated in the mechanism of tumor progression and is associated with poor survival in patients with synovial sarcoma.

Decreased cortisol secretion by adrenal glands perfused with the P-glycoprotein inhibitor valspodar and mitotane or doxorubicin.

The aim of this study was to investigate the role of P-glycoprotein (P-gp) in the adrenal gland. It has been presumed that P-gp, rather than being involved in physiological cortisol secretion, plays a role in protecting the adrenacortical cells from xenobiotics. To explore this a study was performed on perfused bovine adrenal glands. Individual experimental groups were perfused with either a selective P-gp blocker (valspodar) alone, with a xenobiotic (mitotane or doxorubicin) alone or with both valspodar and a xenobiotic. The cumulative amounts of cortisol secreted in each individual group were calculated and the two-sample t-test was used to compare the mean values of cumulative amounts. The mean value of cortisol secreted from the group of adrenals perfused with the P-gp blocker was not significantly different from that of the control group. Treatment with either mitotane or doxorubicin decreased the amount of cortisol secreted but not significantly when compared to the amount of cortisol secreted in basal conditions. However, treatment with the P-gp blocker valspodar in addition to either mitotane or doxorubicin significantly decreased cortisol secreted compared to the amount of cortisol secreted by the glands treated with either mitotane (p=0.009) or doxorubicin (p=0.017) alone. The regressive changes discovered in all experimental groups were most prominent when valspodar was used with either mitotane or doxorubicin. We found that P-gp blockade increases by xenobiotic (mitotane and doxorubicin)-induced damage of adrenocortical cells, which points to a role of P-gp in the protection of adrenal gland from xenobiotics.

Evaluation of microsatellite markers for efficient assessment of high microsatellite instabile colorectal tumors.

Two hundred thirty randomly collected primary colorectal tumors were initially screened for microsatellite instability (MSI) with three highly informative microsatellite markers (BAT26, D2S123 and D5S346). Forty one (17.8%) tumors showed alterations in at least one marker. In further MSI analysis of these 41 MSI tumors with additional 9 markers, 21 tumors (9.6% of 230 analyzed) exhibited MSI at more than 40% and the rest 20 (8.7% of 230 analyzed) tumors exhibited MSI at 8%-20% tested markers. These results support classification of MSI tumors into high MSI tumors (more than 40% unstable loci) and low MSI tumors (less than 20% unstable loci). Based on our results the combination of BAT26 and two out of four other highly informative markers (D2S123, D5S346, BAT25 or BAT40) is recommended for rapid and reliable assessment of high MSI tumors.

Cathepsin B and its inhibitor stefin A in brain tumors.

Cysteine protease cathepsin B (CatB) and its endogenous inhibitor stefin A (StA) play an important role in tumor progression. Increase of CatB expression and lower levels of its inhibitors were associated with tumor malignancy in brain tumors. In this study of 100 patients, CatB was localized by immunostaining to both, tumor and endothelial cells of primary brain tissue. Significant correlation with poor prognosis was found by univariate Cox's regression model. Intense overall immunostaining and immunostaining in endothelial cells alone were prognostic for survival (p=0.003 in both). When comparing CatB expression at mRNA level, we found considerable differences between center and periphery of a tumor as well as between different tumor samples. StA mRNA was only detected in benign, but not in malignant tissues. We suggest that screening of cysteine-protease genes expression can be applied in clinical prognosis of brain tumors.

Papillary thyroid carcinoma with exuberant nodular fasciitis-like stroma in a fine needle aspirate. A case report.

BACKGROUND: Papillary thyroid carcinoma (PTC) with exuberant nodular fasciitis-like stroma (PTC-ES) is a new morphologic variant of conventional PTC. It is characterized by extensive reactive stromal proliferation, which may occupy 60-80% of the tumor. CASE: A 42-year-old female developed a tender, left-sided thyroid mass. The fine needle aspiration biopsy specimen contained, besides diagnostic epithelial features of PTC, many cohesive tissue fragments of cellular stroma. CONCLUSION: A correct cytopathologic diagnosis of PTC-ES can be established if both epithelial and stromal components are present in needle aspirates.

Prognostic factors in follicular carcinoma of the thyroid--a multivariate survival analysis.

AIMS: Multivariate studies of the diagnosis, treatment and prognosis of patients with follicular carcinoma of the thyroid gland are relatively scarce. The aim of our multivariate analysis was to study the factors associated with survival of patients with follicular carcinoma in Slovenia, in an iodine-deficient region. METHODS: This retrospective study was carried out in a group of 154 patients (113 women, 41 men; median age 61 years) with follicular carcinoma of the thyroid treated at the Institute of Oncology in Ljubljana between 1972-1992. Data on patient gender, age, disease history, extent of disease, morphological characteristics, mode of therapy and survival were collected. Statistical correlations between possible prognostic factors and survival were analysed by univariate and Cox's multivariate analysis. RESULTS AND CONCLUSIONS: The 10-year survival of the 154 patients was 60%. Multivariate analysis showed that tumour differentiation, primary tumour size, vascular invasion, distant metastases, regional lymph-node metastases, histological tumour type and age were independent prognostic factors for survival. The best prognosis was found in patients with well-differentiated T1 or T2 tumours, without extensive vascular invasion, without distant or regional metastases and aged under 46 years. Patients with Hürthle-cell type carcinomas had better prognosis than those with the follicular type. The worst prognosis was found in patients with poorly differentiated T4 tumours, with extensive vascular invasion, with distant or regional metastases and aged over 60 years.

Cathepsin B immunohistochemical staining in tumor and endothelial cells is a new prognostic factor for survival in patients with brain tumors.

The cysteine endopeptidase, cathepsin (Cat) B, and its endogenous inhibitor, stefin A, were found relevant for cancer progression of many neoplasms, including human brain tumors. Histological sections of 100 primary brain tumors, 27 benign and 73 malignant, were stained immunohistochemically for Cat B and stefin A. The immunohistochemical staining of Cat B in tumor cells, endothelial cells, and macrophages was scored separately from 0-12. The score in tumor and endothelial cells was significantly higher in malignant tumors compared with benign tumors (P<0.000). A significant correlation between immunostaining of Cat B (scored together for tumor and endothelial cells) and clinical parameters, such as duration of symptoms, Karnofsky score, psycho-organic symptoms, and histological score was demonstrated. Univariate survival analysis indicated that total Cat B score above 8 was a significant predictor for shorter overall survival (P = 0.003). In glioblastoma multiforme, intense Cat B staining of endothelial cells was a significant predictor for shorter survival (P = 0.003). Stefin A immunostaining was weak and detected only in a few benign and some malignant tumors, suggesting that this inhibitor alone is not sufficient in balancing proteolytic activity of Cat B. We conclude that specific immunostaining of Cat B in tumor and endothelial cells can be used to predict the risk of death in patients with primary tumors of the central nervous system.