Spectrum and Frequency of BRAF Mutations in Skin Melanomas in the Dalmatian Region of Croatia.

Mutation of the BRAF oncogene is one of the most common mutations detected in human neoplasia, occurring in 40-60% of all cutaneous melanoma. BRAF is a serine/threonine protein kinase which is an essential part of the mitogen-activated protein kinase (MAPK) pathway. It is physiologically activated by RAS, but in mutated form, due to molecular conformational change, BRAF becomes constitutively active with subsequent persistent activation of downstream cytoplasmic and nuclear proteins (MEK, ERK, ETS), which finally leads to gene expression that promotes cell growth and survival. Inhibition of the altered MAPK pathway by BRAF inhibitors and combined BRAF/MEK inhibitors in BRAF mutated melanoma has become a standard therapeutic approach (1,2). We recently reported the frequency and clinicopathological features of BRAF V600E mutated melanomas in the Dalmatian region of Croatia. This report included 80 cutaneous melanomas with BRAF analyses performed at our institution until the second half of 2017, using a kit which detected only BRAF V600E mutation (3). From the second half of 2017, we started using a kit which detects several types of BRAF mutations along with NRAS mutation. The aim of this report was to determine the spectrum and frequency of different BRAF mutations in a group of skin melanomas in the Dalmatian region of Croatia and to comment on the relationship between type of BRAF mutation and therapeutic response to MAPK pathway inhibition. The analysis included 179 patients with stage 3 and stage 4 cutaneous melanoma with known BRAF/NRAS mutational status. The paraffin blocks were forwarded from four Dalmatian hospitals (Split: 139 cases, Zadar: 17 cases, Sibenik: 13 cases, Dubrovnik: 10 cases). BRAF/NRAS mutation analysis was performed at the Institute of Pathology, Clinical Hospital Center Split, Croatia, in the period from the second half of 2017 to the end of 2022. For DNA extraction analysis, hematoxylin and eosin stained slides from each submitted sample were reviewed by a pathologist, and tumor tissue was identified for analysis. For all tissue specimens, DNA was extracted from sections (10 mm thick) using the cobas® DNA Sample Preparation Kit (Roche Molecular Diagnostics), following the manufacturer's protocol. The amount of genomic DNA was quantified using the Qubit® 2.0 Fluorometer (Life Technologies) and adjusted to a fixed concentration to be added to the amplification/detection mixture. For mutation analysis, the target DNA was amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the Roche BRAF/NRAS mutation test (LSR) kit, according to the manufacturer's protocol. The test results were reported as follows: BRAF exon 11 mutation detected, BRAF V600E/E2/D mutation detected, BRAF V600K mutation detected, BRAF V600R mutation detected, BRAF K601E mutation detected, NRAS (G12X, G13X, A18T, Q61X, other NRAS Ex3/4) mutation detected, mutation not detected, or invalid result (no result was obtained on the cobas test). BRAF mutation was observed in 87 patients (48.6%), NRAS mutation was found in 27 patients (15.1%), while 65 patients (36.3%) were without BRAF/NRAS mutation (Table 1). In the group of BRAF mutated melanomas, 61 cases (70.1%) had V600E/E2/D mutation, 20 cases (23%) had V600K mutation, 3 cases (3.4%) had exon 11 mutation, 2 cases (2.3%) had V600R mutation, and 1 case (1.2%) had K601E mutation (Table 2). The observed frequency of BRAF mutated melanomas in this study was similar to the frequency reported in our previous study (48.6% and 47.5%, respectively) (3). The vast majority were BRAF V600 mutations, while BRAF non-V600 mutations were rare (95.4% and 4.6%, respectively). In the group of BRAF V600 mutations, V600E/E2/D mutations predominated, followed by V600K mutations, while V600R mutations were rare. Greaves et al. reported similar frequency of BRAF V600 mutations in a group of 499 BRAF-mutated cutaneous melanomas, with V600E/E2/D mutations observed in 77.2% cases, followed by V600K mutations observed in 17.2% cases, and V600R mutations observed in 2.6% cases (4). BRAF non-V600 mutations (exon 11 and K601E mutations) were rarely observed in this study, confirming the findings of other authors (4,5). A three-class system of BRAF mutations was recently proposed that takes into account the differences in their kinase activity, with class I containing mutants with high kinase activity and high response rate to BRAF and BRAF/MEK inhibitors. Class II BRAF mutations have lower kinase activity than class I mutants, but higher than wild-type BRAF, showing resistance to BRAF inhibitor monotherapy and sensitivity to MEK and BRAF/MEK inhibitors. Finally, class III BRAF mutations are characterized by low kinase activity and low response rate to targeted therapy (6). BRAF V600 mutations belong to class I mutations, which means that the large majority of BRAF-positive melanomas in this study (95.4%) were sensitive to targeted therapy. However, the sensitivity to targeted therapy is different among different class I BRAF mutations. While large randomized controlled trials on combined BRAF/MEK inhibition showed good overall response (63-68%) and improvement of progression-free survival (PFS) and overall survival (OS) for the melanomas with most prevalent V600E and V600K mutations, Menzer et al. showed lower response rate to MAPK pathway inhibition (45%) in the group of metastatic melanomas with BRAF V600 mutations other than V600E/K. The overall response rate to MAPK pathway inhibition in the same group of melanomas with BRAF non-V600 mutations (class II and III mutations) was only 18% (7). In our group of BRAF mutated skin metastatic melanomas, we found only 6 cases (6.9%) with expected lower response rate to MAPK pathway inhibition: 2 cases with V600R mutation (class I non-V600E/K mutation), 1 case with K601E mutation (class II mutation), and 3 cases with exon 11 mutation (class II and III mutations).

ABO and Rh blood groups, demographics, and comorbidities in COVID-19 related deaths: A retrospective study in Split-Dalmatia County, Croatia.

BACKGROUND: Blood group phenotypes have been associated with COVID-19 susceptibility and severity. This study aimed to examine ABO/Rh blood group distribution in COVID-19-related deaths considering demographics and pathological conditions. METHODS: We conducted a retrospective study at the University Hospital Center Split, Croatia, that included 245 COVID-19 positive individuals that died from April 8, 2020, to January 25, 2021. We extracted data on their blood groups, demographics, and pre-existing comorbidities and compared findings with general population data from blood group donations (n = 101,357) and non-COVID-19 deaths from 2019 (n = 4968). RESULTS: The proportion of dead males was significantly higher than in non-COVID-19 cases (63.7% vs. 48.9%, P < 0.001), while the proportion of older individuals did not differ. The prevailing pre-existing diseases were hypertension (59.6%), diabetes (37.1%), heart failure (28.8%), digestive disorder (26.5%), and solid tumor (21.6%). The ABO distribution in the deceased and donors' group showed significant differences, with the higher prevalence of A/AB group and lower prevalence of 0, but with individual differences significant only for AB and non-AB groups. There was a reduced proportion of females within the deceased with group 0 (P = 0.014) and a higher proportion of AB individuals with coronary heart disease (P = 0.024). CONCLUSION: The study confirmed a higher risk of death in males. The lower proportion of type 0 in deceased individuals was greater in females, implying that group 0 is not necessarily an independent protective factor. Coronary heart disease was identified as a potential risk factor for AB individuals.

BUILDING THE COVID-19 TESTING CAPACITIES IN CROATIA: ESTABLISHING THE INTERDEPARTMENTAL COVID-19 UNIT AT THE SPLIT UNIVERSITY HOSPITAL CENTRE.

This paper presents the chronology, experiences, and challenges in introducing COVID-19 RT-PCR testing in Split, Croatia. We describe the processes from March 12, 2020 to May 26, 2020, starting from the initial knowledge transfer, expert team formation and management, testing implementation, and concluding with the standalone testing facilities, which used automated processes sufficient to meet testing requirements at that time. In the case presented, the COVID-19 unit was organized by joining human and laboratory resources from five clinical departments at the Split University Hospital Centre. Sample preparation procedures and analyses were launched within the restricted time frame while simultaneously training and organizing new laboratory staff and completing equipment requirements. As a result, the process that started with 30 tests per day was constantly improved over time and reached up to 160 tests per day when MagNA Pure was added to automatize RNA extraction at the end of April. At that pace, the cumulative number of samples soon exceeded the first thousand, and by the end of May it exceeded 4000. The case presented provides an example of good practice for crisis response and organization that successfully enabled sufficient COVID-19 testing capacities within the restricted time frame, human and technical resources. Despite limited understanding of COVID-19 at that time, appropriate management, transfer of knowledge, previous experiences in related laboratory and diagnostic work, as well as interdisciplinary and interdepartmental cooperation proved appropriate to overcome the above limitations and ensure adequate healthcare response.

SARS-CoV-2 Antibody Seroprevalence in Industry Workers in Split-Dalmatia and Sibenik-Knin County, Croatia.

OBJECTIVES: To examine seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in industry workers population sample. METHODS: From 23 to April 28, 2020, we conducted serological testing for antibodies (Immunoglobulin G (IgG) and Immunoglobulin M (IgM)) on 1494 factory employees living in the Split-Dalmatia and Sibenik-Knin County (Croatia). RESULTS: We detected antibodies in 1.27% of participants (95% confidence interval [CI] 0.77-1.98%). In Split facility 13/1316 (0.99%, 95% CI 0.53-1.68%) of participants were tested positive, of which 13/1079 (1.20%, 95% CI 0.64-2.05%) of those living outside the facility and 0/237 (0%, 95% CI 0-1.26%) of those living inside the facility. In Knin facility, 6/178 (3.37%, 95% CI 1.25-7.19%) participants were tested positive for antibodies. CONCLUSIONS: The study showed relatively small SARS-CoV-2 antibody seroprevalence in the DIV Group population sample.

Invasive breast carcinoma with hyaline globules ("thanatosomes").

Hyaline globules (thanatosomes) have been observed in many neoplastic and non-neoplastic processes. They represent terminal cytoplasmic structures associated with cytoplasmic blebbing, which are related to cell injury and apoptosis. Thanatosomes are reported rarely in breast tumors. Here we describe a case of invasive breast cancer of no special type with abundant thanatosomes.

Low ERCC1 expression is a good predictive marker in lung adenocarcinoma patients receiving chemotherapy based on platinum in all TNM stages - a single-center study.

BACKGROUND: High ERCC1 expression is thought to be related with resistance to chemotherapy based on platinum. The aim of this study was to present our institutional observations regarding to the association of ERCC1 and overall survival (OS) of the lung adenocarcinoma patients who received chemotherapy based on platinum. MATERIAL/METHODS: A total of 253 lung adenocarcinoma patients in all TNM stages were retrospectively investigated. The diagnosis was based on small biopsy samples obtained during bronchoscopy. Depending on the TNM stage of the disease and clinical condition, patients received only the chemotherapy based on platinum, or in combination with radiotherapy or surgery. Tissue sample for ERCC1 immunohistochemical analysis was sufficient in 129 patients. Low from high ERCC1 expression was separated by the semi-quantitative H-score median. RESULTS: High ERCC1 expression was found in 47.3% patients, and was correlated with higher TNM (p = 0.021), tumor enlargement (p = 0.002), positive lymph nodes (p = 0.001), positive distant metastasis (p = 0.005), and higher relative risk of death (p < 0.001). Furthermore, significance association was observed for low ERCC1 expression and better performance status (ECOG) (p = 0.023). Longer OS was strongly associated with a low ERCC1 expression, not only in the group of patients in TNM stage I-III, who were treated with combination of chemotherapy with surgery or radiotherapy (p = 0.002), but also in the group of patients in TNM stage IV who received only chemotherapy based on platinum (p < 0.001), compared with the patients in the same TNM stage and high ERCC1 expression. CONCLUSIONS: ERCC1 expression in lung adenocarcinoma is a useful prognostic marker and moreover, a useful predictive marker in patients receiving chemotherapy based on platinum in all stages of the disease.

Clinicopathological Characteristics of BRAF V600E Mutated Melanomas in the Dalmatian Region of Croatia.

A high proportion of cutaneous melanomas harbor activating mutations of the BRAF or NRAS genes, which are components of mitogen-activated protein kinase (MAPK) signal transduction pathway. The importance of BRAF V600E mutation in melanoma is not only related to the possibility of the administration of the targeted therapy, but also to the fact that BRAF V600E mutated melanomas have distinct clinicopathological features. We investigated the clinicopathological features of 80 primary skin melanomas with known BRAF V600E mutation status excised in the Dalmatian region of Croatia, with comparison of these features between the mutated and wild-type group. The frequency of BRAF V600E mutation was 47.5%. In comparison with wild-type melanomas, BRAF V600E mutated melanomas were significantly associated with younger age and female sex (P=0.014 and P=0.011, respectively). The mutated melanomas were more often located on the extremities, of a nodular type, ulcerated, and with higher median of mitotic index but without significant difference in comparison with wild-type tumors. There were no differences in the depth of invasion and the presence of lymphovascular invasion, tumor infiltrating lymphocytes, and regression between the investigated groups. The frequency of BRAF V600E mutation in our cohort of primary skin melanomas and the clinicopathological features of mutated tumors were similar to those reported in the literature, except for the higher proportion of women observed in our group with mutation.

Breast fibroadenoma with pseudoangiomatous (PASH-like) stroma.

Pseudoangiomatous stromal hyperplasia (PASH) is a breast stromal change, histologically characterized by anastomosing, slit-like spaces lined by slender myofibroblasts and surrounded by dense collagenous stroma. Mass forming cases clinically and radiologically simulate fibroadenoma. A middle aged women presented with unpalpable breast nodule discovered on ultrasound examination. The ultrasound characteristics were typical for fibroadenoma, while fine-needle aspiration cytology was inconclusive. The histological examination of the lumpectomy specimen showed fibroadenoma with peculiar stromal alteration consistent with pseudoangiomatous stromal hyperplasia. The presented case of fibroadenoma with pseudoangiomatous hyperplasia within its stroma demonstrates the relationship between these two entities not only clinically and radiologically, but also histologically.

Pathobiology of HER 2 positive small invasive (T1a,b) breast cancer: "A wolf in sheep's clothing".

Small invasive breast cancers (cancers with maximum diameter <1cm, T1a,b) become more prevalent form of breast cancer as a result of the introduction of breast cancer mammographic screening programs. Although associated with an excellent prognosis, T1a,b breast cancers are heterogeneous group of tumors with prognostically unfavorable subset of cases, primarily those with axillary lymph node metastases. To determine if the HER2 overexpression is associated with the prognostically unfavorable traditional clinicopathological features in this group of breast cancers, clinicopathological features (age, tumor size, histological type, histological grade, nodal status, hormone receptor status, proliferation index, lymphovascular invasion, ploidy) of 38 HER2 positive T1a,b cancers were compared with those of the control group consisting of 315 HER2 negative T1a,b cancers. The comparison of clinicopathological features was made using χ2 and t-test. HER2 positive T1a,b breast cancers were significantly associated with higher tumor grades (p<0.001), negative hormone receptors (p=0.008), presence of lymphovascular invasion (p=0.025), high proliferation index (p<0.001), and abnormal DNA content (p=0.04). We also noticed the higher frequency of lymph node positive cases in the HER2 positive group of cancers (p=0.05). There were no differences in age, tumor size and histological type between investigated groups. Our group of HER2 positive T1a,b breast cancers was associated with many unfavorable traditional prognostic factors, demonstrating that this subtype of early breast cancer has an aggressive biological phenotype which may have potential benefit from adjuvant chemo and immunotherapy.

Fibroadenoma with "immature-like" type of usual ductal hyperplasia.

We herein report a case of the breast fibroadenoma with foci of so-called immature variant of the conventional ductal hyperplasia. This type of usual ductal hyperplasia is histologically characterised by encircling intraductal proliferation of large cells with pale to amphophilic cytoplasm and large nuclei which vary in shape and in staining quality of the chromatin. We showed here, using the cytokeratin immunohistochemistry, that the proliferating cells were not of immature but rather mature immunohistochemical phenotype. Because of the presented discordance between immature histology and mature immunohistological profile we suggest that this rare type of usual ductal hyperplasia should be called "immature-like".

Flow cytometric DNA hypertetraploidy tends to be more frequent in male than in female breast cancers.

The aim of the study was to explore possible differences in DNA flow cytometric characteristics, particularly differences in distribution of DNA indices of aneuploid clones, between male and female breast cancers. We retrospectively analyzed 31 male breast cancers. Clinicopathological and DNA flow cytometric characteristics of male breast cancers (patient age, tumor size, histological type, histological grade, axillary lymph node status, hormone receptor expression, ploidy, and S-phase fraction) were compared with that of the control group of matched female breast cancers. Hormone receptors and HER-2/neu were investigated immunohistochemically with additional chromogenic in situ hybridization (CISH) analysis of HER-2/neu 2+ cases. Ploidy and S-phase fraction were determined by DNA flow cytometry. Comparison with clinicopathological features was made using χ (2) and t test. Aneuploidy was found in 78% of the cases, with the predomination of hypotetraploid clones (39%), followed by tetraploid (23%) and hypertetraploid clones (16%). We found higher frequency of hypertetraploidy in male breast cancers (16 and 6%, respectively) than in the control group of matched female breast cancers. Clinicopathological features of hypertetraploid male breast cancers did not differ from that of non-hypertetraploid cancers. Higher frequency of hypertetraploidy among male breast cancers might indicate different cytogenetical evolutionary pathway between male and female breast cancer.

Signet ring lobular carcinoma in situ as a part of the "Rosen Triad" (tubular carcinoma, columnar cell hyperplasia, and lobular carcinoma in situ).

OBJECTIVE: The association of tubular carcinoma, columnar cell lesions and lobular carcinoma in situ, also known as the "Rosen Triad", may be encountered in breast biopsies performed for evaluation of mammographically detected microcalcifications. CASE REPORT: A case in which tubular carcinoma and columnar cell hyperplasia were associated with a histologically unusual form of signet ring lobular carcinoma in situ is presented. Signet ring non-invasive lobular carcinoma is classified as high-grade lobular carcinoma in situ, but herein is associated with changes that belong to the molecular pathway of low-grade mammary neoplasia. CONCLUSION: We reported the case of lobular carcinoma in situ associated with columnar cell hyperplasia and tubular carcinoma, in which the lobular carcinoma in situ was presented in a histologically unexpected form comprised predominantly of signet ring cells.

Mammographic screening has failed to improve pathohistological characteristics of breast cancers in Split region of Croatia.

The national breast cancer screening program has been introduced in Croatia in the second half of 2006 with mammography as the screening method. We investigated the impact of screening mammography on the basic pathohistological characteristics of breast cancers retrieved from the database of large community hospital. The data were collected in the period following the initiation of national mamographic screening program (2007-2011), and compared with the data collected in the period before the program introduction (2002-2006) to explore the possible changing trends. In the screening period 1,320 breast cancers were diagnosed, while in the prescreening period 1,204 breast cancers were diagnosed (p=0.02). We found the reduction of mean tumor size (p=0.039), decrease of the diagnosed non-invasive cancers (p=0.001), and the higher percentage of the diagnosed ductal (p=0.0003) and grade 3 (p=0.038) invasive cancers in the screening period. We also noticed higher percentage of diagnosed advanced breast cancers with unknown tumor size in the screening period (p=0.027). We concluded that the implementation of national screening program did not improve the pathohistological features of breast cancers in our region probably due to low response rate to screening invitation.

Near-diploid hyperploidy in early breast cancer (T1a,b) is associated with higher risk of lymph node involvement.

Due to the worldwide implementation of the mammographic screening program early breast cancer (T1a,b) has become more prevalent form of breast cancer. Although T1a,b breast cancers are generally associated with excellent prognosis, some of them, particularly those with lymph node involvement, has unfavourable outcome. Searching for additional prognostic factors, we investigated DNA content of 163 T1a,b cancers measured by DNA flow cytometry, and correlated it with regional lymph node status. T1a,b cancers were divided into four ploidy classes based on their DNA index (DI): hypodiploid (DI < 0.95), diploid (DI 0.95-1.05), low-hyperploid (DI 1.06-1.3), and high-hyperploid (DI > 1.3). Diploid T1a,b cancers were associated with negative lymph node status (p = 0.003). Among aneuploid cancers only low-hyperploid tumors were associated with positive lymph node status (p = 0.03). The histopathological features of low-hyperploid group of T1a,b cancers did not differ from the other three ploidy groups of cancers, except for lower S-phase fraction of tumor cells in low-hyperploid group compared to high-hyperploid group (p = 0.01). Our data showed that near-diploid hyperploid T1a,b cancers are associated with higher risk of lymph node involvement despite similar clinicopathological features shared with other ploidy classes of T1a,b tumors.

Her-2/neu assessment for gastric carcinoma: validation of scoring system.

BACKGROUND/AIMS: Gastric cancer is the second leading cause of cancer mortality in the world. Amplification of HER-2/neu oncogene has become an important biomarker for identifying patients who respond to HER-2 targeting therapy. A number of studies have analyzed HER-2/neu overexpression in gastric carcinoma, and the rate of HER2 positivity is variable, ranging from 6% to 35%. METHODOLOGY: In our study HER-2/neu expression was assessed on 73 samples of primary gastric cancer, using immunohistochemistry. For 19 patients preoperative biopsy samples and resected specimens were available. Additionally, internal ring study was performed to estimate intraobserver variability of IHC scoring among pathologists at our department. RESULTS: HER-2/neu overexpression was found in 10 (13.6%) of the tested samples, and it was more common in intestinal (22.5%) than the diffuse type (3.7%). Not one of the 6 analyzed mixed type tumors showed HER-2/neu expression. For the paired samples (preoperative biopsy samples and resected specimens) the concordance rate for HER-2/neu expression was 94.7%. CONCLUSIONS: According to high concordance rate in paired samples we consider it appropriate to evaluate HER2 expression on biopsy specimens, especially in unresectable cases, and to re-evaluate it on resected specimens if available, due to high heterogeneity of a gastric cancer.

Can we identify the group of small invasive (T1a,b) breast cancers with minimal risk of axillary lymph node involvement? A pathohistological and DNA flow cytometric study.

The goal of this study was to identify a group of small (≤1cm) breast cancers (T1a,b) with a particularly low probability of axillary lymph node metastases, where routine axillary staging may be unnecessary. We retrospectively analyzed 152 T1a,b breast carcinomas with axillary dissection surgically removed at Clinical Hospital Center Split (Croatia) in the period from 1997 to 2006. The analysis included 40 T1a,b cancers with, and 112 T1a,b cancers without axillary lymph node metastases. The basic morphological features of cancers were investigated histologically, while hormone receptors and HER2/neu were investigated immunohistochemically with an additional CISH analysis of HER2/neu 2+ cases. The ploidy and S-phase fraction were determined by DNA flow cytometry. The association of the investigated features with the likelihood of axillary lymph node metastases was analyzed by univariate and multivariate analysis. The univariate analysis showed that lymph node metastases were associated with tumor size (T1a/T1b; p=0.026), histological type (ductal/non-ductal; p=0.014), lymphovascular invasion (p<0.001), HER2/neu expression (p=0.04), ploidy (p=0.027), and combined values of ploidy and S-phase fraction (p=0.025). The lymphovascular invasion was the only independent factor associated with axillary nodal metastases (p=0.01). In the group of T1a,b cancers without lymphovascular invasion, HER2/neu expression (p=0.021) and combined values of ploidy and S-phase fraction (p=0.016) were independent factors associated with axillary lymph node metastases. This study showed that diploid T1a,b cancers with low S-phase fraction, which are also without lymphovascular invasion and HER2/neu amplification, represented the group of cancers with a low probability of axillary lymph node metastases.