Application of morphometry, static DNA ploidy analysis, and steroid receptor expression in diagnosis and prognosis of Libyan breast cancer

The aim of this study was to describe the demographic, clinicopathological, biological and morphometric features of Libyan breast cancer patients. The supporting value of nuclear morphometry and static image cytometry in the sensitivity for detecting breast cancer in conventional fine-needle aspiration biopsies were estimated. The findings were compared with findings in breast cancer in Finland and Nigeria. In addation, the value of ER and PR were evaluated. There were 131 histological samples, 41 cytological samples, and demographic and clinicopathological data from 234 Libyan patients. The Libyan breast cancer is dominantly premenopausal and in this feature it is similar to breast cancer in sub-Saharan Africans, but clearly different from breast cancer in Europeans, whose cancers are dominantly postmenopausal in character. At presention most Libyan patients have locally advanced disease, which is associated with poor survival rates. Nuclear morphometry and image DNA cytometry agree with earlier published data in the Finnish population and indicate that nuclear size and DNA analysis of nuclear content can be used to increase the cytological sensitivity and specificity in doubtful breast lesions, particularly when free cell sampling method is used. Combination of the morphometric data with earlier free cell data gave the following diagnostic guidelines: Range of overlap in free cell samples: 55 μm2 -71 μm2. Cut-off values for diagnostic purposes: Mean nuclear area (MNA) >54 μm2 for 100% detection of malignant cases (specificity 84 %), MNA < 72 μm2 for 100% detection of benign cases (sensitivity 91%). Histomorphometry showed a significant correlation between the MNA and most clinicopathological features, with the strongest association observed for histological grade (p <0.0001). MNA seems to be a prognosticator in Libyan breast cancer (Pearson’s test r = - 0.29, p = 0.019), but at lower level of significance than in the European material. A corresponding relationship was not found in shape-related morphometric features. ER and PR staining scores were in correlation with the clinical stage (p= 0.017, and 0.015, respectively), and also associated with lymph node negative patients (p=0.03, p=0.05, respectively). Receptor-positive (HR+) patients had a better survival. The fraction of HR+ cases among Libyan breast cancers is about the same as the fraction of positive cases in European breast cancer. The study suggests that also weak staining (corresponding to as few as 1% positive cells) has prognostic value. The prognostic significance may be associated with the practice to use antihormonal therapy in HR+ cases. The low survival and advanced presentation is associated with active cell proliferation, atypical nuclear morphology and aneuploid nuclear DNA content in Libyan breast cancer patients. The findings support the idea that breast cancer is not one type of disease, but should probably be classified into premenopausal and post menopausal types.

Libyan breast cancer: Health services and biology. Diagnosis delay and prognostic value of DNA pploidy, S-phase fraction, and Ki-67 and Bcl-2 immunohistochemistry

The aim of this study was to investigate the diagnosis delay and its impact on the stage of disease. The study also evaluated a nuclear DNA content, immunohistochemical expression of Ki-67 and bcl-2, and the correlation of these biological features with the clinicopathological features and patient outcome. 200 Libyan women, diagnosed during 2008–2009 were interviewed about the period from the first symptoms to the final histological diagnosis of breast cancer. Also retrospective preclinical and clinical data were collected from medical records on a form (questionnaire) in association with the interview. Tumor material of the patients was collected and nuclear DNA content analysed using DNA image cytometry. The expression of Ki-67 and bcl-2 were assessed using immunohistochemistry (IHC). The studies described in this thesis show that the median of diagnosis time for women with breast cancer was 7.5 months and 56% of patients were diagnosed within a period longer than 6 months. Inappropriate reassurance that the lump was benign was an important reason for prolongation of the diagnosis time. Diagnosis delay was also associated with initial breast symptom(s) that did not include a lump, old age, illiteracy, and history of benign fibrocystic disease. The patients who showed diagnosis delay had bigger tumour size (p<0.0001), positive lymph nodes (p<0.0001), and high incidence of late clinical stages (p<0.0001). Biologically, 82.7% of tumors were aneuploid and 17.3% were diploid. The median SPF of tumors was 11% while the median positivity of Ki-67 was 27.5%. High Ki-67 expression was found in 76% of patients, and high SPF values in 56% of patients. Positive bcl-2 expression was found in 62.4% of tumors. 72.2% of the bcl-2 positive samples were ER-positive. Patients who had tumor with DNA aneuploidy, high proliferative activity and negative bcl-2 expression were associated with a high grade of malignancy and short survival. The SPF value is useful cell proliferation marker in assessing prognosis, and the decision cut point of 11% for SPF in the Libyan material was clearly significant (p<0.0001). Bcl-2 is a powerful prognosticator and an independent predictor of breast cancer outcome in the Libyan material (p<0.0001). Libyan breast cancer was investigated in these studies from two different aspects: health services and biology. The results show that diagnosis delay is a very serious problem in Libya and is associated with complex interactions between many factors leading to advanced stages, and potentially to high mortality. Cytometric DNA variables, proliferative markers (Ki-67 and SPF), and oncoprotein bcl-2 negativity reflect the aggressive behavior of Libyan breast cancer and could be used with traditional factors to predict the outcome of individual patients, and to select appropriate therapy.

Molecular Markers of Oral Lichen Planus

Oral lichen planus (OLP) is a chronic inflammatory mucosal disease and is detected in between 0.5% - 2.2% of the population. WHO has defined OLP as a potentially precancerous disorder, representing a generalized state associated with a significantly increased risk of cancer. However, only 0.5 – 2.9% of OLP lesions will progress to cancer. Currently, there are no prognostic markers to identify the lesions at increased risk for malignant transformation. The main aim of these studies was to identify cellular and molecular markers in order to understand the pathogenesis of atrophic OLP and its progression towards malignancy. Selected markers for cell proliferation, adhesion, apoptosis, and lymphocytic infiltration were assessed by immunohistochemistry in addition to static cytometry analyses for DNA content. DNA quantification of epithelial cells in 82 biopsy samples derived from atrophic lichen planus showed altered DNA content in 41% of the samples. DNA content was associated with proliferation activity, topoisomerase IIalpha, desmocollin-1 and infection with human papillomavirus. CD27+ and CD38+ lymphocytes were detected in inflammatory cell infiltrate, indicating an abnormal homing of B cells from blood circulation to tissue. Physiologic cell death, apoptosis, is frequently seen in OLP, but its pathways are unknown. Here it was shown that caspases 2 and 12 were up-regulated in OLP, indicating that intracellular apoptosis, rather than an external causal factor, is triggering apoptosis. However, this thesis could not identify any singular prognostic marker of malignancy in OLP. Thus, every OLP patient should receive regular follow-up care to identify cancer risk patients at an early stage.

Securin, Cdc20 and Cdc27 in predicting the outcome of human breast cancer

Deregulated proliferation has been recognized among the most important factors promoting breast cancer development and progression. The aim of the project is to gain understanding of the role of specific cell cycle regulators of metaphase-anaphase transition and evaluate their potential in breast cancer prognostication and treatment decisions. Metaphase-anaphase transition is triggered by activation of anaphase promoting complex (APC) which is activated by a cascade of regulatory proteins, among them securin, Cdc20 and Cdc27. These proteins promote the metaphase–anaphase transition and participate in the timely separation of the chromatids. This study is based on a patient material of approximately 600 breast cancer patients and up to 22 years of follow-up. As the main observation, based on DNA cytometric and immunohistochemical methods, securin, Cdc20 and Cdc27 protein expressions were associated with abnormal DNA content and outcome of breast cancer. In the studied patient material, high securin expression alone and in combination with Cdc20 and Cdc27 predicted up to 9.8-fold odds for aneuploid DNA content in human breast cancer. In Kaplan–Meier analyses, high expression of securin systematically indicated decrease in breast cancer survival as compared to low expression cases. The adverse effect of high securin expression was further strengthened by combining it with Cdc20 or Cdc27 expressions, resulting in up to 6.8-fold risk of breast cancer death. High securin and Cdc20 expression was also associated with triple-negative breast cancer type with high statistical significance. Securin, Cdc20 or Cdc27 have not previously been investigated in a clinically relevant large breast cancer patient material or in association with DNA ploidy. The present findings suggest that the studied proteins may serve as potential biomarkers for identification of aggressive course of disease and unfavourable outcome of human breast cancer, and that they may provide a future research aim for understanding abnormal proliferation in malignant disease.