Prostate cancer epidemiology in a rural area of North Western Greece.

Epirus is a rural area of North-Western Greece. We reviewed data from 4 hospitals for 4.975 patients who underwent prostate biopsy in Epirus in the twelve year period from 1999 to 2010. Two six-year periods were compared (1999-2004 and 2004-2010). All cases of prostate cancer confirmed by biopsy were recorded and age-standardized incidence rates per 100,000 males were calculated. We also recorded the clinical stage for patients diagnosed in our hospital and correlated this with PSA and Gleason scores. Percentage of positive prostate biopsies was also calculated. There were a total of 1714 new cases during 1999-2010 and the mean annual age-adjusted incidence was 34/100,000. The mean incidences during 1999-2004 and 2005-2010 were 26/100,000 and 42/100,000, respectively. The mean age at diagnosis was 74. The most common Gleason score was 6 and the prevalent clinical stage was T2. Median PSA at diagnosis was 10.8 ng/ml. There was a significant difference between stage cT4 and all other stages regarding PSA value (p=0.000). A positive correlation was found between Gleason score and PSA (p=0.013). These results are in accordance with the incidence rise recorded in neighboring countries of South-East Europe. However we should keep in mind the risk of overdiagnosis and the detection of low-risk cancers that would not have caused morbidity or death during a man's lifetime anyway.

Expression of Bax protein in gastric carcinomas. A clinicopathological and immunohistochemical study.

BACKGROUND AND STUDY AIMS: Reduced Bax protein expression has been shown to be a negative prognostic factor in patients with breast, ovarian, colorectal, esophageal and pancreatic cancer. Our aim was to immunohistochemically study Bax protein expression in gastric carcinomas and correlate its expression with clinicopathological parameters and prognosis. PATIENTS AND METHODS: Immunohistochemistry was performed, using a monoclonal antibody against bax, in paraffin-embedded tumor specimens from 47 cases of gastric cancer. RESULTS: Positive staining for the Bax protein was found in 20/47 (42.4%) adenocarcinomas examined. Negative Bax protein expression in tumour cells was correlated with lymph node metastasis (P < 0.05), and degree of differentiation (p < 0.05). Univariate analysis showed that the variables with a significant negative impact on survival were: high TNM tumour stage, depth of penetration in the gastric wall, lymph node involvement, and Bax protein expression. Multivariate analysis showed that the only variable with an impact on survival was Bax protein expression (p < 0.05, Relative Risk: 3.34). Kaplan-Meier curves showed that the 5-year survival was 36.8% in cases with positive compared with 16% in cases with negative Bax protein expression (p = 0.0427). CONCLUSION: Negative Bax expression in gastric cancer is associated with de-differentiation, lymph node metastases, and poor clinical prognosis. Bax protein expression might play an important role in the development and phenotypic differentiation of gastric carcinomas and tumor progression.

Surface topography and ultrastructural changes of mucinous carcinoma breast cells.

Mucinous carcinoma of the breast (MCB) is histologically classified into 2 groups: (1) pure MCB and (2) mixed MCB. Pure MCB carries a better diagnosis than mixed MCB. This research relates to the cell surface topography and ultrastructure of the cells in the above cases and aims to find the differences between them, by means of two methods: scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For the SEM examination, it was necessary to initially culture the MCB tissues and then proceed with the usual SEM method. In contrast, for the TEM technique, MCB tissues were initially fixed followed by the classic TEM method. The authors found the topography of pure MCB cases to be without nodes. The cell membrane was smooth, with numerous pores and small ruffles that covered the entire cell. The ultrastructural appearance of the same cases was with a normal cell membrane containing abundant collagen fibers. They also had many small vesicles containing mucin as well as secretory droplets. In contrast the mixed MCB had a number of lymph nodes and their cell surface topography showed stronger changes such as microvilli, numerous blebs, ruffles and many long projections. Their ultrastructure showed very long microvilli with large cytoplasmic inclusions and extracellular mucin collections, electron-dense material vacuoles, and many important cytoplasmic organelles. An important fact is that mixed MCB also contains areas of infiltrating ductal carcinoma. These cells of the cytoplasmic organelles are clearly responsible for the synthesis, storage, and secretion of the characteristic mucin of this tumor type. Evidently, this abnormal mucin production and the abundance of secretory granules along with the long projections observed in the topographical structure might be responsible for transferring tumor cells to neighboring organs, thus being responsible for metastatic disease.

In breast carcinoma dysadherin expression is correlated with invasiveness but not with E-cadherin.

Reduction/loss of E-cadherin is associated with the development and progression of many epithelial tumours. Dysadherin, recently characterised by members of our research team, has an anti-cell-cell adhesion function and downregulates E-cadherin in a post-transcriptional manner. The aim of the present study was to study the role of dysadherin in breast cancer progression, in association with the E-cadherin expression and the histological type. We have selected ductal carcinoma, which is by far the most common type and lobular carcinoma, which has a distinctive microscopic appearance. Dysadherin and E-cadherin expression was examined immunohistochemically in 70 invasive ductal carcinomas, no special type (NST), and 30 invasive lobular carcinomas, with their adjacent in situ components. In ductal as well as in lobular carcinoma dysadherin was expressed only in the invasive and not in the in situ component, and this expression was independent of the E-cadherin expression. Specifically, all 10 (100%) Grade 1, 37 out of 45(82.2%) Grade 2 and six out of 15 (40%) Grade 3 invasive ductal carcinomas showed preserved E-cadherin expression, while 'positive dysadherin expression' was found in six out of 10 (60%) Grade 1, 34 out of 45(75.5%) Grade 2 and all 15 (100%) Grade 3 neoplasms. None of the 30 infiltrating lobular carcinomas showed preserved E-cadherin expression, while all the 30 infiltrating lobular carcinomas exhibited 'positive dysadherin expression'. Dysadherin may play an important role in breast cancer progression by promoting invasion and, particularly in lobular carcinomas, it might also be used as a marker of invasion.

E-cadherin adhesion molecule and syndecan-1 expression in various thyroid pathologies.

UNLABELLED: Cadherins and syndecans are transmembrane glycoproteins implicated in cell-cell and cell-matrix adhesion. Impairment of cadherin and syndecan mediated adhesion is likely to constitute one of the main factors leading to the reduced cell-cell and cell-matrix adhesion characteristics of tumor cells and play a pivotal role in the acquisition of invasive and metastatic proprieties by neoplastic epithelial cells. AIM: To elucidate the role and alterations of syndecan-1 expression in comparison with those of E-cadherin in normal and pathological thyroid glands (TG). METHODS: A total of 55 TG carcinomas, 40 TG adenomas, 40 cases of hyperplastic TG disorders and 20 cases of normal TG autopsy samples, were evaluated by immunohistochemistry. The staining intensity, and localization of syndecan-1 and E-cadherin in sequential sections were examined, and semi-quantified. RESULTS: Immunostaining of syndecan-1 and E-cadherin was strong in normal follicular TG epithelial cells, and located mainly in basolateral membrane. No significant change was seen in either molecule in hyperplastic TG disorders compared with TG adenomas. A significant reduction in expression of both syndecan-1 and E-cadherin was seen in well-differentiated TG carcinomas as compared with normal TG epithelium (p = 0.0001 and p = 0.032, respectively). Similarly, there was a significant reduction of both molecules expression in poorly differentiated and anaplastic TG carcinomas compared to well differentiated tumors (syndecan-1: p = 0.0037; and E-cadherin: p = 0.075). CONCLUSION: Decreased E-cadherin and syndecan-1 expression along with decreasing cellular differentiation may be involved in the complex mechanism of progression of TG pathology.

DCIS histopathology from a historical perspective.

Ductal carcinoma in situ (DCIS) represents a biologically and morphologically heterogeneous disease. It is characterized by a proliferation of presumably epithelial malignant cells confined within the lumens of the mammary ducts, without evidence of invasion beyond the basement membrane into the adjacent breast stroma. With the widespread use of screening mammography, a dramatic change has occurred in the frequency, management and types of DCIS detected. Historically, there has been some confusion regarding the definition of DCIS and the terminology associated with the histological types of DCIS. In this review, DCIS histopathology from a historical point of view is presented.

Expression of matrix metalloproteinase-9 (gelatinase B) in benign, premalignant and malignant laryngeal lesions.

The matrix metalloproteinases (MMPs) are a family of proteolytic zinc-containing enzymes, which are responsible for the breakdown of the extracellular matrix components in pathological and physiological conditions. They are involved in basement membrane disruption, stroma and blood vessel penetration, metastasis and more recently there is evidence that they participate in tumor growth and angiogenic events. Matrix metalloproteinase 2 and 9 (MMP 2 and 9) belong to the gelatinases, a subgroup of MMPs, and have the capacity to degrade the triple helix type IV collagen of basal lamina of the basement membrane. With the present study, we tried to demonstrate the expression of MMP-9 immunohistochemically, comparatively in benign, premalignant and malignant lesions of the larynx. We studied 154 laryngeal lesions including 55 squamous cell carcinomas, 8 in situ carcinomas, 54 cases of dysplasia (of low and intermediate grade), 13 papillomas and 24 cases of keratosis. Overexpression of MMP 9 was observed in 74.4% and 50% in invasive and in situ squamous cell carcinomas respectively. In dysplastic cases, in papillomas and in keratoses the percentage of overexpression was 62.9%, 61.53% and 54.16% respectively and the expression of MMP-9 was significantly higher in invasive squamous cell carcinomas compared to dysplasias (p=0.000004). Also significantly higher was the expression of MMP-9 in dysplastic cases compared to papillomas (p=0.023). The MMP-9 expression was related neither to survival nor to the other available clinicopathological parameters (tumor size, grade, clinical stage, lymph node status and patient age). In conclusion, our study indicates that the expression of MMP-9 is up-regulated in a stepwise fashion, with two main steps, the first one, when a dysplastic lesion evolves and the next one, when the dysplasia progresses to invasive carcinoma.

The enigmatic nature of apocrine breast lesions.

Epithelial cells of fetal breast glandular structures, at the third trimester of pregnancy (28 weeks), produce GCDFP-15, in the absence of specific apocrine morphology. Apocrine epithelium of the breast may be a normal process of differentiation rather than a result of metaplasia, and it has been demonstrated that it is estrogen-receptor, progesterone-receptor and bcl-2 negative, but androgen-receptor (AR) positive. The significance of AR expression in apocrine epithelium is uncertain. Apocrine epithelium is seen in a wide spectrum of breast entities, ranging from benign lesions to invasive carcinoma. Breast cancer accounts 32% of all cancer cases among women and is the most common type of cancer in women. Little is known about breast carcinogenesis. Widely, it is accepted that breast cancer, like most other type of cancer, is being developed through the accumulation of genetic aberrations. Apocrine epithelium may reflect instability of the breast epithelium, creating an environment favouring further oncogenic alterations. In the last decade, several lines of evidence support the idea that some breast benign epithelial apocrine lesions are clonal lesions and may be considered as truly pre-malignant or precursors of breast carcinoma. Apocrine changes in many cases do not present any diagnostic difficulty; on the other hand, apocrine proliferations with cytologic atypia can be particularly difficult and challenging. The purpose of this study is to collect and highlight the areas of consensus in the literature as well as the controversial areas concerning the apocrine epithelium of the breast.

Clinicopathological study of metallothionein immunohistochemical expression, in benign, borderline and malignant ovarian epithelial tumors.

Metallothioneins (MTs) are a family of cystein-rich metal-binding proteins, which are expressed in normal cells during fetal and postnatal life but also in a variety of human neoplasms. MT expression in human tumors has been linked to resistance to anticancer drugs and differentiation and progression in some types of tumors. This study examined the immunohistochemical expression of MTs in benign, borderline and malignant tumors of ovarian surface epithelium and the possible correlations with clinicopathological parameters and survival. A total of 87 cases with diagnosis of ovarian surface epithelial tumors were included. Specifically, 21 cases of benign cystadenomas (11 serous and 10 mucinous), 14 borderline (low malignant potential tumors, 8 mucinous and 6 serous) and 52 cases of ovarian cancer were analysed. Immunohistochemical expression of MT (cut-off level > 10% of tumor cells) was clearly associated with malignancy. A statistically significant correlation was found between the expression of MT in cancer cases and benign tumors (p < 0.0001) and cancer cases and borderline tumors p = 0.003. In cancer cases a difference was observed between grade I and III (p = 0.002). There was no correlation of MT overexpression with survival in the small number of ovarian carcinoma patients where it was analysed. MT constitutes a marker that characterizes aggressiveness and a high malignant potential in ovarian epithelial tumors. In diagnostic problems MT may help distinguish between benign, borderline and malignant tumors.

The MAPK-AP-1/-Runx2 signalling axes are implicated in chondrosarcoma pathobiology either independently or via up-regulation of VEGF.

AIMS: To investigate whether and how the JNK/ERK-AP-1/-Runx2 signalling pathways and vascular endothelial growth factor (VEGF) are engaged in the pathogenesis of cartilaginous tumours. Chondrosarcoma is the third most common primary skeletal malignancy. Nevertheless, the molecular events underlying its pathogenesis remain elusive. JNK/ERK MAPKs and their downstream effectors, c-Jun and c-Fos (AP-1), are involved in chondroblastic differention/proliferation. These proteins interact with the Runx2 transcription factor, which is also implicated in chondroblast biology. VEGF, a key angiogenic factor, is up-regulated in human chondrosarcomas. METHODS AND RESULTS: Normal cartilage and neoplastic cells from 45 chondrosarcomas and 21 enchondromas were investigated immunohistochemically. We evaluated the cellular levels of JNK2, p-JNK2 (phosphorylated/activated JNK2), its main substrate, c-Jun, pc-Jun (phosphorylated/activated c-Jun) and c-Fos. Moreover, the levels of p-ERK (phosphorylated/activated ERK), Runx2 and VEGF were assessed. Positive immunostaining for all proteins was observed in the majority of the examined chondrosarcomas and in a small fraction of enchondromas. The expression levels of all proteins were positively and significantly correlated with each other. These levels were substantially augmented in high-grade compared with low-grade chondrosarcomas and in low-grade tumours compared with benign enchondromas, implying a potential use as molecular markers for prediction of high-grade neoplasms. CONCLUSIONS: The JNK/ERK-AP-1/-Runx2 signal transduction 'network' is associated with chondroblastic malignant transformation and chondrosarcoma development, either separately or through coordinated induction of VEGF.

Involvement of dysadherin and E-cadherin in the development of testicular tumours.

Testicular neoplasms are comprised of a variety of histologically different forms, and their pathogenesis has not been elucidated. Dysadherin is a recently described cell membrane glycoprotein, which has an anticell-cell adhesion function and downregulates E-cadherin. In this study, we examined immunohistochemically the expression of E-cadherin and dysadherin in 120 testicular neoplasms (37 seminomas-26 classic, five spermatocytic and six anaplastic-, 45 embryonal carcinomas, 10 mixed germ cell tumours, two yolk sac tumours, 10 mature and eight immature teratomas and eight non-Hodgkin B-cell lymphomas), clinical stage I. The intensity, the expression pattern and the percentage of neoplastic cell staining was recorded and correlated with the histologic type and vascular/lymphatic invasion. Dysadherin was not expressed in non-neoplastic germ cells, neither in CIS/ITGCNU, but it was highly expressed in all types of germ cell tumours, that demonstrated either embryonic phenotype or somatic differentiation, in most terminally differentiated neoplasms, and in all lymphomas. Dysadherin expression did not correlate with vascular invasion. Increased dysadherin expression was correlated with aberrant E-cadherin expression in most tumours. In 17% of embryonal carcinomas colocalisation of dysadherin and membranous E-cadherin staining was noted. This is the first report on dysadherin expression and its association with E-cadherin in testicular tumours. Since dysadherin is not normally expressed in non-neoplastic testis, it is conceivable that it plays a role in the neoplastic transformation of germ cells. In testicular tumours, as in other neoplasms, dysadherin downregulates E-cadherin expression, at least in part.

Cell cycle proteins in laryngeal cancer: role in proliferation and prognosis.

A study of laryngeal carcinomas was performed in order to analyze (a) the expression of p53/p21, cyclin D1/cyclin E, p21/p27 (b) the relation of normal and abnormal protein expression, with the proliferation status, as determined by the expression of Ki67 and PCNA and (c) the correlation of our findings with prognosis. We performed a retrospective analysis of 57 cases of squamous cell carcinomas of the larynx. We applied monoclonal antibodies against p53, p21, p27, cyclin D1, cyclin E, Ki67 and PCNA, using streptavidin-biotin method. Analysis of the p53/p21 proteins, revealed abnormalities in 25/37 cases (67.57%), while 12/37 (32.43%) cases displayed normal phenotype (p53-/p21-). Analysis of cyclins revealed overexpression in 17/48 cases (35.42), while the majority 31/48(64.58%) displayed normal phenotype (cyclin D1-/cyclin E-). Concerning CDKIs expression, the majority 30/50(60%) presented high levels of both inhibitors (p21+/p27+). Cases with simultaneous overexpression of CDKIs demonstrated significantly higher levels of Ki67 protein (p = 0.05). Analysis of p53/p21, cyclin D/cyclin E, p21/p27 patterns showed no association between the presence of one or two alterations and prognosis. In conclusion, we demonstrated that p53 tumor suppressor pathway is frequently disrupted in laryngeal cancer. Furthermore, levels of CDKIs, although they act as cell cycle activity blockers, are not reliable markers for the estimation of laryngeal neoplastic cells growth fraction.

Immunohistochemical study of angiogenesis and proliferative activity in epiretinal membranes.

Formation of epiretinal membranes (ERMs) is a serious complication of retinal diseases, the most important being proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). In this study, our goal was to (i) calculate the microvessel density (MVD), (ii) evaluate vascular endothelial growth factor (VEGF) expression and (iii) correlate angiogenesis with the proliferative activity as expressed by the expression of Ki67 marker, in both membrane types. We performed immunohistochemistry in 14 PVR and eight PDR membranes, using antibodies against CD34, VEGF, Ki67 and glial fibrillary acidic protein. PDR membranes presented higher average count of microvessels compared with PVR membranes (p = 0.0015). No differences were observed concerning VEGF expression (p = 0.1). The expression of Ki67 was not correlated with microvessel number or VEGF expression. Our study confirms the presence of vascularisation in PDR membranes, as well as the presence of VEGF even in avascular PVR membranes, suggesting that immunoreactivity for VEGF may not be accompanied by angiogenesis.

Clinical correlation of bone marrow microvessel density in essential thrombocythemia.

The aim of this study was to investigate the prognostic relevance of angiogenesis expressed as bone marrow microvessel density (MVD) in 40 patients with essential thrombocythemia (ET). Bone marrow MVD was visualized in paraffin tissue sections using immunohistochemical staining for CD34 protein. Increased MVD of bone marrow specimens was not significantly correlated with clinical (sex, age, microvascular disturbances, liver and spleen size, complications, treatment or no before biopsy), laboratory (peripheral blood count), bone marrow histopathological parameters (cellularity, megakaryocyte clumping and reticulin fibrosis) and overall survival. These preliminary findings suggest that angiogenesis has no prognostic value in patients with ET.

Immunohistochemical study of extracellular matrix components in epiretinal membranes of vitreoproliferative retinopathy and proliferative diabetic retinopathy.

PURPOSE: The migration, proliferation, differentiation, and adhesion of cells and other cellular functions are influenced by the surrounding extracellular matrix in normal and wound healing conditions. The formation of epiretinal membranes, a wound healing process, is a serious complication of retinal diseases, the most important being proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). In the present study, the authors investigated the expression of various extracellular matrix components and in particular tenascin, fibronectin, laminin, collagen IV, and MMP-3 glycoprotein as well as the expression of glial fibrillary acidic protein in each type of epithelial membrane in order to elucidate the role of these molecules in the formation of these two types of membranes. METHODS: The authors performed immunohistochemistry in 14 PVR and 14 PDR membranes, using antibodies against the above mentioned extracellular matrix components. Tenascin and fibronectin were observed as major components in the extracellular matrix, while laminin and collagen type IV were detected as minor components in both types of membranes. A higher fibronectin expression in PVR compared with PDR membranes was found (p=0.0035). A positive relationship of its expression with the proliferative activity (p=0.15) and collagen type IV expression (p<0.0001) was also observed. RESULTS: Tenascin expression was positively correlated with glial fibrillary acidic protein positive cells in PDR membranes (p=0.04). Collagen type IV localized around vessels was observed with high levels in PDR membranes (p=0.0031). CONCLUSIONS: The results indicated that the extracellular matrix components seem to be involved in PVR and PDR, contributing to tissue remodeling and perhaps by different pathogenetic pathways, which could reflect different stages of development in these two types of membranes.

The role of fine-needle aspiration biopsy in the management of patients with thyroid nodules.

Fine-needle aspiration biopsy (FNAB) of the thyroid gland is the most cost-effective examination in the evaluation of thyroid nodules. The aim of this study was to present our experience from all patients who underwent thyroid FNA in the University Hospital of Ioannina, Greece, in the period 1993-2003, and its value in the diagnostic management of patients with thyroid nodules. FNA was performed in 900 patients of whom 753 were females and 147 males. The cases were classified according to diagnosis into five groups: benign/negative 628, primary carcinoma 28, metastatic carcinoma 5, suspicious/indeterminate 60 and non-diagnostic 179. Cytological findings were compared with histopathological findings and the statistical analysis in our data yielded the following results: sensitivity 92.1%, specificity 93.2%. These results are in accordance with the already published data in the international literature. In cases of differential diagnosis between adenomatoid hyperplasia and follicular neoplasia, four cases were diagnosed as hot nodules. In the benign group, three cases were diagnosed as nodular hyperplasia with cystic degeneration on FNA, but, after surgical treatment, histologically were diagnosed as papillary carcinomas. In the group of suspicious/indeterminate, two cases were diagnosed as suspicious for follicular neoplasia on FNA and, after surgical treatment, were diagnosed histopathologically as medullary carcinomas. In conclusion, we suggest that routine measurement of serum calcitonin is useful and mandatory in the detection of medullary carcinoma among patients with nodular thyroid diseases. Taking into consideration the clinical data can minimize false-positive and false-negative rates. We conclude that FNA is an effective screening test in the evaluation of the necessity for surgical treatment in patients with thyroid nodules.

FNAC: its role, limitations and perspective in the preoperative diagnosis of breast cancer.

Fine-needle aspiration cytology (FNAC) was first described and performed in 1930. Thirty years later, it gained acceptance first in Europe and about a decade later in North America. The method is generally considered as a rapid, reliable, safe diagnostic tool to distinguish non-neoplastic from neoplastic breast lesions. In developed countries, in the last 20 years, mammographic screening programmes, which have been used extensively, are designed to detect the earliest possible breast cancer. The FNAC report is extremely important because it gives the necessary information for the management of patients, in order to proceed with more invasive diagnostic methods or surgical treatment, and to decide what kind of operation to perform. In the preoperative phase, FNAC has taken a fundamental role of both palpable and nonpalpable lesions, using ultrasound or stereotactic guidance. New developed techniques, breast biopsy instrumentation (ABBI) and mammotome have the advantage of complete removal of breast lesions, but this is not possible in all the examined cases. In developing countries, economical restrictions, low budget for health care and screening programmes put the patients at a disadvantage because of the high cost of sophisticated diagnostic methods, thus we recommend that FNAC be used as a routine diagnostic method because of its low cost compared with the others and this policy maximizes the availability of health care to women with breast cancer. We conclude that FNAC plays an important and essential role in the management of patients with breast lesions and also offers a great potential for prediction of patient outcome, disease response to therapy and assessment of risk of developing breast cancer. The reliability and efficiency of the method depends on the quality of the samples and the experience of the medical staff that performs the aspiration.

hMSH2 is the most commonly mutated MMR gene in a cohort of Greek HNPCC patients.

Germline mutations in genes encoding proteins involved in DNA mismatch repair are responsible for the autosomal dominantly inherited cancer predisposition syndrome hereditary nonpolyposis colorectal cancer (HNPCC). We describe here analysis of hMLH1 and hMSH2 in nine Greek families referred to our centre for HNPCC. A unique disease-causing mutation has been identified in seven out of nine (78%) families. The types of mutations identified are nonsense (five out of seven) (hMLH1: E557X, R226X; hMSH2: Q158X, R359X and R711X), a 2 bp deletion (hMSH2 1704_1705delAG) and a 2.2 kb Alu-mediated deletion encompassing exon 3 of the hMSH2 gene. The majority of mutations identified in this cohort are found in hMSH2 (77.7%). Furthermore, four of the mutations identified are novel. Finally, a number of novel benign variations were observed in both genes. This is the first report of HNPCC analysis in the Greek population, further underscoring the differences observed in the various geographic populations.