Spatial immunophenotyping of the tumour microenvironment in non-small cell lung cancer.

INTRODUCTION: Immune cells in the tumour microenvironment are associated with prognosis and response to therapy. We aimed to comprehensively characterise the spatial immune phenotypes in the mutational and clinicopathological background of non-small cell lung cancer (NSCLC). METHODS: We established a multiplexed fluorescence imaging pipeline to spatially quantify 13 immune cell subsets in 359 NSCLC cases: CD4 effector cells (CD4-Eff), CD4 regulatory cells (CD4-Treg), CD8 effector cells (CD8-Eff), CD8 regulatory cells (CD8-Treg), B-cells, natural killer cells, natural killer T-cells, M1 macrophages (M1), CD163+ myeloid cells (CD163), M2 macrophages (M2), immature dendritic cells (iDCs), mature dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). RESULTS: CD4-Eff cells, CD8-Eff cells and M1 macrophages were the most abundant immune cells invading the tumour cell compartment and indicated a patient group with a favourable prognosis in the cluster analysis. Likewise, single densities of lymphocytic subsets (CD4-Eff, CD4-Treg, CD8-Treg, B-cells and pDCs) were independently associated with longer survival. However, when these immune cells were located close to CD8-Treg cells, the favourable impact was attenuated. In the multivariable Cox regression model, including cell densities and distances, the densities of M1 and CD163 cells and distances between cells (CD8-Treg-B-cells, CD8-Eff-cancer cells and B-cells-CD4-Treg) demonstrated positive prognostic impact, whereas short M2-M1 distances were prognostically unfavourable. CONCLUSION: We present a unique spatial profile of the in situ immune cell landscape in NSCLC as a publicly available data set. Cell densities and cell distances contribute independently to prognostic information on clinical outcomes, suggesting that spatial information is crucial for diagnostic use.

Generalized perforating granuloma annulare associated with latent tuberculosis successfully treated with isoniazid: case report and review.

Generalized perforating granuloma annulare (GPGA) is a very rare form of granuloma annulare, with only 31 reported cases to the best of our knowledge. Furthermore, GPGA is a chronic disease that mimics many diseases, with no known exact etiology, resulting in a lack of specific clinical criteria leading to a lack of guidelines for diagnosis and therapy. In GPGA, papules are the predominant lesions followed by central crusting/scaling or umbilication; pustules, plaques, annular lesions or nodules are less frequent. We report a 66-year-old woman who presented with a 7-month history of mostly asymptomatic generalized infiltrated, flesh-colored to red-brown umbilicated or crusted papules. Histopathological findings were compatible with perforating granuloma annulare. Diagnostic workup revealed latent tuberculosis. To the best of our knowledge, this is the second published case of GPGA associated with latent tuberculosis and the first one that was successfully treated by isoniazid monotherapy. From our case we can speculate and support the theory that GPGA is a phenotypic granulomatous response to multiple etiologies and/or antigenic stimulation and that testing for tuberculosis should be seriously considered in the evaluation of patients with GPGA.

Cutaneous polyarteritis nodosa in three patients: disease course and our experience leading to faster remission.

We present three new cases of cutaneous polyarteritis nodosa with a follow-up ranging from 38 to 49 months, describing their clinical and histological findings, as well as treatment options leading to sustained remission. All patients met the criteria for diagnosis. The presence of extracutaneous symptoms and laboratory analysis differed among our patients, as did various elements of the workup in comparison to published studies. We concluded that dapsone alone, or in combination with systemic steroids, proved superior and highly effective despite being less frequently used. More aggressive therapy for shorter intervals could lead to quicker remission of cutaneous lesions and symptoms without chronic relapses, which are commonly noted.

Miliaria Scarlatinosa, A Peculiar and Rare Form of Scarlet Fever - A Case Report.

Scarlet fever typically presents with distinctive erythematous papular rash following pharyngitis. Atypical forms may develop, making the diagnosis difficult. We present the case of a girl with fever, and unusual vesicular skin eruption (miliaria scarlatinosa) preceded by a skin infection, without mucosal changes. Leukocyte count, C-reactive protein, and antistreptolysin O-titer were elevated. Bacteriological swabs of the skin injury revealed Streptococcus pyogenes. Histopathology was compatible with scarlet fever exanthema. Intramuscular penicillin and topical wound care induced complete remission. It is of great importance to be aware of uncommon clinical presentations of scarlet fever in order to establish a timely diagnosis and prevent potential complications.

Treatment of COVID 19-Repurposing drugs commonly used in dermatology.

For the last two decades, the outbreaks of diseases caused by coronaviruses and intermittent worldwide public health emergences have reminded us that they still represent a severe threat to global health. The recent outbreak of corona virus disease 19 (COVID-19) highlighted the urgent need for effective treatment, and initiated rapid search for therapies, able to counter the most severe disease effects. Many aspects of COVID-19 pathogenesis are unknown, but complex interplay of direct viral damage and immune response dysregulation is underline. Intensive research is undergoing for therapeutic targets of virus and high-efficiency and low toxicity targeted drugs. There is no available specific antiviral treatment of this disease, therefore repurposing of drugs already available for the treatment of other viral and autoimmune diseases has been a part of research efforts. Well known anti-inflammatory properties of chloroquine and hydroxychloroquine, agents widely used in dermatology, made them potential candidates for the treatment of COVID-19. We review pathogenesis and clinical characteristic of COVID-19, as well as treatment options that have been under evaluation in past several months. In addition, we focus more on chloroquine and hydroxychloroquine, their pharmacological properties, clinical utility, and current recommendations for their use in COVID-19.

Keratoacanthoma centrifugum marginatum associated with mechanical trauma: Response to acitretin-A case report and review of the literature.

Keratoacanthoma centrifugum marginatum (KCM) is a very rare variant of keratoacanthoma, characterized with progressive centrifugal growth, central healing, and atrophy. Due to its rarity and lack of distinctive histopathological features, KCM often raises diagnostic and therapeutic challenge. We present a case of a 76-year-old Caucasian woman with a single large tumor on her right shin that responded to oral retinoids. The patient presented history of local trauma. The tumor developed over the course of 20 months from a scar. To the best of our knowledge, this is the fifth case of KCM associated with mechanical trauma as a possible triggering factor.

Linear IgA dermatosis of the childhood-Report of an amoxicillin-induced case.

Linear IgA dermatosis (LAD) is a rare autoimmune disorder in children. A 9-year-old boy was presented with blisters on the intact skin (face, body, arms, hands, soles, perigenital and perianal area) after amoxicillin treatment. Systemic corticosteroids and dapsone treatment for 6 weeks was successful. Clinical and immunofluorescence examinations are most important for differentiation of LAD and other drug-induced bullous dermatoses. They enable an early introduction of proper therapy.

Poikilodermatous mycosis fungoides with CD30-positive large cell transformation successfully treated by brentuximab vedotin.

We present a patient with a 33-year history of poikilodermatous mycosis fungoides (MF) who subsequently developed CD30-positive large cell transformation. After 6 years of conventional MF treatment, side effects of therapy and/or concomitant diseases prevented the previously applied treatment modalities. The CD30-directed antibody-cytotoxic drug conjugate (brentuximab vedotin) was introduced and followed by quick and excellent therapeutic response.

PRMT1 expression in renal cell tumors- application in differential diagnosis and prognostic relevance.

BACKGROUND: Protein arginine methyltransferase-1 (PRMT1) is associated with the progression of various tumor types and the process of epithelial to mesenchymal transition (EMT). However, the expression of PRMT1 in renal cell tumors (RCT) is unknown. METHODS: We evaluated PRMT1 immunohistochemical (IHC) expression on tissue microarray (TMA) of 208 specimens of RCT, including clear cell renal cell carcinomas (ccRCC), papillary RCC type I and II (pRCC I and II), chromophobe RCC (chRCC), renal oncocytomas (RO), collecting duct carcinomas - Bellini (CDC) and multilocular cystic renal cell neoplasms of low malignant potential (MLCRN-LMP). Moreover, a subset of ccRCC, pRCC, chRCC, RO were also studied using conventional sections. PRMT1 expression in tumor tissue was compared to the IHC expression of EMT-related transcription factors (ZEB1, RUNX1, and TWIST1) and cell surface markers (ß-catenin, N- and E-cadherin). Additionally, qRT-PCR expression of PRMT1 in ccRCC, pRCC, and chRCC was evaluated and the results were compared to the mRNA PRMT1 transcript profiling data in The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) cohort. RESULTS: PRMT1 immunoreactivity was observed in the majority of ccRCC, RO, all MLCRN-LMP, but in a minority of chRCC (p = 0.044), and it was associated with low grade and low stage ccRCC (p = 0.014; p = 0.044, respectively). ZEB1 immunoreactivity was noted in all RO, in minority of chRCC and neither of MLCRN-LMP (p < 0.001). The majority of PRMT1-negative ccRCC was negative to ZEB1 and showed cytoplasmic expression of TWIST1 (p = 0.028; p < 0.001, respectively). PRMT1 positive ccRCC mostly expressed RUNX1 (p = 0.019). PRMT1 and ZEB1 expression were associated with better cancer-specific survival in patients with ccRCC (p = 0.029; p = 0.009, respectively). In multivariate analysis, ZEB1 expression was an independent prognostic factor for cancer-specific survival (hazard ratio [HR], 0.367; p = 0.026). Significant IHC heterogeneity was observed in PRMT1, ZEB1 and TWIST1 expression (p < 0.001). Homogenous loss of PRMT1 was associated with high grade and high stage ccRCC, while the homogenous loss of PRMT1 and ZEB1 was more frequent in patients who died of ccRCC (p = 0.017; p = 0.040; p = 0.044; p = 0.009, respectively). Relative mRNA-PRMT1 expression in both cohorts was down-regulated in tumor tissue compared to non-tumor parenchyma (p = 0.009). Unlike in our samples, mRNA-PRMT1 expression in the TCGA cohort was not correlated to ccRCC tumor stage or grade. PRMT1, ZEB1, and TWIST1 expression were not associated with EMT related aberrant ß-catenin expression, a gain of N-cadherin or loss of E-cadherin expression. Only RUNX1 was associated with a gain of N-cadherin (p = 0.003). CONCLUSIONS: IHC expression of PRMT1 may be characteristic for low grade and low stage ccRCC, while the homogenous loss of PRMT1 may be significant for high grade and high stage ccRCC. Both, PRMT1 and/or ZEB1 expression, could be associated with better survival of the patients with ccRCC.

Expression of Minichromosome Maintenance Proteins in Actinic Keratosis and Squamous Cell Carcinoma.

Minichromosome maintenance (MCM) proteins are a group of proteins involved in DNA replication and cell-cycle regulation. Because they are associated with DNA through G1 into S phase, MCM proteins are potentially specific indicators of cell proliferation that could be valuable markers of dysplasia, and preinvasive and invasive malignant tumors. To analyze MCM protein expression patterns in actinic keratosis (AK), Bowen disease (BD), and cutaneous squamous cell carcinoma (SCC), we performed immunohistochemical staining of MCM2, -5, and -7 on tissue microarray blocks from 91 AK, 50 BD, and 174 SCC samples. The distribution and semiquantitatively assessed number of positive cells were analyzed in relation to the type of the lesion and the SCC prognostic parameters (grade, diameter, and thickness). Basal expression of all 3 proteins was observed more frequently in AK, whereas the distribution in BD was predominantly diffuse (P<0.001). All 3 proteins showed peripheral distribution in most well-differentiated SCC and diffuse distribution in poorly differentiated tumors (P<0.001). Using the 50% cut-off value, there was a statistically significant difference among AK, BD, and SCC (P<0.001). In addition, all MCM proteins showed highly significant differences (P<0.001) between well-differentiated SCC and both moderately and poorly differentiated SCC. The diffuse distribution and 50% cut-off value of positive cells revealed statistically significant associations of all MCM proteins with SCC thicker than 6 mm. Our results suggest a role for MCM proteins in the progression of in situ keratinocytic lesions and their association with high-risk features in SCC.

Targeted molecular profiling reveals genetic heterogeneity of poromas and porocarcinomas.

The genetic landscape of rare benign tumours and their malignant counterparts is still largely unexplored. While recent work showed that mutant HRAS is present in subsets of poromas and porocarcinomas, a more comprehensive genetic view on these rare adnexal neoplasms is lacking. Using high-coverage next generation sequencing, we investigated the mutational profile of 50 cancer-related genes in 12 cases (six poromas and six porocarcinomas). Non-synonymous mutations were found in two-thirds of both poromas and porocarcinomas. Hotspot HRAS mutations were identified in two poromas (p.G13R and p.Q61R) and one porocarcinoma (p.G13C). While in poromas only few cases showed single mutated genes, porocarcinomas showed greater genetic heterogeneity with up to six mutated genes per case. Recurrent TP53 mutations were found in all porocarcinomas that harboured mutated genes. Non-recurrent mutations in porocarcinomas were found in several additional tumour suppressors (RB1, APC, CDKN2A, and PTEN), and genes implicated in PI3K-AKT and MAPK signalling pathways (ABL1, PDGFRA, PIK3CA, HRAS, and RET). UV-associated mutations were found in TP53, APC, CDKN2A, PTEN, and RET. In conclusion, our study confirms and extends the spectrum of genetic lesions in poromas and porocarcinomas. While poromas exhibited only few mutations, which did not involve TP53, the majority of porocarcinomas harboured UV-mediated mutations in TP53 with some of these cases showing considerable genetic heterogeneity that may be clinically exploitable.

Expression of p300 and p300/CBP associated factor (PCAF) in actinic keratosis and squamous cell carcinoma of the skin.

p300 and p300/CBP-associated factor (PCAF) are histone modifiers and transcriptional co-factors involved in a number of cell processes. We investigated their expression patterns in 79 actinic keratoses (AK), 45 cases of Bowen's disease (BD), and 168 invasive squamous cell carcinomas of the skin (SCC). Using tissue microarray and immunohistochemistry, we evaluated p300 and PCAF expression in relation to the type of the lesion and SCC prognostic parameters (grade, diameter, thickness and level of invasion). High nuclear expression of p300 (>60% of positive cells) (p=0.001) and absent cytoplasmic expression (p=0.026) were more frequent in SCC compared to AK and BD, respectively. Cytoplasmic expression of p300 was associated with the SCC invasion of subcutaneous fat and deeper tissues (p=0.049). Diffuse distribution of cells with p300 nuclear expression was more commonly seen in BD and SCC compared to AK (p<0.001), in moderately- and poorly-differentiated SCC compared to well-differentiated SCC (p<0.001), in tumors thicker than 6mm (p<0.001), and in deeply invading tumors (p=0.001). More frequent loss of PCAF nuclear expression was observed in SCC than in AK and BD (p<0.001). Diffuse distribution of cells with PCAF cytoplasmic expression was more common in BD and SCC compared to AK (p<0.001), and in poorly-differentiated SCC compared to well- and moderately-differentiated SCC (p<0.001). Our results suggest that increase in nuclear expression of p300, as well as the presence of cytoplasmic but loss of nuclear expression of PCAF, could play an important role in the development and progression of cutaneous SCC.

Expression of G1/S-cyclins and cyclin-dependent kinase inhibitors in actinic keratosis and squamous cell carcinoma.

BACKGROUND: Actinic keratosis (AK) and Bowen's disease (squamous cell carcinoma in situ, SCCIS) are pre-invasive stages in the development of squamous cell carcinoma (SCC). METHODS: Immunohistochemical study of cyclin D1, cyclin E, p16(INK4a) and p21(Cip1) (/Waf1) in AK (53 cases), SCCIS (16 cases) and SCC (40 cases), in relation to the type of the lesion and SCC prognostic parameters (grade, diameter and thickness). RESULTS: Diffuse cyclin D1 distribution was more frequent in SCCIS and SCC than in AK (p = 0.03) and similar pattern was observed for p16(INK4a) . For cyclin E, central distribution dominated in SCC compared with the AK (p = 0.001) and SCCIS (p = 0.03). p21(Cip1) (/Waf1) displayed suprabasal distribution more frequently in AK than in SCCIS (p = 0.001) and SCC (p = 0.0004). Semiquantitative assessment showed more positive cells in AK (p = 0.04) and SCCIS (p = 0.04) than in SCC for cyclin E. SCC with diameter over 20 mm and those thicker than 6 mm revealed higher labeling index with p16(INK4a) and p21(Cip1) (/Waf1) , respectively. CONCLUSIONS: Our results suggest different alterations for p16(INK4a) and p21(Cip1) (/Waf1) in AK, SCCIS and SCC. Immunostaining distribution showed closer correlation with the type of the lesion, whereas percentage of positive cells displayed better association with the SCC prognostic parameters.

Necrosis and apoptosis in Trichinella spiralis-mediated tumour reduction.

It is known that infection with different pathogens, including helminths, can alter the progression of malignant or other diseases. We studied the effect of chronic Trichinella spiralis infection or muscle larvae excretory-secretory (ES L1) antigens on the malignant tumour growth in the mouse melanoma model system in vivo and in vitro. Our results confirmed that chronic infection with T. spiralis possesses the capacity to slow down the progression of tumour growth, resulting in an impressive reduction in tumour size. We found that the phenomenon could, at least partially, be related to a lower level of tumour necrosis compared to necrosis present in control animals with progressive malignancy course. An increased apoptotic potential among the low percentage of cells within the total tumour cell number in vivo was also observed. ES L1 antigen, as a parasitic product that is released during the chronic phase of infection, reduced the survival and slightly, but significantly increased the apoptosis level of melanoma cells in vitro. Our results imply that powerful Trichinella anti-malignance capacity does not rely only on necrosis and apoptosis but other mechanisms through which infection or parasite products manipulate the tumor establishment and expansion should be considered.