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Purpose: From April to September 2020, Poland was minimally affected by COVID-19 compared to other EU countries. We aimed to investigate the risks of false reverse transcription polymerase chain reaction (RT-PCR) results during the first wave (compared to later waves), that rises when cycle threshold (Ct) of positive result is close to limit of detection (LOD)., Materials/methods: We analyzed Ct values of SARS-CoV-2 positive RT-PCR results of 7726 patients in Poland from April-September 2020. SARS-CoV-2 positive RT-PCR results of 14,534 patients in the 2nd-3rd wave and 10,861 patients in the 4th-5th pandemic waves were used. Statistical analysis was based on one-way analysis of variance. To verify, 95% confidence intervals with Bonferroni correction were computed. Incidence of SARS-CoV-2 variants in Poland was analyzed using Whole Genome Sequencing from 923 (3.6%) patients., Results: The mean Ct of RT-PCR positive test results analyzed ranged between 22.89 and 26.71 depending on the month of the results collection. The differences between months were significant (p ​< ​0.001). Differences in Ct were observed between age groups, with younger patients displaying higher Ct values, however, major trends over time were paralleled between age groups., Conclusions: The mean Ct of the tested RT-PCR positive test results was lower than 35 which is considered an upper borderline for reliable positive results of the assay. Therefore, most COVID-19 cases recorded in Poland from April to September 2020 were detected with minor risks of inaccuracy. Data from a single center exhibited greater consistency for both virus Ct level and SARS-CoV-2 virus variant identification., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2022 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

Fibronectin (FN) plays an essential role in the host's response to infection. In previous studies, a significant decrease in the FN level was observed in sepsis; however, it has not been clearly elucidated how this parameter affects the patient's survival. To better understand the relationship between FN and survival, we utilized innovative approaches from the field of explainable machine learning, including local explanations (Break Down, Shapley Additive Values, Ceteris Paribus), to understand the contribution of FN to predicting individual patient survival. The methodology provides new opportunities to personalize informative predictions for patients. The results showed that the most important indicators for predicting survival in sepsis were INR, FN, age, and the APACHE II score. ROC curve analysis showed that the model's successful classification rate was 0.92, its sensitivity was 0.92, its positive predictive value was 0.76, and its accuracy was 0.79. To illustrate these possibilities, we have developed and shared a web-based risk calculator for exploring individual patient risk. The web application can be continuously updated with new data in order to further improve the model.

Bing-Neel syndrome (BNS) is a rare presentation of Waldenström macroglobulinemia (WM). BNS is a consequence of the central nervous system (CNS) involvement by lymphoplasmacytic lymphoma (LPL) and, rarely, the peripheral nervous system. The data on BNS are extremely scarce. Therefore, we performed a multicenter retrospective analysis of BNS patients diagnosed and treated in centers aligned with the Polish Lymphoma Research Group. The analysis covers the years 2014-2021. Eleven patients were included, 55% females and the median age at BNS diagnosis was 61 years. The median time from WM to BNS was 3.5 years; 27% of patients did have a diagnosis of WM and BNS made simultaneously or within 30 days from each other. Isolated parenchymal involvement was the least frequent (20%). Patients were treated with different regimens, mostly able to cross the blood-brain barrier, including 18% treated with ibrutinib first line. The cumulative objective response to treatment was 73%. With the median follow-up of 20 months (95% CI, 2-32), the 36-month estimates were: overall survival (OS) 47%, progression-free survival (PFS) 33%, and cumulative incidence of BNS-associated death 41%. The performance status according to ECOG was significant for PFS (HR = 7.79) and the hemoglobin concentration below 11 g/dL was correlated with PFS. To conclude, BNS is a very rare manifestation of WM. It is associated with a poor outcome with most patients succumbing to BNS.

Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls (HCs). In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analyzed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could distinguish patients from healthy controls. A similar finding was obtained when comparing patients with non-infectious or unknown disease trigger with healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at their disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4&#95;0529 and EBNA6&#95;0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. To confirm this finding, a follow-up study will be conducted in a separate cohort of patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sepúlveda, Malato, Sotzny, Grabowska, Fonseca, Cordeiro, Graça, Biecek, Behrends, Mautner, Westermeier, Lacerda and Scheibenbogen.)

We describe new functionalities that have been added to an existing and widely used software solution in Polish public healthcare. The system automatically evaluates a number of medical scores. It provides epidemiologic monitoring concerning infectious diseases; classifies a particular patient to a specific risk group; and detects anomalies. Moreover, an analysis of a prescribed drug is performed.

(1) Background: There is a constant search for new prognostic factors that would allow us to accurately determine the prognosis, select the type of treatment, and monitor the patient diagnosed with uveal melanoma in a minimally invasive and easily accessible way. Therefore, we decided to evaluate the prognostic role of its pigmentation in a clinical assessment. (2) Methods: The pigmentation of 154 uveal melanomas was assessed by indirect ophthalmoscopy. Two groups of tumours were identified: amelanotic and pigmented. The statistical relationships between these two groups and clinical, pathological parameters and the long-term survival rate were analyzed. (3) Results: There were 16.9% amelanotic tumours among all and they occurred in younger patients (p = 0.022). In pigmented melanomas, unfavourable prognostic features such as: epithelioid cells (p = 0.0013), extrascleral extension (p = 0.027), macronucleoli (p = 0.0065), and the absence of BAP1 expression (p = 0.029) were statistically more frequently observed. Kaplan−Meier analysis demonstrated significantly better overall (p = 0.017) and disease-free (p < 0.001) survival rates for patients with amelanotic tumours. However, this relationship was statistically significant for lower stage tumours (AJCC stage II), and was not present in larger and more advanced stages (AJCC stage III). (4) Conclusions: The results obtained suggested that the presence of pigmentation in uveal melanoma by indirect ophthalmoscopy was associated with a worse prognosis, compared to amelanotic lesions. These findings could be useful in the choice of therapeutic and follow-up options in the future.

Alpha-enolase (ENO1) is a glycolytic metalloenzyme, and its overexpression occurs in numerous cancers, contributing to cancer cell survival, proliferation, and maintenance of the Warburg effect. Patients with an overexpression of ENO1 have a poor prognosis. The aim of the present study was to investigate the prognostic significance of ENO1 in surgical resections from 112 melanoma patients and to assess its expression and enzymatic activity in normoxia and hypoxia in several melanoma cell lines. Overexpression of ENO1 in tumor cells from patients was correlated with unfavorable prognosticators such as Breslow thickness, Clark level, mitotic activity, and the presence of ulceration. The expression of ENO1 also positively correlated with a greater thickness of the neoplastic infiltrate and a worse long-term prognosis for patients with cutaneous melanoma. We report significantly higher expression of ENO1 in melanoma cell lines in comparison to normal melanocytes. To conclude, our in vitro and clinical models showed that overexpression of ENO1 promotes invasiveness of melanoma cells and correlates with aggressive clinical behavior. These observations open the way to further search of a potential prognostic and therapeutic target in cutaneous melanoma.

LncRNAs have arisen as new players in the world of non-coding RNA. Disrupted expression of these molecules can be tightly linked to the onset, promotion and progression of cancer. The present study estimated the usefulness of 14 lncRNAs (HAGLR, ADAMTS9-AS2, LINC00261, MCM3AP-AS1, TP53TG1, C14orf132, LINC00968, LINC00312, TP73-AS1, LOC344887, LINC00673, SOX2-OT, AFAP1-AS1, LOC730101) for early detection of non-small-cell lung cancer (NSCLC). The total RNA was isolated from paired fresh-frozen cancerous and noncancerous lung tissue from 92 NSCLC patients diagnosed with either adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC). The expression level of lncRNAs was evaluated by a quantitative real-time PCR (qPCR). Based on Ct and delta Ct values, logistic regression and gradient boosting decision tree classifiers were built. The latter is a novel, advanced machine learning algorithm with great potential in medical science. The established predictive models showed that a set of 14 lncRNAs accurately discriminates cancerous from noncancerous lung tissues (AUC value of 0.98 ± 0.01) and NSCLC subtypes (AUC value of 0.84 ± 0.09), although the expression of a few molecules was statistically insignificant (SOX2-OT, AFAP1-AS1 and LOC730101 for tumor vs. normal tissue; and TP53TG1, C14orf132, LINC00968 and LOC730101 for LUAD vs. LUSC). However for subtypes discrimination, the simplified logistic regression model based on the four variables (delta Ct AFAP1-AS1, Ct SOX2-OT, Ct LINC00261, and delta Ct LINC00673) had even stronger diagnostic potential than the original one (AUC value of 0.88 ± 0.07). Our results demonstrate that the 14 lncRNA signature can be an auxiliary tool to endorse and complement the histological diagnosis of non-small-cell lung cancer.

Salvage autologous hematopoietic stem cell transplantation (auto-HSCT) constitutes a therapeutic option for a group of well-selected patients with relapsed multiple myeloma (MM). However, if an insufficient number of stem cells were harvested and stored before the first auto-HSCT, stem cells need to be remobilized. Patients diagnosed with MM who following relapse after auto-HSCT, had remobilization and afterward, auto-HSCT with remobilized cells were included in this retrospective analysis. Thirty-three patients, 61% males, the median age 61 years, were included. With a median follow-up of 1.8 years, 2-year progression-free survival was 56.2%, non-relapse mortality 4.8%. The 2-year cumulative incidence of t -MDS was 4.9%. Factors important for the outcome were: the quality of response, previous radiotherapy, the time between the first and salvage auto-HSCT. To conclude, salvage auto-HSCT performed with cells procured after the previous auto-HSCT can be efficacious in relapsed MM, especially if a sufficiently long response had been obtained to the first auto-HSCT(s).

The sudden outbreak and uncontrolled spread of COVID-19 disease is one of the most important global problems today. In a short period of time, it has led to the development of many deep neural network models for COVID-19 detection with modules for explainability. In this work, we carry out a systematic analysis of various aspects of proposed models. Our analysis revealed numerous mistakes made at different stages of data acquisition, model development, and explanation construction. In this work, we overview the approaches proposed in the surveyed Machine Learning articles and indicate typical errors emerging from the lack of deep understanding of the radiography domain. We present the perspective of both: experts in the field - radiologists and deep learning engineers dealing with model explanations. The final result is a proposed checklist with the minimum conditions to be met by a reliable COVID-19 diagnostic model., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Purpose: We analyzed the SARS-CoV-2 genome using our integrated genome analysis system and present the concept of a comprehensive approach to monitoring and surveillance of emerging variants., Material/methods: A total of 69 SARS-CoV-2 positive samples (with Ct value ​≤ ​28) were tested. Samples included in this study were selected from 7 areas of eastern Poland. All samples were sequenced on an Illumina MiSeq platform using a 300-cycle MiSeq Reagent Kit v2. BWA was used for reads mapping on the reference SARS-CoV-2 sequence. SAMTools were used for post-processing of reads to genome assembly. Pango lineage and Nexstrain were used to identify variants and amino acid mutations. Statistical analysis was performed with R 4.0.2., Results: This study shows the first confirmed case of SARS-CoV-2 in Poland with the lineage B.1.351 (known as 501Y.V2 South African variant), as well as another 18 cases with epidemiologically relevant lineage B.1.1.7, known as British variant. Supplementary analysis of SARS-CoV-2 sequences deposited in GISAID shows that the share of a new variant can change rapidly within one month. In addition, we show a complete, integrated concept of a networked system for analyzing the variability of the SARS-CoV-2 genome, which, used in the present study, generated data and a variant report within 6 days., Conclusion: The analyzed viral genomes showed considerable variability with simultaneous clear distinction of local clusters of genomes showing high similarity. Implementing real-time monitoring of new SARS-CoV-2 variants in Poland is urgently needed, and our developed system is available to be implemented on a large scale., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

Objectives: Relapse of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) belongs to the major causes of treatment failure., Methods: Retrospective multicenter analysis of patients diagnosed with AML or MDS who had hematological relapse after allo-HSCT and were treated with azacitidine for this indication., Results: Twenty-three patients receiving azacitidine as the first treatment of relapse (Group&#95;1) and 8 patients receiving azacitidine after other treatment of relapse (Group&#95;2) were included. There were 68% males, median age at initiation of azacitidine was 53 years (15-66). Median time to relapse was 3.5 months and 6.3 months in Group&#95;1 and Group&#95;2, respectively; median time from relapse to azacitidine 0.2 and 2.3 months. Azacitidine 75 mg/m 2 , days 1-7, was administered in 78% and 75% of patients in Group&#95;1 and Group&#95;2, concomitant DLI in 48% and 50%. With median follow-up of 4.7 and 13.6 months, the median overall survival was 5.9 and 9.5 months. 17% and 37.5% patients proceeded to salvage allo-HSCT, with median OS of 11.6 months and not reached respectively., Conclusions: Azacitidine treatment for hematological relapse is associated with poor outcome; nevertheless, a proportion of patients may benefit from it, including patients receiving subsequent salvage allo-HSCT., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The association of immune markers and clinicopathologic features and patient outcome has not been extensively studied in Merkel cell carcinoma (MCC). We correlated tumoral PD-L1 and IDO1 expression, and intratumoral CD8+ and FoxP3+ lymphocytes count with clinicopathologic variables, Merkel cell polyomavirus (MCPyV) status, and patient outcomes in a series of 132 MCC. By univariate analyses, tumoral PD-L1 expression >1% and combined tumoral PD-L1 >1% and high intratumoral FoxP3+ lymphocyte count correlated with improved overall survival (OS) ( p = 0.016, 0.0072), MCC-specific survival (MSS) ( p = 0.019, 0.017), and progression-free survival (PFS) ( p = 0.043, 0.004, respectively). High intratumoral CD8+ and FoxP3+ lymphocyte count correlated with longer MSS ( p = 0.036) and improved PFS ( p = 0.047), respectively. Ulceration correlated with worse OS and worse MSS. Age, male gender, and higher stage (3 and 4) significantly correlated with worse survival. MCPyV positivity correlated with immune response. By multivariate analyses, only ulceration and age remained as independent predictors of worse OS; gender and stage remained for shorter PFS. Tumoral PD-L1 expression and increased density of intratumoral CD8+ lymphocytes and FoxP+ lymphocytes may represent favorable prognosticators in a subset of MCCs. Tumoral PD-L1 expression correlated with intratumoral CD8+ and FoxP3+ lymphocytes, which is supportive of an adaptive immune response.

Background: Merkel cell carcinomas of unknown primary (MCC-UPs) are defined as deep-seated tumors without an associated cutaneous tumor. Although the distinction has important clinical implications, it remains unclear whether these tumors represent primary tumors of lymph nodes or metastatic cutaneous primaries., Methods: We compared the immunohistochemical profiles of four groups of MCCs (Merkel cell polyomavirus (MCPyV)-positive UP, MCPyV-negative UP, MCPyV-positive known primary (KP), and MCPyV-negative KP) using B-cell and pre-B-cell markers, cell cycle regulating proteins, follicular stem cell markers, and immune markers, and performed next generation and Sanger sequencing., Results: Virus-positive and virus-negative MCC-UPs exhibited an immunoprofile similar to virus-positive and virus-negative primary cutaneous MCCs, respectively. MCC-UP tumors (both virus-positive and -negative) were immunogenic with similar or even higher tumoral PD-L1 expression and intratumoral CD8 and FoxP3 infiltrates in comparison to MCPyV-positive cutaneous tumors. In addition, similar to primary cutaneous MCCs, MCPyV-negative MCC-UPs exhibited UV signatures and frequent high tumor mutational burdens, whereas few molecular alterations were noted in MCPyV-positive MCC-UPs., Conclusions: Our results showed distinct UV-signatures in MCPyV-negative tumors and high immunogenicity in MCPyV-positive tumors. Although additional studies are warranted for the MCPyV-positive cases, our findings are supportive of a cutaneous metastatic origin for MCPyV-negative MCC-UP tumors.

(1) Background: Poly(ADP-ribose) polymerase 1) (PARP1) is a pleiotropic enzyme involved in several cellular processes, e.g., DNA damage repair, regulation of mitosis, and immune response. Little is known about the role of PARP1 in melanoma development and progression. We aimed to investigate the prognostic significance of PARP1 expression in cutaneous melanoma through evaluation of mRNA and protein levels of PARP1 in normal melanocytes and melanoma cell lines, as well as in patients' tissue material from surgical resections. (2) Methods: An in vitro model was based on two types of normal human melanocytes (HEMn-DP and HEMn-LP) and four melanoma cell lines (A375, WM1341D, Hs294T, and WM9). PARP1 mRNA gene expression was estimated using real-time polymerase chain reaction (RT-PCR), whereas the protein level of PARP1 was evaluated by fluorescence confocal microscopy and then confirmed by Western Blotting analysis. The expression of PARP1 was also assessed by immunohistochemistry in formalin-fixed paraffin-embedded tissues of 128 primary cutaneous melanoma patients and correlated with follow-up and clinicopathologic features. (3) Results: The in vitro study showed that melanoma cells exhibited significantly higher PARP1 expression at mRNA and protein levels than normal melanocytes. High PARP1 expression was also associated with the invasiveness of tumor cells. Elevated nuclear PARP1 expression in patients without nodal metastases strongly correlated with significantly shorter disease-free survival ( p = 0.0015) and revealed a trend with shorter cancer-specific overall survival ( p = 0.05). High PARP1 immunoreactivity in the lymph node-negative group of patients was significantly associated with higher Breslow tumor thickness, presence of ulceration, and a higher mitotic index ( p = 0.0016, p = 0.023, and p < 0.001, respectively). In patients with nodal metastases, high PARP1 expression significantly correlated with the presence of microsatellitosis ( p = 0.034), but we did not confirm the prognostic significance of PARP1 expression in these patients. In the entire analyzed group of patients (with and without nodal metastases at the time of diagnosis), PARP1 expression was associated with a high mitotic index ( p = 0.001) and the presence of ulceration ( p = 0.036). Moreover, in patients with elevated PARP1 expression, melanoma was more frequently located in the skin of the head and neck region ( p = 0.015). In multivariate analysis, high PARP1 expression was an independent unfavorable prognosticator in lymph node-negative cutaneous melanoma patients. (4) Conclusions: In vitro molecular biology approaches demonstrated enhanced PARP1 expression in cutaneous melanoma. These results were confirmed by the immunohistochemical study with clinical parameter analysis, which showed that a high level of PARP1 correlated with unfavorable clinical outcome. These observations raise the potential role of PARP1 inhibitor-based therapy in cutaneous melanoma.

Introduction: Screening sinonasal evaluation is routinely performed before allogeneic hematopoietic cell transplantation (allo-HCT), however, data supporting such evaluation is inconsistent. O b j e c t i v e s: Assessment of the utility of screening sinonasal evaluation with computed tomography (CT)., Methods: A retrospective analysis of acute leukemia patients who underwent allo-HCT, for whom screening sinonasal CT scans were reevaluated, and for whom Lund-Mackay score (LMS) was calculated. R e s u l t s: Forty-eight patients, the median age at allo-HCT 38 years (18-58), 52% males, were included. 79% had acute myeloid leukemia (AML), 21% acute lymphoblastic leukemia (ALL). Conditioning intensity was myeloablative in 96% of patients, 21% of patients received total body irradiation. 19% of patients had a history of sinusitis before allo-HCT. Screening sinus CT was performed a median of 22 days before allo-HCT. The median LMS was 1 point (0- 10). The severity of sinus abnormalities was: no abnormalities (31%), mild (67%), moderate (2%), severe (0%). Mucosal thickening was the most frequent abnormality (69%). Eleven patients experienced sinusitis after a median of 93 days (11-607) after allo-HCT. 1-year cumulative incidence of sinusitis was 22%. No threshold of LMS and no type of sinus abnormalities were correlated with sinusitis development after allo-HCT. Mild sinus disease at screening did not negatively impact survival in comparison to no sinus disease. C o n c l u s i o n s: Despite the fact, that majority of analyzed patients had either no or mild sinus disease at screening a significant proportion of patients developed sinusitis after allo-HCT. Evaluation of LMS before allo-HCT did not help predict the development of sinusitis after the procedure.

Aim: The evaluation of the prognostic role of nuclear pseudoinclusions (NPIs) and nuclear grooves (NGs) in UM., Patients and Methods: We examined the presence of NPIs and NGs in hematoxylin and eosin-stained tissue sections from 164 removed eyeballs with uveal melanoma (UM) and analyzed statistical relationships with clinical and pathological parameters and the long-term survival rate., Results: We observed NPIs in 38% and NG in 21% of all UM. The presence of NPIs was significantly positively correlated with epithelioid type, marked pleomorphism, and the presence of multinucleated giant cells, macro-nucleoli and multiple nucleoli. Patients with UM with NPIs had a significantly reduced overall survival rate (p<0.0001). The presence of NGs was significantly inversely correlated with marked pleomorphism, and the presence of multinucleated giant cells, macro-nucleoli and multiple nucleoli. Kaplan-Meier analysis demonstrated significantly better overall (p<0.01) and disease-free (p<0.05) survival rates for patients with NGs., Conclusion: The obtained results suggest that the presence of NGs in UM is associated with a better prognosis, as opposed to the presence of NPIs, which means the prognosis is worse., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the sole potential cure for paroxysmal nocturnal hemoglobinuria (PNH); however, the data on its utility in PNH are limited. This retrospective analysis of patients with PNH who underwent allo-HSCT in 11 Polish centers between 2002 and 2016 comprised 78 patients with PHN, including 27 with classic PNH (cPNH) and 51 with bone marrow failure-associated PNH (BMF/PNH). The cohort was 59% male, with a median age of 29 years (range, 12 to 65 years). There was a history of thrombosis in 12% and a history of hemolysis in 81%, and 92% required erythrocyte transfusions before undergoing allo-HSCT. No patient received eculizumab, and 26% received immunosuppressive treatment. The median time from diagnosis to allo-HSCT was 12 months (range, 1 to 127 months). Almost all patients (94%) received reduced-toxicity conditioning, 66% with treosulfan. The stem cell source was peripheral blood in 72% and an identical sibling donor in 24%. Engraftment occurred in 96% of the patients. With a median follow-up of 5.1 years in patients with cPNH and 3.2 years in patients with BMF/PNH, 3-year overall survival (OS) was 88.9% in the former and 85.1% in the latter (P = not significant [NS]). The 3-year OS for patients with/without thrombosis was 50%/92% (P = NS) in the cPNH group and 83.3%/85.3% (P = NS) in the BMF/PNH group. The 3-year OS for in the BMF/PNH patients with/without hemolysis was 93.9%/62.9% (hazard ratio, .13; P = .016). No other factors impacted OS. After allo-HSCT, the frequency of the PNH clone was reduced to 0%, <1%, and <2.4% in 48%, 48%, and 4% of cPNH patients and in 84%, 11%, and 5% of BMF/PNH patients, respectively. The frequency of acute graft-versus-host disease (GVHD) grade II-IV was 23%, and the cumulative 1-year incidence of extensive chronic GVHD was 10.8% in the BMF/PNH group and 3.7% in the cPNH group. Allo-HSCT is a valid option for PNH patients, effectively eliminating the PNH clone with satisfactory overall survival and acceptable toxicity. Reduced-toxicity conditioning with treosulfan is effective and safe in patients with cPNH and BMF/PNH., (Copyright © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Renal impairment (RI) is one of the multiple myeloma (MM)-defining events for initiating therapy. After induction therapy, high-dose chemotherapy followed by autologous peripheral blood stem cell transplant (ASCT) remains the standard of care for transplant-eligible patients with MM. According to the International Myeloma Working Group (IMWG), the organ criterion for kidney damage is defined by a serum creatinine concentration (CrC) > 2 mg/dL or estimated glomerular filtration rate (eGFR) < 40 mL/min. In this long-term study, we evaluated the impact of CrC and eGFR calculated by the Modification of Diet in Renal Disease equation on progression-free and overall survival using a lower threshold than the IMWG criteria., Patients and Methods: We studied the longitudinal outcomes as measured by progression-free survival and overall survival in 59 transplant-eligible patients with MM: 38 patients with normal renal function and 21 patients with RI defined as a CrC higher than upper limit of normal (≥ 1.1 mg/dL), eGFR < 60 mL/min, treated with ASCT from 1998 to 2004., Results: The risk of disease progression and death following ASCT increased by 16.5% (P = .005) and 19% (P < .0009) per 1 mg/dL of CrC, respectively. The thresholds for the association of renal insufficiency and negative outcomes were CrC > 1.4 mg/dL and eGFR < 55mL/min., Conclusions: We observed a negative correlation between minimal renal insufficiency and long-term outcomes. Management of patients with even marginally increased CrC and/or decreased eGFR not fulfilling IMWG RI criteria requires more concentrated effort to reverse even minimal renal insufficiency., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aim: To assess the prognostic significance of nucleolar morphological parameters in a large cohort of patients with uveal melanoma., Patients and Methods: The presence, size and number of nucleoli of cancer cells were assessed in haematoxylin and eosin (HE)-stained slides of 164 formalin-fixed paraffin-embedded primary uveal melanoma tissue specimens. The results were correlated with clinicopathological features and patient survival., Results: The presence of macronucleoli and multiple nucleoli significantly correlated with the epithelioid type of uveal melanoma, high mitotic rate, and marked pleomorphism. There was a positive correlation between the presence of macronucleoli as well as the number of nucleoli and the largest tumour basal diameter. The increased nucleolus count in tumour cells positively correlated with primary tumour (pT) staging. The presence of both prominent and multiple nucleoli was associated with significantly reduced overall and disease-free survival., Conclusion: Histological assessment of nucleolar morphology in routine HE staining would be a helpful low-cost method to obtain reliable prognostic information., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: RhoA and its downstream effectors Rho-associated coiled-coil kinases (ROCK) 1 and 2 are central controllers of cytoskeleton dynamics, and therefore influence cell shape, adhesion and migration. Since modulation of these processes holds promise for an effective anticancer strategy, effects of ROCK inhibition have been evaluated in a number of malignancies., Materials and Methods: Using immunohistochemistry, ROCK1 and ROCK2 expression was semi-quantitatively assessed in 129 patient-derived primary melanomas., Results: There was a striking predilection for low melanocytic expression of both kinases in thick, ulcerated and mitogenic tumors, as well as in nodular histological type. ROCK1 and -2 expression in tumor-infiltrating lymphocytes (TILs) was preferentially down-regulated in advanced and aggressive tumors. Moreover, diminished ROCK2 reactivity in melanoma cells and TILs was associated with shorter melanoma-specific and recurrence-free survival., Conclusion: This is the first analysis of ROCK1 and -2 protein expression in clinical melanoma samples and the results indicated the suppression of ROCK signaling in melanocytes of aggressive and late-stage tumors. Functional models that more accurately represent the clinical setting are necessary to dissect the role of ROCK1 and -2 in melanoma. Additionally, our study indicates that ROCK activity in TILs may be involved in the pathogenesis of cancer, and thus merits further investigations., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Summary: Krüppel-associated box domain zinc finger protein (KRAB ZNF) explorer is a web-based tool for comprehensive characterization of the mRNA expression status of KRAB-ZNF transcription factors in the data from The Cancer Genome Atlas study. Key functionalities cover a comparative analysis of KRAB-ZNF expression between normal and cancer tissues, an association of KRAB-ZNF expression with various pathological features, including survival analysis, association with global DNA methylation status, analyses of KRAB-ZNF isoform expressions and analysis of KRAB-ZNF levels in normal tissues., Availability and Implementation: KRAB ZNF explorer is available at http://mi2.mini.pw.edu.pl: 8080/KRAB&#95;ZNF/. The source code for shiny application is available at: https://github.com/MI2DataLab/KRAB&#95;ZNF/tree/master/app and the source code for analyses and precalculations are available at: https://github.com/MI2DataLab/KRAB&#95;ZNF/tree/master/work., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Identification of genomic biomarkers is an important area of research in the context of drug discovery experiments. These experiments typically consist of several high dimensional datasets that contain information about a set of drugs (compounds) under development. This type of data structure introduces the challenge of multi-source data integration. High-Performance Computing (HPC) has become an important tool for everyday research tasks. In the context of drug discovery, high dimensional multi-source data needs to be analyzed to identify the biological pathways related to the new set of drugs under development. In order to process all information contained in the datasets, HPC techniques are required. Even though R packages for parallel computing are available, they are not optimized for a specific setting and data structure. In this article, we propose a new framework, for data analysis, to use R in a computer cluster. The proposed data analysis workflow is applied to a multi-source high dimensional drug discovery dataset and compared with a few existing R packages for parallel computing.

Introduction: Uveal melanoma (UM) is the most common primary eye tumour in adults. Distant metastases are seen in 50% of cases regardless of treatment, which contributes to high mortality rates. Polo-like kinase-1 (PLK-1) is a protein regulator of mitotic entry and cytokinesis. Increased PLK-1 expression has been shown in different tumours, which makes its inhibition a potential treatment target. To date, no study has been published to discuss the prognostic role of PLK-1 expression in patients with uveal melanoma., Material and Methods: We assessed by immunohistochemistry PLK-1 expression in uveal melanoma cells collected in 158 patients treated by primary enucleation. We determined the correlation between PLK-1 levels evaluated by the immunoreactivity scale (IRS) method and detailed clinical as well as histological parameters. Additionally, we determined the association between PLK-1 expression levels and long-term prognosis., Results: Elevated PLK-1 expression in tumour cells, defined as IRS > 2, was observed in 70% (111/158) of cases, whereas low expression or no expression was seen in the remaining 30% (47/158) of patients. There was a significant correlation between low PLK-1 expression and a higher clinical tumour stage (pT, p = 0.04) as well as a higher AJCC prognostic stage group (p = 0.037). We observed an inverse correlation between PLK-1 expression and tumour cell pigment content (p = 0.0019). There was no correlation between PLK-1 expression and other histological parameters such as mitotic rate or histological subtype. The Kaplan-Meier's analysis demonstrated that low PLK-1 expression was associated with significantly reduced overall survival (p = 0.0058). A similar trend, albeit not significant, was observed for disease-free survival (p = 0.088)., Conclusions: Downregulated PLK-1 expression is a negative prognostic factor in uveal melanoma. It warrants further, multicentre research on prognostic role of PLK-1 expression and possibility of PLK-1 inhibition in uveal melanoma.

Idiopathic scoliosis (IS) etiology remains unclear, but strong genetic background is suggested. Previously reported TIMP2 study indicates an association of genic rs8179090 with IS progression in a Han Chinese population. However, there has been a lack of investigation into intragenic TIMP2 polymorphisms in IS patients. We recruited 100 Caucasian females with IS and 100 controls. Patients were subdivided accordingly to: progression rate, curve severity, joint mobility, and curve pattern. Allele-specific-polymerase chain reaction based on fluorescence resonance energy transfer was applied to evaluate nine TIMP2 polymorphisms. Distribution of genotype and allele frequency in only one polymorphism (rs11658743) differed in case-control study. Four of the polymorphisms (rs2277700, rs11077401, rs2376999, and rs4789934) showed non-equal distributions either in genotype or/and allele distributions in the patients of different progression rates. The rs11077401 was related to curve severity patients distinction and the rs8179090 distinguished patients with different joint mobility level. Two polymorphisms either differed statistically in case of curve patterns subgrouping (rs8068674 and rs8179090) or showed a slight tendency toward significance in the recessive model of allele distributions (rs9916809 and rs8179090). The remaining two polymorphisms (rs2377005, rs11658743) showed no association with either clinical or radiographic IS characteristics. The influence of the G allele of the rs8179090 on the clinical course of IS has not yet been confirmed. We identified four TIMP2 polymorphisms (rs11077401, rs2376999, rs2277700, and rs4789934) that were associated with a higher risk of the progressive IS form. Further genetic association studies based on suggested clinical criteria would be necessary to validate TIMP2 polymorphisms associated with the curve progression. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2217-2225, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

RhoA GTPase is physiologically involved in the formation of stress fibers, cellular contractility and polarity, maintenance of cell cycle and transcriptional control. During tumorigenesis, it plays roles in cancer cell proliferation, apoptosis, adhesion, invasion and metastasis. While RhoA seems to act as a tumor promotor in most malignancies, data regarding its function in skin melanoma are fragmentary and conflicting. We aimed to clarify the clinical significance of RhoA expression in melanoma by immunohistochemical evaluation of 134 primary tumors and subsequent statistical analysis with clinicopathological profiles of patients. Increased RhoA expression was associated with thinner tumors, higher grade of tumor-infiltrating lymphocytes and lack of disease recurrence. Moreover, we observed a trend towards higher RhoA expression in cases without concurrent metastases. Recurrence-free survival and melanoma-specific survival of patients with high RhoA-expressing tumors were significantly prolonged. Multivariable regression model adjusting for melanoma thickness and status of regional lymph nodes confirmed independent prognostic value of RhoA immunoreactivity. In summary, we found associations between RhoA expression and histopathological phenotype of primary tumors as well as patient survival which suggest a suppressive role of RhoA in skin melanoma., Competing Interests: None.

Introduction : Seroma formation (SF) is the most common postoperative complication after mastectomy and axillary surgery. The aim of this study was to assess its incidence and risk factors following a modified radical mastectomy in breast cancer patients. Materials and methods : 271 patients who underwent a modified radical mastectomy (250 with traditional electrocautery and 21 with an ultrasonic scalpel) were studied. The SF rate was calculated and its association with patient-related factors, surgical features and postoperative variables was assessed and statistically analyzed with P <0.05 as a significance threshold. Results : SF was observed in 18% of patients. Patient's age, operating time and number of removed axillary lymph nodes did not significantly differ between SF and non-SF patients. Patients BMI, total drainage amount, number of days with drain were higher and postoperative hospital stay was significantly longer in SF patients ( P <0.001 each). The dissection instrument was also an important risk factor: SF developed in 20% of patients operated with electrocautery and in none with an ultrasonic scalpel ( P <0.05). The association between surgical instrument and the number of removed lymph nodes, patient's age and BMI was not significant. Dissection with an ultrasonic scalpel resulted in a statistically significant lower total drainage amount. However, it was also related to a significantly longer operating time ( P <0.001 each). Conclusions : Risk of SF after a modified radical mastectomy is significantly higher in patients with obesity. Despite longer operating time, using an ultrasonic scalpel is a valuable option in those cases because it lowers the total drainage amount and seems to be an important protective factor against SF., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) patients, including those treated with azacitidine, are at increased risk for serious infections. The aim of our study was to identify patients with higher infectious risk at the beginning of azacitidine treatment., Patients and Methods: We performed a retrospective evaluation of 298 MDS/CMML/AML patients and included in the analysis 232 patients who completed the first 3 cycles of azacitidine therapy or developed Grade III/IV infection before completing the third cycle., Results: Overall, 143 patients (62%) experienced serious infection, and in 94 patients (41%) infection occurred within the first 3 cycles. The following variables were found to have the most significant effect on the infectious risk in multivariate analysis: red blood cell transfusion dependency (odds ratio [OR], 2.38; 97.5% confidence interval [CI], 1.21-4.79), neutropenia <0.8 × 10 9 /L (OR, 3.03; 97.5% CI, 1.66-5.55), platelet count <50 × 10 9 /L (OR, 2.63; 97.5% CI, 1.42-4.76), albumin level <35 g/dL (OR, 2.04; 97.5% CI, 1.01-4.16), and Eastern Cooperative Oncology Group performance status ≥2 (OR, 2.19; 97.5% CI, 1.40-3.54). Each of these variables is assigned 1 point, and the combined score represents the proposed Azacitidine Infection Risk Model. The infection rate in the first 3 cycles of therapy in lower-risk (0-2 score) and higher-risk (3-5 score) patients was 25% and 73%, respectively. The overall survival was significantly reduced in higher-risk patients compared with the lower-risk cohort (8 vs. 29 months)., Conclusion: We selected a subset with high early risk for serious infection and worse clinical outcome among patients treated with azacitidine., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Overexpression of SMAD7-a hallmark inhibitor of transforming growth factor β (TGFβ) signaling-has been documented and related with adverse prognosis in a number of epithelial malignancies, suggesting that it may be responsible for resistance to TGFβ-induced growth arrest of cancer cells. The involvement of SMAD7 in development and progression of malignant melanoma is unclear, and its expression has not been characterized so far at the protein level in clinical melanoma tissue samples. We evaluated SMAD7 expression in 205 skin melanoma primary tumors by immunohistochemistry and correlated the findings with clinicopathological profiles of patients. Melanocytic SMAD7 was evidenced in 204 cases, and the expression pattern was predominantly nuclear. High expression of SMAD7 was positively associated with several features of tumor aggressiveness, for example, presence of ulceration (P < 0.001), higher tumor thickness (P < 0.001), and mitotic rate (P < 0.001), but not presence of regional or distant metastases. Moreover, high SMAD7 expression independently predicted unfavorable outcome: melanoma-specific survival (hazard ratio = 3.16, P < 0.001) and recurrence-free survival (hazard ratio = 2.88, P < 0.001). Taken together, our results underline the importance of TGFβ signaling in cancer and define SMAD7 as a marker of aggressive tumor behavior and adverse clinical outcomes in melanoma patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We retrospectively analyzed long-term disease outcome of 350 elderly Hodgkin Lymphoma (eHL) patients treated with ABVD/ABVD-like regimen enrolled in the PLRG-R9 study between 2001 and 2013 in Poland. Complete remission was reported for 73% of early (ES) and 61% advanced stage (AS) patients. Nine (10%) ES and 56 (20%) AS patients have died. With the median follow-up of 36 (1-190) months, 3-year EFS and OS was 0.74 (95%CI: 0.63-0.85) and 0.90 (95%CI: 0.82-0.98) for ES; 0.51 (95%CI: 0.44-0.57), and 0.81 (95%CI: 0.75-0.86) for AS patients, respectively. For ES patients, Cox regression revealed ECOG <2 and age >70 as predictive for inferior OS and EFS. For AS patients, the most predictive for OS was the presence of cardiovascular disorders (CVD) (p = .00044), while for EFS four factors were significantly associated with a poor outcome: ECOG< 2, age >70 years, CVD and extranodal disease. In conclusion, CVD significantly impacts outcomes of ABVD-treated advanced eHL patients.

Epithelial-mesenchymal transitions (EMTs) have been recently implicated in the process of cancer progression. The aim of this study was to assess how the preoperative expression patterns of EMT biomarkers correlate with the risk of postoperative invasion in ductal carcinoma in situ (DCIS) found on stereotactic breast biopsies. N-cadherin, Snail1, and secreted protein acidic and rich in cysteine (SPARC) immunoreactivity was observed in 8%, 62%, and 38% of tumors, respectively. Snail1 and SPARC expressions were significantly related to N-cadherin expression and to each other. The postoperative upgrading rate was associated with a positive preoperative expression of all biomarkers. Significance of Snail1 and SPARC persisted in multivariate analysis, but the impact of SPARC on invasion was more significant. When these two EMT triggers were considered together, the risk of invasion did not significantly differ between the subtypes of DCIS with single positive expression (SPARC-/Snail1+ vs. SPARC+/Snail1-). However, it was significantly lower in single-positive DCIS when compared to lesions of a double-positive profile (SPARC+/Snail1+). Moreover, there were no cases in the double-negative DCIS (SPARC-/Snail1-), with foci of infiltrating cancer found postoperatively in residual postbiopsy lesions. In contrast, DCIS with a combined high SPARC and Snail1 expression (intermediate or strong) had an invasive component in 66⁻100% of tumors.

Atypical chronic myeloid leukaemia (aCML) belongs to myelodysplastic/myeloproliferative neoplasms. Because of its rarity and changing diagnostic criteria throughout subsequent classifications, data on aCML are very scarce. Therefore, we at the Polish Adult Leukemia Group performed a nationwide survey on aCML. Eleven biggest Polish centres participated in the study. Altogether, 45 patients were reported, among whom only 18 patients (40%) fulfilled diagnostic criteria. Among misdiagnosed patients, myelodysplastic/myeloproliferative syndrome unclassifiable and chronic myelomonocytic leukaemia were the most frequent diagnoses. Thirteen patients were male, median age 64.6 years (range 40.4-80.9). The median parameters at diagnosis were as follows: white blood cell count 97 × 10 9 /L (23.8-342) with immature progenitors amounting at 27.5% (12-72), haemoglobin 8.6 g/dL (3.9-14.9), and platelet count 66 × 10 9 /L (34-833). Cytoreductive treatment was used in all patients, and 2 patients underwent allogeneic hematopoietic stem cell transplantation. The median overall survival was 14.1 months (95% CI, 7.2), with median acute myeloid leukaemia-free survival of 13.3 months (95% CI, 3.6-22.6). Cumulative incidence of acute myeloid leukaemia transformation after 1 year in aCML group was 12.5% (95% CI, 0%-29.6%). To conclude, aCML harbours a poor prognosis. Treatment options are limited, with allogeneic hematopoietic stem cell transplantation being the only curative method at present, although only a minority of patients are transplant eligible. Educational measures are needed to improve the quality of diagnoses., (Copyright © 2018 John Wiley & Sons, Ltd.)

Nuclear pseudoinclusions (NPIs) are classically found in papillary thyroid carcinoma and meningioma. Although NPIs have been described in melanocytic lesions, there is no systematic analysis of potential relationship between NPIs and other clinicopathological characteristics of melanoma. We examined the presence of NPIs in H&E-stained tissue sections form 96 melanomas and analyzed statistical associations with important clinicopathological parameters and tissue immunoreactivity for selected proteins involved in epithelial-mesenchymal transition (SPARC, N-cadherin), cell adhesion and mobility (ALCAM, ADAM-10), regulation of mitosis (PLK1), cell survival (FOXP1) and functioning of Golgi apparatus (GOLPH3, GP73). NPIs were observed in 20% of melanomas and their presence correlated with high mitotic rate and ulceration of the tumor, but not with Breslow thickness, histologic type, or presence of metastases. We observed a significant correlation with shorter cancer-specific survival, but not disease-free survival. Presence of NPIs was related to high expression of GOLPH3 in melanoma cells, whereas their absence was linked to enhanced immunoreactivity of GOLPH3 in tumor-associated macrophages. NPIs are not an uncommon finding in skin melanoma and their diagnostic and prognostic utility could be helpful in the daily routine histopathological practice. The possible explanation of NPI generation is associated with enhanced activity of Golgi apparatus in melanoma cells., (© 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Aim: Incurred sample reanalysis (ISR) helps ensure the reliability of pharmacokinetic studies. An appropriate graph may facilitate the evaluation of an unmatched reanalyses or a failed ISR test., Methods: We evaluated different ways of visualizing multidimensional ISR data using an extended cumulative ISR plot., Results: 3D and 4D cumulative ISR plots enable comprehensive data analysis using a single plot. We propose to use color for percentage difference classes in bar and XY-scatter plots. For the latter the shape of symbols may represent analyte concentration class, study phase, analyst or subject., Conclusion: The extended 3D and 4D cumulative ISR plots facilitate in-study monitoring and post-study inspection of data. It helps find the root cause of unmatched ISR, thus increasing reliability of bioanalytical data.

Background: The nucleolus is an organelle that is an ultrastructural element of the cell nucleus observed in H&E staining as a roundish body stained with eosin due to its high protein content. Changes in the nucleoli cytomorphology were one of the first histopathological characteristics of malignant tumors. The aim of this study was to assess the relationship between the cytomorphological characteristics of nucleoli and detailed clinicopathological parameters of melanoma patients. Moreover, we analyzed the correlation between cytomorphological parameters of nucleoli and immunoreactivity of selected proteins responsible for, among others, regulation of epithelial-mesenchymal transition (SPARC, N-cadherin), cell adhesion and motility (ALCAM, ADAM-10), mitotic divisions (PLK1), cellular survival (FOXP1) and the functioning of Golgi apparatus (GOLPH3, GP73)., Methods: Three characteristics of nucleoli - presence, size and number - of cancer cells were assessed in H&E-stained slides of 96 formalin-fixed paraffin-embedded primary cutaneous melanoma tissue specimens. The results were correlated with classical clinicopathological features and patient survival. Immunohistochemical analysis of the above mentioned proteins was described in details in previous studies., Results: Higher prevalence and size of nucleoli were associated with thicker and mitogenic tumors. All three nucleolar characteristics were related to the presence of ulceration. Moreover, microsatellitosis was strongly correlated with the presence of macronucleoli and polynucleolization (presence of two or more nucleoli). Lack of immunologic response manifested as no TILs in primary tumor was associated with high prevalence of melanoma cells with distinct nucleoli. Interestingly, in nodular melanoma a higher percentage of melanoma cells with prominent nucleoli was observed. In Kaplan-Meier analysis, increased prevalence and amount, but not size of nucleoli, were connected with shorter cancer-specific and disease-free survival., Conclusion: (1) High representation of cancer cells with distinct nucleoli, greater size and number of nucleoli per cell are characteristics of aggressive phenotype of melanoma; (2) higher prevalence and size of nucleoli are potential measures of cell kinetics that are strictly correlated with high mitotic rate; and (3) high prevalence of cancer cells with distinct nucleoli and presence of melanocytes with multiple nucleoli are features associated with unfavorable prognosis in patients with cutaneous melanoma.

Reprogramming to induced pluripotent stem cells (iPSCs) and differentiation of pluripotent stem cells (PSCs) are regulated by epigenetic machinery. Tripartite motif protein 28 (TRIM28), a universal mediator of Krüppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate both processes; however, the exact mechanism and identity of participating KRAB-ZNF genes remain unknown. Here, using a reporter system, we show that TRIM28/KRAB-ZNFs alter DNA methylation patterns in addition to H3K9me3 to cause stable gene repression during reprogramming. Using several expression datasets, we identified KRAB-ZNFs (ZNF114, ZNF483, ZNF589) in the human genome that maintain pluripotency. Moreover, we identified target genes repressed by these KRAB-ZNFs. Mechanistically, we demonstrated that these KRAB-ZNFs directly alter gene expression of important developmental genes by modulating H3K9me3 and DNA methylation of their promoters. In summary, TRIM28 employs KRAB-ZNFs to evoke epigenetic silencing of its target differentiation genes via H3K9me3 and DNA methylation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

It remains a formidable challenge to characterize the diverse complexes of RNA binding proteins and their targets. While crosslink and immunoprecipitation (CLIP) methods are powerful techniques that identify RNA targets on a global scale, the resolution and consistency of these methods is a matter of debate. Here we present a comparative analysis of LIN28-pre-let-7 UV-induced crosslinking using a tandem mass spectrometry (MS/MS) and deep sequencing interrogation of in vitro crosslinked complexes. Interestingly, analyses by the two methods diverge in their identification of crosslinked nucleotide identity - whereas bioinformatics and sequencing analyses suggest guanine in mammalian cells, MS/MS identifies uridine. This work suggests the need for comprehensive analysis and validation of crosslinking methodologies.

Background and Objectives: Transcription factor Snail1 is a key inducer of epithelial-mesenchymal transition (EMT), a biological process implicated in the cancer progression and metastasis. The aim of the study was to investigate Snail1 expression in DCIS found on breast biopsy and assess its predictive value for the final invasion., Methods: A total of 209 patients with histologically diagnosed pure DCIS entered the study. Snail1 reactivity was evaluated with immunohistochemistry in tumor tissue from stereotactic vacuum-assisted biopsy of suspicious microcalcifications., Results: Snail1 staining was observed in 62% of tumors: weak, intermediate, and strong in 27%, 21%, and 14% of lesions, respectively. Positive Snail1 expression was significantly rarer in DCIS presenting as powdery microcalcifications, when compared with crushed stone-like and casting-type and was more common in DCIS with comedonecrosis. Correlation with other features was not significant. None of standard parameters significantly influenced the upgrading rate. In contrast, in uni- and multivariate analysis the risk of postoperative invasion was significantly associated with positive Snail1 immunoreactivity. Moreover, there was a significant stepwise increase of upgrading rate according to Snail1 expression in DCIS cells: weak 9%, intermediate 26%, and strong 55%, respectively., Conclusions: Snail1 can reflect the invasive potential of DCIS and help identify its more aggressive subtypes., (© 2017 Wiley Periodicals, Inc.)

Utilizing the TCGA PANCAN12 dataset we discovered that cancer patients with mutations in TP53 tumor suppressor and overexpression of MDM2 oncogene exhibited decreased survival post treatment. Interestingly, in the case of breast cancer patients, this phenomenon correlated with high expression level of several molecular chaperones belonging to the HSPA, DNAJB and HSPC families. To verify the hypothesis that such a genetic background may promote chaperone-mediated chemoresistance, we employed breast and lung cancer cell lines that constitutively overexpressed heat shock proteins and have shown that HSPA1A/HSP70 and DNAJB1/HSP40 facilitated the binding of mutated p53 to the TAp73α protein. This chaperone-mediated mutated p53-TAp73α complex induced chemoresistance to DNA damaging reagents, like Cisplatin, Doxorubicin, Etoposide or Camptothecin. Importantly, when the MDM2 oncogene was overexpressed, heat shock proteins were displaced and a stable multiprotein complex comprising of mutated p53-TAp73α-MDM2 was formed, additionally amplifying cancer cells chemoresistance. Our findings demonstrate that molecular chaperones aid cancer cells in surviving the cytotoxic effect of chemotherapeutics and may have therapeutic implications., Competing Interests: CONFLICTS OF INTEREST The authors claim no conflicts of interest.

Aim: Incurred sample reanalysis (ISR) contributes to the reliability of pharmacokinetic studies. Despite regulatory guidelines having adopted ISR methodology, graphical presentation of data has been overlooked., Materials & Methods: Different graphs were tested for datasets including limited, standard and large numbers of ISR pairs. The datasets covered both passed and failed cases., Results: We have developed a combination of complementary plots enabling the visual inspection of ISR data quality: %difference versus mean concentration and cumulative ISR plot. The former shows individual ISR datapoints and concentration-dependent trends, while the latter presents the contribution of individual pairs to the overall result as well as time-dependent trends., Conclusion: The proposed visualization of ISR data shows at a glance whether acceptance criteria for each sample and whole experiment are met or not. Standardized graphical presentation of ISR outcomes may increase quality of bioanalytical data.

ATP-dependent chromatin remodeling complexes are important regulators of gene expression in Eukaryotes. In plants, SWI/SNF-type complexes have been shown critical for transcriptional control of key developmental processes, growth and stress responses. To gain insight into mechanisms underlying these roles, we performed whole genome mapping of the SWI/SNF catalytic subunit BRM in Arabidopsis thaliana, combined with transcript profiling experiments. Our data show that BRM occupies thousands of sites in Arabidopsis genome, most of which located within or close to genes. Among identified direct BRM transcriptional targets almost equal numbers were up- and downregulated upon BRM depletion, suggesting that BRM can act as both activator and repressor of gene expression. Interestingly, in addition to genes showing canonical pattern of BRM enrichment near transcription start site, many other genes showed a transcription termination site-centred BRM occupancy profile. We found that BRM-bound 3΄ gene regions have promoter-like features, including presence of TATA boxes and high H3K4me3 levels, and possess high antisense transcriptional activity which is subjected to both activation and repression by SWI/SNF complex. Our data suggest that binding to gene terminators and controlling transcription of non-coding RNAs is another way through which SWI/SNF complex regulates expression of its targets., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated the role of TRIM28 protein in the regulation of breast cancer stem cells (CSC) populations and tumorigenesis in vitro and in vivo. Downregulation of TRIM28 expression in xenografts led to deceased expression of pluripotency and mesenchymal markers, as well as inhibition of signaling pathways involved in the complex mechanism of CSC maintenance. Moreover, TRIM28 depletion reduced the ability of cancer cells to induce tumor growth when subcutaneously injected in limiting dilutions. Our data demonstrate that the downregulation of TRIM28 gene expression reduced the ability of CSCs to self-renew that resulted in significant reduction of tumor growth. Loss of function of TRIM28 leads to dysregulation of cell cycle, cellular response to stress, cancer cell metabolism, and inhibition of oxidative phosphorylation. All these mechanisms directly regulate maintenance of CSC population. Our original results revealed the role of the TRIM28 in regulating the CSC population in breast cancer. These findings may pave the way to novel and more effective therapies targeting cancer stem cells in breast tumors.

GOLPH2 and GOLPH3 are Golgi-related proteins associated with aggressiveness and progression of a number of cancers. Their prognostic significance in melanoma has not yet been analyzed. We performed immunohistochemical analysis for GOLPH2 and GOLPH3 in 20 normal skin, 30 benign nevi and 100 primary melanoma tissue samples and evaluated their expression in three compartments: cancer cells, tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs). High levels of both proteins in melanoma cells were associated with characteristics of aggressive disease, and shorter disease-free survival (DFS) and cancer-specific overall survival (CSOS). On the contrary, increased numbers of GOLPH2-positive and GOLPH3-positive TAMs were observed in thinner, non-ulcerated tumors, with brisk lymphocytic reaction and absent lymphangioinvasion. Distant metastases were not observed among patients with high numbers of GOLPH2-positive TAMs. Increased expression of either protein in TAMs was related to prolonged CSOS and DFS. Similarly, GOLPH3-expressing CAFs were more frequent in thin melanomas with low mitotic rate, without ulceration and lymphangioinvasion. Moreover, increased GOLPH3-positive CAFs correlated with the absence of regional or distant metastases, and with longer CSOS and DFS. GOLPH2 expression was not observed in CAFs. Our results suggest that GOLPH2 and GOLPH3 play a role in melanoma progression and are potential targets for molecular-based therapies., Competing Interests: The authors declare no conflict of interest.

Background and Objectives: Secreted protein, acidic and rich in cysteine (SPARC) is able to play an important role in cancer invasion due to de-adhesive properties and impact on stromal remodeling. The aim of study was to investigate SPARC expression in preoperatively diagnosed breast DCIS and to assess its predictive value for the final invasion., Methods: A total of 209 patients with DCIS found on stereotactic vacuum-assisted biopsy of suspicious microcalcifications were studied prospectively., Results: SPARC staining was positive in luminal epithelial cells, stromal fibroblasts, and myoepithelial cells in 38%, 62%, and 61% of tumors, respectively. Neither patient age nor pattern of microcalcifications were related to SPARC expression. High nuclear grade and comedonecrosis were associated with strong immunoreactivity of SPARC in stromal fibroblasts and myoepithelial cells while not in luminal epithelial cells. Rate of postoperative invasion was significantly increased in DCIS with strong SPARC staining with regard to all investigated cells. None of standard parameters significantly influenced the upgrading risk. In multivariate analysis most significant and independent predictive factors were strong SPARC expression in luminal epithelial cells, and stromal fibroblasts., Conclusions: SPARC can be a new biomarker helpful to identify more aggressive DCIS and for prediction of invasive disease on final pathology. J. Surg. Oncol. 2016;114:548-556. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Aim: To develop a multivariable survival model based on histopathological parameters that would provide the best possible estimate of the risk of death in cutaneous melanoma patients., Materials and Methods: Breslow thickness and Infiltrating Lymphocytes (BILL(CD8)) is based on two key parameters, namely Breslow thickness and tumor-infiltrating lymphocytes (TILs) shown to be the most important prognostic factors using logistic regression model. The next step was to apply a decision tree method to analyze the impact of the percentage of CD8+ lymphocytes on the risk of cancer death., Results: A patient is given two points if Breslow thickness is >2 mm and one point if TILs are not found in the tumor. The probability of death over a 4-year follow-up is 75% if the patient's score is 3 points; in patients with 0 point score the risk of death is 0%. A significant positive impact of high percentage of CD8+ lymphocytes on the risk of death was shown in the subgroup of melanoma patients with Breslow thickness >2 mm where the risk of death is only 28%. In the case of low percentage of CD8+ lymphocytes in melanomas with Breslow thickness over 2 mm, the risk is as high as 67%., Conclusion: Breslow thickness and TILs, with the evaluation of the CD8+ lymphocytes, may be used for precise prognostic stratification of patients and may help intensify the follow-up, diagnose a relapse sooner and apply proper treatment., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Hypertension is a multifactorial disease caused by environmental, metabolic and genetic factors, but little is currently known on the complex interplay between these factors and blood pressure. The aim of the present study was to assess the potential impact of obesity, and angiotensin-converting enzyme (ACE) I/D polymorphism and endothelial nitric oxide synthase gene (NOS3) 4a/4b, G894T and -T786C variants on the essential hypertension. The study group consisted of 1,027 Caucasian adults of Polish nationality (45.5 ± 13.6 years old), of which 401 met the criteria for hypertension. Body weight, height and blood pressure were measured and data on self-reported smoking status were collected. Fasting blood glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides were determined by standard procedures. The ACE I/D polymorphism and three polymorphisms in NOS3 gene (4a/4b, G894T, -T786C) were detected by the PCR method. Multivariable logistic regression demonstrated that age above 45 years, diabetes, dyslipidemia, smoking and male sex are important risk factors for hypertension and no significant influence of variants in ACE and NOS3 genes on this risk was recognized. Obese subjects had a 3.27-times higher risk (OR = 3.27, 95% CI: 2.37 - 4.52) of hypertension than non-obese, and in obese the NOS3 894T allele was associated with 1.37 fold higher risk of hypertension (P = 0.031). The distribution of NOS3 G894T genotypes supported the co-dominant (OR = 1.35, P = 0.034, Pfit = 0.435) or recessive (OR = 2.00, P = 0.046, Pfit = 0.286), but not dominant model of inheritance (P = 0.100). The study indicates that in obese NOS3 G894T polymorphism may enhance hypertension risk. However, in the presence of such strong risk factors as age, diabetes and smoking, the impact of this genetic variant seems to be attenuated. Further studies are needed to reveal the usefulness of G894T polymorphism in hypertension risk assessment in obese.

Background: There is an ongoing debate about factors affecting the maintenance of a healthy lifestyle especially in the population without coronary artery disease (CAD) symptoms and with one or several risk factors. The study was aimed at describing self-reported health-related behaviors and dietary habits in patients with metabolic syndrome (MetS)., Methods: Consecutive patients with an outpatient diagnosis of MetS admitted to our cardiology department underwent clinical examination and cardiovascular risk assessment based on the SCORE scale. Self-reported intensity of pro-healthy behaviors was described using the Health Behavior Inventory (HBI) developed by Juczynski. Diet quality was assessed using the 24-h dietary recall method, diet history questionnaire and the Healthy Eating Index-2010 (HEI)., Results: A total of 113 patients were recruited (90 males, mean age 48 ± 9 years) including 85% of patients with at least moderate cardiovascular risk (SCORE ≥ 1%). Central obesity was confirmed in 100%, family history of CAD in 75%, LDL exceeding 115 mg/dL in 68% of the patients. A total of 66% of the patients had already been on antihypertensive and 30% on lipid-lowering treatment without previous counselling on lifestyle modification. Most patients reported high or medium level health-related behaviors (23% and 45%, respectively). However, 91% led sedentary lifestyle and none of the patients followed cardioprotective diet recommendations. According to the HEI, 73% required partial and 27% complete diet modification., Conclusions: There is a significant discrepancy between health perception and medical recommendations in patients with MetS. Effective patient education, taking into account a revision of the patient's knowledge on the principles of prophylaxis, may form the fundament for the changes in patient behavior, and cardiovascular risk reduction.

Although some hypotheses that attempt to explain the variation in supraorbital region morphology in modern humans have been proposed, this issue is still not well understood. In this study, the craniofacial size and spatial models were tested using a sample of modern human crania from geographically diverse populations, and the co-occurrence of the degrees of glabella (GL) and superciliary arch (ST) expression were analyzed. The two supraorbital structures were examined by visual assessment, and eight quantitative variables were included in the three-way ANOVA, canonical variates analysis and partial rank correlation. The influences of sex and the region of origin of the cranial samples on the relationships between the examined variables and the degrees of supraorbital structures expression were also considered. The results only partially supported the craniofacial size and spatial models and suggested that GL and ST experienced separate influences during development. In the sample of all crania, the neurocranial size more strongly influenced the morphological variation of the ST than of the GL, and sex influenced both of these structures the most. The results suggest that sex may be the main factor (having an influence independent of the other traits) on the morphological variation of the GL and ST., (© 2014 Wiley Periodicals, Inc.)

The differentiation-inducing potential of side-chain modified analogs of vitamins D, compared to the reference compound, 1,25-dihydroxyvitamin D3, was studied in blast cells from patients with acute myeloid leukemia and in cell lines. Analogs PRI-1906 and PRI-1907 showed increased cell-differentiation activities, PRI-1907 even at a very low concentration. Our study revealed a high variability of individual patients' blasts in their susceptibility to vitamin D analogs. The blasts of the patients with normal karyotype and with mutated NPM1 reacted to analogs with stronger differentiation than the blasts of the remaining patients, while the blasts with mutated FLT3 receptor reacted with weaker differentiation than the remaining blasts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)