Lauri Talve

Loss of expression of the p16INK4/CDKN2 gene in cutaneous malignant melanoma correlates with tumor cell proliferation and invasive stage

The G1/S checkpoint of the cell cycle is regulated by p16, p53 and RB tumor suppressor genes. Loss of expression of the p16INK4 tumor suppressor protein, the product of the CDKN2 gene, has been associated with a wide variety of human malignancies. Mutations, loss of heterozygosity and deletions of the CDKN2 locus have been reported in sporadic and familial cutaneous malignant melanomas (CMM). To investigate the role of the alterations of p16 expression in melanoma, we evaluated by immunohistochemistry the p16 expression and cell proliferation in 79 primary CMM and 10 benign melanocytic nevi (BMN). Forty‐six melanomas (58%) and all BMN were found to be p16 positive; 33 melanomas (42%) were considered p16 negative. The extent of invasion according to Clark was significantly higher in p16‐negative tumors than in p16‐positive tumors. Cell proliferation as expressed by the proportion of positive cells in Ki‐67 immunostaining was found to be significantly higher in p16‐negative tumors than in p16‐positive tumors, although there was no significant difference in the mitotic index between p16‐positive and p16‐negative tumors. In p16‐positive tumors, the number of Ki‐67‐positive cells correlated with the mitotic index; in p16‐negative tumors, there was no correlation between these parameters. Our data suggest that loss of p16 expression is more common in advanced melanomas, and that G1/S checkpoint regulation is disrupted in p16‐negative melanomas. Our results show that loss of p16 expression is a common event in primary melanomas, which further substantiates the role of p16 as a major tumor suppressor. Int. J. Cancer 74:255‐259, 1997.

Analysis of G1/S checkpoint regulators in metastatic melanoma

We have analyzed the expression of the CDKN1A (p21CIP1), CDKN1B (p27Kip1), TP53, RB1 and MDM2 proteins and tumor cell proliferation by immunohistochemical staining in 59 cases of metastatic melanoma. The genomic status of the CDKN2A (INK4‐ARF, p16/p14ARF), CDKN2B (p15) and CDKN2C (p18) genes was determined by PCR‐SSCP (single‐strand conformation polymorphism) in 46 of these cases. These results were correlated with various clinico‐pathological parameters, including the outcome of combined chemoimmunotherapy. We found positive correlations between the expression of CDKN1A and MDM2 (r = 0.5063, P = 0.001), between the expression of CDKN1B and RB1 (r = 0.5026, P = 0.001), and between RB1 expression and tumor cell proliferation (0.5564, P < 0.001). Two mutations in the CDKN2A (p16) gene were detected, including a novel base change AAC→ATC (Asn to Ile) at codon 71, that also changes the codon 85 of the alternative reading frame gene p14ARF from CAA to CAT (Gln to His). Homozygous deletion at exon 2 of the CDKN2A (INK4‐ARF) gene was detected in six cases. In seven cases, the 540C→G polymorphism in the 3′UTR of the CDKN2A (p16) gene was found in linkage disequilibrium with the 74C→A polymorphism in intron 1 of the CDKN2B gene (P < 0.0001). These cases had significantly lower expression of the TP53 protein (P = 0.0032). Both 540C→G and 580C→T polymorphisms in the 3′UTR of the CDKN2A (p16) gene were associated with significantly shorter progression time from primary to metastatic disease (P = 0.0071). We conclude, that although none of the analyzed cell cycle regulators could be singled out as a major prognostic factor, G1/S checkpoint abnormalities remain one of the most significant factors in the development of malignant melanoma. Genes Chromosomes Cancer 28:404–414, 2000.

Prognostic Value of the Expression of Tumor Suppressor Genes p53, p21, p16 and pRb, and Ki-67 Labelling in High Grade Astrocytomas Treated with Radiotherapy

Cumulative inactivation of tumor suppressor genes and/or amplification of oncogenes lead to progressively more malignant astrocytic tumors. We have analyzed the significance of tumor suppressor genes p53, p21, p16 and retinoblastoma protein (pRb) and proliferative activity for survival in 77 high grade astrocytic tumors.After operation, the patients – 25 anaplastic astrocytomas (AA) and 52 glioblastomas (GBs) – were treated with similar radiotherapy. The expression of the suppressor genes and the proliferative activity were analyzed immunohistochemically.p53 immunopositivity was found in 44% of AAs and 46% of GBs. Tumors with aberrant p53 expression had lower proliferation indices than p53 immunonegative tumors. Neither p53 expression nor p21 immunonegativity (52% of AAs and 48% of GBs) correlated with survival. p16 immunostaining was negative in 16% of AAs and in 44% of GBs, and it correlated inversely with survival in both uni- and multivariate analyses. pRb immunostaining was negative only in 8% of both AAs and GBs and the absence of p16 and pRb were mutually exclusive.Ki-67 labelling index (LI) was significantly higher in GBs (26.8%) than in AAs (20.3%), and in multivariate analysis it was an independent prognostic factor for survival. In 48% of AAs Ki-67 LI exceeded 20% and this subset of AAs had similar prognosis as GB.In high grade astrocytic tumors p16 immunonegativity was an independent indicator of poor prognosis in addition to the previously established patients age, histopathology and Ki-67 LI. Furthermore, there was a subset of AAs with a high proliferation rate (>20%) in which the histopathological hallmarks of GB were lacking, but which had similarly dismal prognosis as GB.

Nuclear morphometry, immunohistochemical staining with Ki-67 antibody and mitotic index in the assessment of proliferative activity and prognosis of primary malignant melanomas of the skin

Nuclear morphometry, immunohistochemical staining with Ki‐67 antibody and mitotic index were studied in primary cutaneous malignant melanomas. The number of Ki‐67 positive cells/200 tumor cells did not correlate with any nuclear morphometrical parameters, and it only approached but did not reach significant correlation with melanoma thickness according to Breslow. The nuclear area, short axis and long axis correlated with melanoma thickness, but the nuclear axis ratio (which reflects the sphericity of nuclei) and melanoma thickness did not show significant correlation. Mitotic index was higher in thick melanomas and in melanomas with high Ki‐67 positivity, large nuclear area, long nuclear short axis, and small nuclear axis ratio. In Coxs stepwise proportional hazard model, melanoma thickness and the nuclear axis ratio were significant independent prognostic factors for patient survival, while the nuclear area, short axis and long axis, gender, age, Clark level, mitotic index and Ki‐67 positivity lacked significant independent prognostic value.

Immunohistochemically Detectable Bcl-2 Expression in Metastatic Melanoma: Association with Survival and Treatment Response

Objective: The antiapoptotic protein Bcl-2 is supposed to influence the treatment responsiveness of different malignancies. In the present study the prognostic and predictive significance of Bcl-2 expression for survival and response to an administered therapy was explored in patients with metastatic melanoma. Also, the correlation between Bcl-2 expression and proliferation activity of tumor cells was defined to examine the regulatory role of Bcl-2 in proliferation. Materials and Methods: Sixty metastatic melanomas obtained from patients treated with chemoimmunotherapy were examined by immunohistochemistry with anti-Bcl-2 and anti-Ki-67 (MIB-1) antibodies. Proliferation activity was expressed in percentages as MIB-1 index. Results: The presence of Bcl-2 immunoreactivity was associated with a significantly lower MIB-1 index (p = 0.016), and a longer disease-free survival (p = 0.004). The lack of Bcl-2 expression was related to a higher response rate to therapy in comparison to a diffuse and focal pattern of Bcl-2 expression (p = 0.017). Although the presence of Bcl-2 immunoreactivity as such did not correlate with survival after the initiation of chemoimmunotherapy, the focal Bcl-2 expression pattern was strongly associated with a worse prognosis compared to a diffuse expression or a lack of Bcl-2 staining (p < 0.0001). Conclusions: Our results support the role of Bcl-2 in the regulation of cell proliferation and suggest that an increase of metastatic potential and progression of malignant melanoma is associated with a loss of Bcl-2 expression. The lack of Bcl-2 expression could be a predictor of the response to chemoimmunotherapy, whereas the Bcl-2 expression pattern, possibly indicating the heterogeneity of the tumors, might be a potential prognostic factor for survival after the initiation of therapy.

The extracellular matrix in skin tumor development – a morphological study

The development of cancer involves epithelial‐stromal interactions. Alterations in the synthesis and deposition of type I and III collagens are related to the tumor morphology. Skin carcinogencsis in experimental animals provides a reliable model for the development of neoplasia. Ultraviolet (UV) irradiation is the main etiological factor for epidermal dysplasia and malignant tumors in man, but also for dermal degeneration. Non‐neoplastic dermal changes and skin tumors induced by ultraviolet irradiation and 7,12‐dimethylbenz(a)anthra‐cene were investigated in various mouse strains with different susceptibilities to tumor formation. UVB irradiation resulted in an increased immunoreactivity of collagens in the dermis, in comparison with 7,12‐dimethylbenz(a)anthxacene. Increased synthesis and deposition of type I and III collagens were found in the stroma adjacent to benign alterations. In well‐differentiated squamous cell carcinomas, a similar induction of collagen synthesis and deposition was observed. The destruction of fibrillary structures was more pronounced during the decrease of differentiation from moderately to poorly differentiated squamous cell carcinomas. Anaplastic carcinomas with spindle cell morphology displayed a delicate meshwork of reticular fibers and collagen III, and abnormal expression of mRNA for collagens in some malignant cells with epithelial characteristics. The underlying stroma reacts to the development of epithelial tumors in a reproducible way, which is related to the carcinogenic agent involved.

Progesterone receptor negativity is an independent risk factor for relapse in patients with early stage endometrioid endometrial adenocarcinoma

OBJECTIVE In endometrioid endometrial adenocarcinoma (EEA), the currently established prognostic factors in clinical guidelines are stage and grade. Many guidelines include lymphovascular invasion (LVI) and tumor size as prognostic factors. Although several studies have associated lack of estrogen (ER) and progesterone receptor (PR) expression with reduced outcome, the prognostic use of these markers is uncommon. Better prognostication of clinical behavior would be useful in patients with early stage (I-II) disease. In this study we evaluated ER and PR as prognostic factors in EEA, and compared their expression with other potential biomarkers and clinical parameters. METHODS Tissue microarrays were constructed from 182 patients with stages I-II EEA. ER, PR, p53, Ki-67, PTEN, MLH and HER-2 expression were assessed by immunohistochemical staining and HER-2 was confirmed with SISH. The results were correlated with clinicopathologic parameters and to disease-free survival. RESULTS Eleven patients (6%) developed recurrent disease during a median follow up time of 62.8 months. In univariate analysis FIGO grade (p=0.019), positive expression of p53 (p=0.010) and negative PR expression (p=0.001) were associated with a shorter disease-free survival. In multivariate analysis only negative PR expression (p=0.019) was significantly associated with a shorter disease-free survival. LVI and tumor size where not of prognostic value. CONCLUSIONS Lack of PR expression is a strong, independent risk factor for tumor recurrence in patients with stages I-II endometrioid endometrial cancer. The use of this easily measurable biomarker as a prognostic factor in the clinical context should be considered and tested in a larger patient population.

MIB-1 Immunoreactivity Correlates with Blood Vessel Density and Survival in Disseminated Malignant Melanoma

The purpose of our current work was to evaluate the prognostic significance of tumor cell proliferation in advanced metastatic melanomas and to investigate a possible correlation between the proliferation index and blood vessel density in metastatic tissue. Sixty patients with disseminated malignant melanoma treated with four-drug chemotherapy combined with interferon-α were included in this study. Proliferative activity and vascularity in metastatic tissues were examined by immunohistochemistry with anti-Ki-67 (MIB-1) and anti-CD31 antibody, respectively. A significant relationship between MIB-1 index and blood vessel number was detected (rho = 0.323, p = 0.013). In survival analysis, the overall survival and disease-free survival were significantly longer (58 and 38 vs. 38 and 17 months) for patients with low MIB-1 immunoreactivity (p = 0.012 and p = 0.023, respectively). Likewise, the low MIB-1 labeling index was associated with the prolonged survival calculated from the initiation of the chemoimmunotherapy (12 vs. 7 months, p = 0.032). In multivariate Cox’s proportional hazard analysis, MIB-1 positivity was an independent prognostic factor both for overall survival and for survival after beginning of the chemoimmunotherapy (p = 0.016 and p = 0.029).

Head and neck cutaneous melanoma: A retrospective observational study on 146 patients

Background. Sentinel node biopsy (SNB) is a novel staging technique in cutaneous melanoma, but it is more challenging in the head and neck (H&N) than in the trunk and extremities. The aim of this study was to investigate the utility of SNB in patients with clinical stage I-II H&N cutaneous melanoma, with emphasis on disease outcome. Patients and methods. Twenty five patients with H&N melanoma of >1.0 mm in Breslow depth underwent SNB and were compared to 121 historic H&N melanoma patients, who had either undergone routine prophylactic neck dissection or had been observed without any invasive nodal staging. Results. Sixteen percent of the SNB patients were sentinel-positive and there have been no false-negative cases. In the Kaplan-Meier analysis, there were no significant differences between the study groups in melanoma-specific overall survival. Among the entire cohort, melanoma-specific overall survival rate was 67.1% at 5 years and 61.9% at 10 years. Predictive factors for worsen survival were nodal micrometastases, male sex, scalp location, thick primary lesion and ulceration. Discussion. SNB is a reliable and mini-invasive approach for the nodal staging of H&N cutaneous melanoma. Traditional neck dissection is recommended only for therapeutic purposes in clinically node-positive or sentinel-positive patients.

Previous pregnancy is a favourable prognostic factor in women with localised cutaneous melanoma

Abstract Background. The influence of pregnancy on survival in melanoma has been a controversial issue. Objective. In this retrospective study we investigated whether pregnancy (overall or temporally melanoma-related) has any effect on melanoma progression or patient outcome. Methods. Patient data were collected from Turku University Hospital records concerning all women in fertile age (15–45 years) and diagnosed with melanoma between January 1, 1990 and December 31, 2009. We collected data on melanoma characteristics, treatment, pregnancies and patient outcomes. Results. Of the 334 patients, 248 (74%) had been pregnant in some point during their life while 55 (17%) were nulliparous. The history of pregnancies could not be verified in 31 women (9%). Progression of melanoma to advanced stage was found in 58 (17%) of these women. Altogether, 35 women (14%) with at least one pregnancy had disease progression in contrast to 14 women (26%) with no pregnancies (p =0.049). Women with at least one pregnancy had a 94% probability to survive from melanoma compared to nulliparous women of whom only 83% survived (p =0.041). In Multivariate (COX) analysis pregnancy was a favourable factor for disease-specific survival (DSS) (HR 3.75; 95% CI 1.24–11.34; p =0.019) when adjusted for age (HR 1.064; 95% CI 1.00–1.13; p =0.50), localisation and stage (p =0.040), and Breslow (HR 1.32; 95% CI 1.10–1.58; p =0.002). However, when ulceration of the primary tumour was included in the multivariate model, Breslow remained as the only independent predictor of DSS (HR 1.58; 95% CI 1.34–1.86; p =0.0001) and pregnancy was dropped from the stepwise backward model in the step preceding the last one (p =0.081). Conclusion. Pregnancy is not a risk factor for disease recurrence or progression in melanoma patients, but instead can exert some favourable influence on prognosis.