Pekka J. Klemi

Florodeoxyglucose imaging: A method to assess the proliferative activity of human cancer in vivo. Comparison with DNA flow cytometry in head and neck tumors

Thirteen patients with malignant head and neck tumors were studied before they were treated with (18F) fluorodeoxyglucose (FDG) imaging and DNA flow cytometry (FCM). The nuclear DNA content and the percentage of proliferative cells (S+G2/M) were compared with the FDG uptake; FDG was retained in the primary tumor and/or neck metastasis in all patients. The accumulation of FDG did not correlate with histologic grade of the tumors, but there was a clear correlation (r = 0.86, P > 0.001) between the proportion of the cells in S+G2/M phases of the cell cycle and the intensity of FDG accumulation. The uptake of FDG by the tumor also correlated with the percentage of S‐phase cells (r = 0.82, P > 0.001). The result suggests that enhanced glucose metabolism, measured by FDG uptake, is associated with the proliferative activity of the tumor. Thus, imaging with FDG may offer a new method to assess the aggressiveness of human cancer growth in vivo.

Association between syndecan-1 expression and clinical outcome in squamous cell carcinoma of the head and neck.

Syndecans are a family of cell-surface heparan sulphate proteoglycans which are involved in cell-matrix interactions and growth factor binding. Syndecan-1 binds basic fibroblast growth factor (bFGF) and several components of the extracellular matrix. Syndecan-1 expression is induced during keratinocyte differentiation and reduced during the formation of squamous cell carcinomas (SCCs). The purpose of this study was to examine the association of syndecan-1 expression with prognostic factors and clinical outcome in SCC of the head and neck. Frozen sections of 29 primary SCCs were analysed for syndecan-1 expression using immunohistochemical methods. Intermediate or strong staining for syndecan-1 was associated with a smaller primary tumour size (P = 0.0005) and higher histological grade of differentiation (P = 0.006) than negative or weakly positive staining. In a univariate analysis, syndecan-1-positive tumours were associated with higher overall (P = 0.001) and recurrence-free survival (P = 0.003) than those tumours with no or little syndecan-1 expression. The results suggest that syndecan-1 could be an important prognostic factor of SCC of the head and neck. Further studies on the prognostic significance of syndecan-1 expression in SCCs are warranted.

Collagenase-3 (MMP-13) is Expressed by Tumor Cells in Invasive Vulvar Squamous Cell Carcinomas

Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by invading tumor cells in squamous cell carcinomas (SCCs) of the head and neck. Here, we have further elucidated the role of MMP-13 in tumor invasion by examining its expression in invasive malignant tumors of the female genital tract. Using in situ hybridization, expression of MMP-13 mRNA was detected in 9 of 12 vulvar SCCs, primarily in tumor cells, but not in intact vulvar epithelium, in cervical SCCs (n = 12), or in endometrial (n = 11) or ovarian adenocarcinomas (n = 8). MMP-13 expression was especially abundant in vulvar carcinomas showing metastasis to lymph nodes and was associated with expression of membrane type 1 MMP by tumor cells and gelatinase-A (MMP-2) by stromal cells, as detected by immunohistochemistry. MMP-13 mRNAs were detected in 9 of 11 cell lines established from vulvar carcinomas and in 4 of 6 cell lines from cervical carcinomas, whereas endometrial (n = 10) and ovarian (n = 9) carcinoma cell lines were negative for MMP-13 mRNA. No correlation was detected between MMP-13 expression and p53 gene mutations in vulvar SCC cell lines. However, MMP-13 expression was detected in 5 of 6 vulvar and cervical SCC cell lines harboring HPV 16 or 68 DNA. These results show that MMP-13 is specifically expressed by malignantly transformed squamous epithelial cells, including vulvar SCC cells, and appears to serve as a marker for their invasive capacity.

The prognostic significance of nuclear DNA content in invasive breast cancer--a study with long-term follow-up.

The nuclear DNA content of 351 breast carcinomas was determined by flow cytometry from paraffin-embedded tissue to assess the prognostic significance of DNA ploidy, the DNA index (DI) and the S-phase fraction (SPF). The minimum follow-up of the patients was 22 years, and they were all from a defined urban population. DNA ploidy correlated with histological type and grade, mitotic count and nuclear pleomorphism (P less than 0.0001), and also with axillary nodal status (P = 0.0005), tumour necrosis (P = 0.001), primary tumour size (P = 0.03), menopausal status (P = 0.004) and the presence of distant metastases at the time of the diagnosis (P = 0.04). Survival corrected for intercurrent deaths of the patients with a diploid tumour was better than that of the patients with a non-diploid tumour (P = 0.0001, 48% vs 28% at 25 years). SPF had prognostic significance in both axillary node positive and negative patients, but ploidy and DI only in the node negative group, and their significance was greater in post-menopausal than in premenopausal patients. Axillary nodal status, primary tumour size, histological grade and the type of tumour margin circumscription were the most important independent prognostic factors in Coxs multivariate analysis, and SPF had independent prognostic value, whereas ploidy and DI did not. It is concluded that DNA ploidy, DI and SPF have long-term prognostic significance in breast cancer.

Syndecan-1: A New Prognostic Marker in Laryngeal Cancer

The cell adhesion molecule syndecan-1 expression is induced during keratinocyte differentiation and reduced during the formation of squamous cell carcinomas (SCCs). A significant correlation between decreased syndecan-1 expression in head and neck SCC measured from frozen sections with immuohistochemical methods and clinical outcome are reported. The clinical relevance of the cellular proliferation marker Ki-67 is controversial in SCC of the head and neck. The purpose of this study was to determine the expression of syndecan-1 and Ki-67 in SCC of the larynx and correlate the results with known prognostic factors and clinical outcome. Paraffin-embedded samples of 100 patients with laryngeal SCC (44 glottic, 36 supraglottic, 20 transglottic) treated at Turku University Central Hospital were re-examined and divided into four histological grades of differentiation, four grades of keratinisation, and four grades of 104-9 (syndecan-1) immunostaining. The mitotic index was analysed as the number of mitoses per volume corrected high power fields. The relative number of Ki-67 positive cells was evaluated. The patients mean age was 64 years and the 5-year survival was 69%. In univariate analysis, intermediate or strong staining for syndecan-1 was associated with higher overall survival than those tumours with no or little syndecan-1 expression (p = 0.048). Nodal status (p = 0.0001), tumour size (p = 0.0004) and localisation (p = 0.0008), general condition (p = 0.0001), histological grade (p = 0.02) and patient age (p = 0.03) correlated with overall survival whereas the Ki-67 index (p = 0.093), mitotic index (p = 0.23) and grade of keratinisation (p = 0.90) failed to do so. The results suggest that syndecan-1 could be a useful prognostic factor in SCC of the larynx.

Influence of cellular DNA content on survival in differentiated thyroid cancer

Cellular DNA content was measured using a novel flow cytometric method to analyze paraffin‐embedded tissue blocks from 125 patients with differentiated thyroid cancer. DNA aneuploidy was found in 20 (24%) of the 82 papillary, 20 (56%) of the 36 follicular, and in four (57%) of the seven medullary carcinomas. Aneuploidy was found to be more common in the elderly (P < 0.002), in moderately and poorly differentiated carcinomas (P < 0.004), and in tumors infiltrating beyond the thyroid capsule (P < 0.03). Patients with an aneuploid tumor had less favorable cumulative survival (P < 0.0001) than patients with diploid cancer. However, in papillary and follicular carcinomas, multivariate analysis using stepwise Cox model showed age at diagnosis, follicular type, and tumor invasion beyond the thyroid capsule to be more important independent prognostic factors. Increasing probability of DNA aneuploidy with increasing age explains partially why prognosis of differentiated thyroid carcinoma is poor in older patients.

Salivary gland cancer in Finland 1991–96: an evaluation of 237 cases

Conclusion In this material consisting of various salivary gland carcinomas, stage I, male gender and age were the most powerful predictors of patient outcome. Objectives To retrieve the records of all salivary gland cancer (SGC) patients diagnosed in Finland between 1991 and 1996 and to evaluate the incidence, histological type and location of SGC, the treatment given and the outcome. Material and methods The records for all SGCs (n=286) diagnosed in Finland between 1991 and 1996 and reported to the Finnish Cancer Registry were retrieved. The histological re-evaluation and retrospective study involved 237 SGC patients. Results The study population consisted of 125 males and 112 females. The mean age was 59 years (males 61 years, females 58 years). Follow-up was at least 5 years. The commonest tumor location was the parotid gland (n=152; 64%), followed by the minor salivary glands (n=46; 19%), the submandibular gland (n=38; 16%) and the sublingual gland (n=1; 0.4%). The most frequent histological types of SGC were adenoid cystic carcinoma (n=65; 27%), mucoepidermoid carcinoma (n=45; 19%) and acinic cell carcinoma (n=41; 17%). Surgery, either alone or in combination with other treatment modalities, was used in 209 cases (88%). Radiotherapy was given to 136 patients (57%), 13 of whom (5%) did not undergo surgery. The 5-year overall survival rate was 56.5%, and for stages I–IV it was 78%, 25%, 21% and 23%, respectively (p<0.001; log-rank test). Of the commonest tumor types, the best 5-year relative survival rate was for patients with acinic cell carcinoma (96%), followed by those with mucoepidermoid (79%) and adenoid cystic carcinoma (74%).

Metalloelastase (MMP-12) expression by tumour cells in squamous cell carcinoma of the vulva correlates with invasiveness, while that by macrophages predicts better outcome

Human metalloelastase (MMP‐12) has been implicated in elastin degradation and macrophage migration in many pathological conditions. It also generates angiostatin, thus having a potential to prevent tumour angiogenesis. It has previously been shown that transformed epithelial cells express MMP‐12 in skin cancer. The aim of this study was further to elucidate the role of metalloelastase in squamous cell cancer (SCC) progression. By in situ hybridization, expression of MMP‐12 mRNA was detected in 28/33 vulvar SCC samples in CD‐68‐positive macrophages, while 10 samples had positive cancer cells. By immunohistochemistry, MMP‐12 protein was seen in the same area as the mRNA. MMP‐12 mRNA expression in tumour cells correlated with more aggressive histology (p = 0.0099). In contrast, macrophage‐derived MMP‐12 mRNA was more abundant in well‐differentiated grade I than grade III tumours (p = 0.01). However, the level of MMP‐12 mRNA, regardless of its origin, did not correlate with metastasis or patient survival. No significant correlation was found between macrophage‐derived MMP‐12 mRNA and a low amount of blood vessels, as quantitated after von Willebrand staining. In agreement with vulvar SCCs in vivo, MMP‐12 was expressed in cultured SCC cells by northern and western blot analysis. In HaCaTs and epithelial MCF‐10f cells, MMP‐12 mRNA was induced by transforming growth factor‐β1 (TGF‐β1) and tumour necrosis factor‐α (TNF‐α) as measured by quantitative RT‐PCR (TaqMan). Two MMPs capable of generating angiostatin in vivo, matrilysin (MMP‐7) and gelatinase B (MMP‐9), were also examined in these tumours. MMP‐7 mRNA was mainly expressed by epithelial tumour cells, particularly in less differentiated tumours. MMP‐9 was usually expressed by neutrophils and macrophages; epithelial protein was predominantly found in grade II/III tumours. These results suggest a dual role for MMP‐12 in tumour progression. Copyright

DNA index and S‐phase fraction and their combination as prognostic factors in operable ductal breast carcinoma

The prognostic significance of DNA ploidy, DNA index (DI), and S‐phase fraction (SPF) and their various combinations were studied together with 16 other clinicopathologic factors in 222 patients with operable invasive ductal breast carcinoma. The patients have been followed for a minimum of 22 years after the diagnosis or until death. Nuclear DNA content was determined by flow cytometry from paraffin‐embedded tissue. Patients with DNA diploid cancer (n = 57, 26%) had better survival rate corrected for intercurrent deaths than patients with nondiploid cancer (P = 0.002), and also, a small SPF (±14%, calculated in 134 cases) was associated with a favorable outcome in a univariate analysis (P = 0.01). The prognostic value of the DI and SPF was increased if they were combined. The most effective combination was obtained if diploid cancers were grouped together with DNA aneuploid cancers with a DI < 2.1 and an SPF < 14%. This combination had considerable prognostic value in a univariate analysis (P = 0.0002) and had independent prognostic value (P < 0.04) in Coxs multivariate analysis together with the primary tumor size (P < 0.001) in axillary node negative patients but not in axillary node positive patients. in the whole series the presence of axillary nodal metastases (P < 0.001), high mitotic count (P < 0.001), a large primary tumor size (P = 0.001), poorly circumscribed tumor margin (P = 0.005), and slight or absent tubule formation (P = 0.05) were the only independent prognostic factors in a multivariate analysis.

Clinical significance of abnormal nuclear DNA content in serous ovarian tumors

The nuclear DNA content of 160 serous ovarian neoplasms was determined by flow cytometry from paraffin‐embedded tissue. Three (11%) of the 27 histologically benign, seven (16%) of the 43 borderline malignant, and 59 (66%) of the 90 malignant neoplasms were aneuploid (P < 0.0001). None of the patients with an aneuploid benign or borderline malignant tumor died from cancer, but in carcinomas the DNA index (DI) was a more important prognostic factor in a multivariate analysis than age at diagnosis, stage, histologic grade or ploidy (diploid versus aneuploid). A DI of 1.3 was the most effective value hi predicting prognosis; patients with carcinoma with the DI more than 1.3 had inferior survival compared with those with the DI less than 1.3 (P = 0.002). Carcinomas with the DI more than 13 were more common in patients older than 60 years at diagnosis (P = 0.0002), and were associated with a low grade of differentiation (P = 0.008) but not with stage. It is concluded that DNA aneuploidy may occur in benign and borderline malignant serous ovarian tumors and that the DI is a highly valuable and objective prognostic parameter in serous ovarian carcinomas.