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The oncogene PIM2 is upregulated in several malignancies but has never been investigated in mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). PIM2 is a well-known oncogene and is regulated by cell signaling pathways like the JAK/STAT- and NF-kB-pathway, key regulators in the pathogenesis of CTCL. The aim of this study was to examine the role of PIM2 in MF. PIM2 gene expression was measured in 81 formalin-fixed paraffin-embedded skin biopsies from patients with MF and 46 control biopsies from healthy skin (HS) and benign inflammatory skin disease (BID). Validation of PIM2 protein expression was performed on selected biopsies with immunohistochemical staining. We found a significant difference in gene expression levels between both early stage MF and HS (p < 0.0001), and BID (p < 0.0001). In addition, the PIM2 gene expression was higher in advanced-stage MF compared to early stage disease (p = 0.0001). No significant difference in gene expression levels was found between patients with and without disease progression. In conclusion, we found PIM2 expression is significantly increased in MF compared to controls, and in advanced-stage MF compared to early stage MF. These findings could potentially have diagnostic value in discriminating early stage MF from BID., (© 2024 Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

A frequently used treatment strategy in locally advanced rectal cancer (RC) is neoadjuvant therapy followed by surgery. Patients treated with neoadjuvant therapy achieve varying pathological response, and currently, predicting the degree of response is challenging. This study examined the association between digitally assessed histopathological features in the diagnostic biopsies and pathological response to neoadjuvant therapy, aiming to find potential predictive biomarkers. 50 patients with RC treated with neoadjuvant chemotherapy and/or radiotherapy followed by surgery were included. Deep learning-based digital algorithms were used to assess the epithelium tumor area percentage (ETP) based on H&E-stained slides, and to quantify the density of CD3 + and CD8 + lymphocytes, as well as the CD8 + /CD3 + lymphocyte percentage, based on immunohistochemically stained slides, from the diagnostic tumor biopsies. Pathological response was assessed according to the Mandard method. A good pathological response was defined as tumor regression grade (TRG) 1-2, and a complete pathological response was defined as Mandard TRG 1. Associations between the ETP and lymphocyte densities in the diagnostic biopsies and the pathological response were examined. The density of CD8 + lymphocytes, and the CD8 + /CD3 + lymphocyte percentage, were associated with both good and complete response to neoadjuvant therapy, while the density of CD3 + lymphocytes was associated with complete response. The ETP did not correlate with response to neoadjuvant therapy. It is well-known that infiltration of lymphocytes in colorectal cancer is a prognostic biomarker. However, assessment of CD8 + and CD3 + lymphocytes in the diagnostic tumor biopsies of patients with RC may also be useful in predicting response to neoadjuvant therapy., Competing Interests: Declaration of competing interest HH and BR are employed at Visiopharm, Hørsholm, A/S. The other authors report no conflict of interest related to the work described., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Local excision of early colon cancers could be an option in selected patients with high risk of complications and no sign of lymph node metastasis (LNM). The primary aim was to assess feasibility in high-risk patients with early colon cancer treated with Combined Endoscopic and Laparoscopic Surgery (CELS)., Methods: A non-randomized prospective feasibility study including 25 patients with Performance Status score ≥ 1 and/or American Society of Anesthesiologists score ≥ 3, and clinical Union of International Cancer Control stage-1 colon cancer suitable for CELS resection. The primary outcome was failure of CELS resection, defined as either: Incomplete resection (R1/R2), local recurrence within 3 months, complication related to CELS within 30 days (Clavien-Dindo grade ≥ 3), death within 30 days or death within 90 days due to complications to surgery., Results: Fifteen patients with clinical T1 (cT1) and ten with clinical T2 (cT2) colon cancer and without suspicion of metastases were included. Failure occurred in two patients due to incomplete resections. Histopathological examination classified seven patients as having pT1, nine as pT2, six as pT3 adenocarcinomas, and three as non-invasive tumors. In three patients, the surgical strategy was changed intraoperatively to conventional colectomy due to tumor location or size. Median length of stay was 1 day. Seven patients had completion colectomy performed due to histological high-risk factors. None had LNM., Conclusions: In selected patients, CELS resection was feasible, and could spare some patients large bowel resection., (© 2023. The Author(s).)

Research Question: The human leukocyte antigen (HLA) class Ib molecules HLA-F and HLA-G are implicated in pregnancy success, but how do HLA-G and HLA-F genetic polymorphisms impact recurrent implantation failure (RIF)?, Design: Prospective cohort study at a fertility clinic including a cohort of 84 women experiencing RIF and 35 IVF controls to assess the influence of HLA-G haplotypes and diplotypes and HLA-F single nucleotide polymorphisms (SNP) on RIF., Results: Over-representation trends for HLA-F SNP genotypes rs1362126, rs2523405 and rs2523393, previously linked with a short time-to-pregnancy, were detected in female control groups compared with RIF patients with no identified pathology linked to infertility. The HLA-G promoter haplotype PROMO-G010101b/c linked with the HLA-G 3'-untranslated region (3'UTR) haplotype UTR-4, which previously has been associated with positive IVF outcome and pregnancy success, was less frequent in the RIF group. For RIF patients carrying the UTR-4 haplotype, the odds ratio (OR) was 0.27 (95% CI 0.12-0.66; P = 0.0044, P c  = 0.026). The HLA-G PROMO-G010104-UTR-3 haplotype was associated with an increased risk of RIF. For RIF patients carrying the UTR-3 haplotype, the OR was 5.86 (95% CI 1.52-26.23; P = 0.0115, P c  = 0.069)., Conclusions: These results show that specific HLA-G haplotypes based on the promoter region and the 3'UTR are either associated with an increased risk of reduced fertility, including the manifestation of RIF, and lower chance of achieving pregnancy, or with a reduced risk of experiencing RIF., (Copyright © 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Aims: Digital image analysis (DIA) is used increasingly as an assisting tool to evaluate biomarkers, including human epidermal growth factor receptor 2 (HER2) in invasive breast cancer (BC). DIA can assist pathologists in HER2 evaluation by presenting quantitative information about the HER2 staining in APP assisted reading (AR). Concurrently, the HER2-low category (HER2-1+/2+ without HER2 gene amplification) has gained prominence due to newly developed antibody-drug conjugates. However, major inter- and intraobserver variability have been observed for the entity. The present quality assurance study investigated the concordance between DIA and AR in clinical use, especially concerning the HER2-low category., Methods and Results: HER2 immunohistochemistry (IHC) in 761 tumours from 727 patients was evaluated in tissue microarray (TMA) cores by DIA (Visiopharm HER2-CONNECT) and AR. Overall concordance between HER2-scores were 73% (n = 552, weighted-κ: 0.66), and 88% (n = 669, weighted-κ: 0.70), when combining HER2-0/1+. A total of 205 scores were discordant by one category, while four were discordant by two categories. A heterogeneous HER2 pattern was relatively common in the discordant cases and a pitfall in the categorisation of HER2-low BC. AR more commonly reassigned a lower HER2 score (from HER2-1+ to HER2-0) within the HER2-low subgroup (n = 624) compared with DIA., Conclusion: DIA and AR display moderate agreement with heterogeneous and aberrant staining, representing a source of discordance and a pitfall in the evaluation of HER2., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: Superficial spreading melanomas (SSMs) are the most common type of melanoma and cause the majority of skin cancer deaths. More than 50% of cases harbor a mutation in the BRAF gene that activates the mitogen-activated protein kinase (MAPK) cancer signaling pathway. BRAFV600E is the most common BRAF mutation, and it represents an important biomarker that guides treatment selection. However, the relationship between the BRAFV600E gene expression and intratumoral protein distribution, on one side, and clinicopathological factors and patient outcomes, on the other, is not fully described. Additionally, whether MAPK cancer signaling activation in melanoma is due to increased biochemical activity of BRAFV600E, increased mRNA levels, or both requires further investigation. Here, we addressed these questions by examining expression patterns of BRAFV600E in primary treatment-naive melanomas and correlating them to clinicopathological factors and patient outcomes., Methods: In 166 SSM cases, we performed immunohistochemical staining to investigate the protein expression of BRAFV600E, and we measured BRAF mRNA levels using NanoString nCounter system., Results: Ninety-seven (49%) melanomas stained positive for BRAFV600E, with nearly 100% intratumoral homogeneity observed. Positive BRAFV600E expression was significantly associated with nonrecurrent disease and was found to be an independent predictor of better prognosis in univariate and multivariable analyses. Furthermore, presence of tumor-infiltrating lymphocytes, sentinel lymph node biopsy negativity, and low Breslow thickness were all independent predictors of better prognosis. We observed no difference in the BRAF mRNA levels in BRAFV600E-negative and BRAFV600E-positive melanomas, respectively. Validation in a larger publicly available cohort confirmed that there is only a weak correlation (Spearman 0.4) between BRAFV600E mRNA and protein levels and no differences in mRNA between BRAFV600E mutated and non-mutated patients., Conclusion: Our findings indicated that BRAFV600E is homogeneously present throughout the whole tumor and is associated with nonrecurrent disease and better survival in primary melanoma. We also showed that BRAFV600E mutation does not result in higher transcriptional levels, suggesting that activation of the MAPK signaling pathway in BRAFV600E mutated patients can be attributed to the increased biochemical activity caused by the mutation., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Our understanding of the regulatory processes of reepithelialization during wound healing is incomplete. In an attempt to map the genes involved in epidermal regeneration and differentiation, we measured gene expression in formalin-fixed, paraffin-embedded standardized epidermal wounds induced by the suction-blister technique with associated nonwounded skin using NanoString technology. The transcripts of 139 selected genes involved in clotting, immune response to tissue injury, signaling pathways, cell adhesion and proliferation, extracellular matrix remodeling, zinc transport and keratinocyte differentiation were evaluated. We identified 22 upregulated differentially expressed genes (DEGs) in descending order of fold change ( MMP1 , MMP3 , IL6 , CXCL8 , SERPINE1 , IL1B , PTGS2 , HBEGF , CXCL5 , CXCL2 , TIMP1 , CYR61 , CXCL1 , MMP12 , MMP9 , HGF , CTGF , ITGB3 , MT2A , FGF7 , COL4A1 and PLAUR ). The expression of the most upregulated gene, MMP1 , correlated strongly with MMP3 followed by IL6 and IL1B . rhIL-1β, but not rhIL-6, exposure of cultured normal human epidermal keratinocytes and normal human dermal fibroblasts increased both MMP1 mRNA and MMP-1 protein levels, as well as TIMP1 mRNA levels. The increased TIMP1 in wounds was validated by immunohistochemistry. The six downregulated DEGs ( COL7A1 , MMP28 , SLC39A2 , FLG1 , KRT10 and FLG2 ) were associated with epidermal maturation. KLK8 showed the strongest correlation with MKI67 mRNA levels and is a potential biomarker for keratinocyte proliferation. The observed gene expression changes correlate well with the current knowledge of physiological reepithelialization. Thus, the gene expression panel described in this paper could be used in patients with impaired healing to identify possible therapeutic targets.

Study Question: Is human leukocyte antigen (HLA)-F protein expressed in mid-secretory endometrium, and are its expression levels influenced by HLA-F gene polymorphisms and correlated with the abundance of uterine natural killer (uNK) cells and anti-inflammatory M2 macrophages?, Summary Answer: HLA-F protein is expressed in mid-secretory endometrium, and levels are correlated with immune cell infiltration, plasma progesterone concentrations and HLA-F single-nucleotide polymorphisms (SNPs), however, women experiencing recurrent implantation failure (RIF) show differences when compared to women attending their first IVF treatment., What Is Known Already: The immunomodulatory HLA class Ib molecules HLA-G and HLA-F are expressed on the extravillous trophoblast cells and interact with receptors on maternal immune cells. Little is known regarding HLA-F expression in endometrial stroma and HLA-F function; furthermore, HLA-F and HLA-G SNP genotypes and haplotypes have been correlated with differences in time-to-pregnancy., Study Design, Size, Duration: Primary endometrial stromal cell (ESC) cultures (n = 5) were established from endometrial biopsies from women attending IVF treatment at a fertility clinic. Basic HLA-F and HLA-G protein expression by the ESCs were investigated. A prospective controlled cohort study was performed including 85 women with a history of RIF and 36 control women beginning their first fertility treatment and with no history of RIF. In some analyses, the RIF group was divided into unknown cause, male infertility, female infertility, and both female and male infertility. Endometrial biopsies and blood samples were obtained the day equivalent to embryo transfer in a hormone-substituted cycle., Participants/materials, Setting, Methods: HLA protein expression by ESCs was characterized using flow cytometry and western blot. In the cohort study, the specific immune markers HLA-F and HLA-G, CD56 and CD16 (NK cells), CD163 (M2 macrophages), FOXP3 (regulatory T cells) and CD138 (plasma cells) were analysed by immunohistochemistry and a digital image analysis system in endometrial biopsies. Endometrial receptivity was assessed by an endometrial receptivity array test (the ERA® test). Endometrial biopsies were examined according to modified Noyes' criteria. SNPs at the HLA-F gene and HLA-G haplotypes were determined., Main Results and the Role of Chance: HLA-F protein is expressed in the endometrium at the time of implantation. Furthermore, the HLA-F protein levels were different according to the womeńs HLA-F SNP genotypes and diplotypes, which have previously been correlated with differences in time-to-pregnancy. Endometrial HLA-F was positively correlated with anti-inflammatory CD163+ M2 macrophage infiltration and CD56+ uNK cell abundance for the entire cohort. However, this was not the case for CD56+ in the female infertility RIF subgroup. HLA-F levels in the endometrial stroma were negatively correlated with plasma progesterone concentrations in the RIF subgroup with known female infertility. Conversely, HLA-F and progesterone were positively correlated in the RIF subgroup with infertility of the male partner and no infertility diagnosis of the woman indicating interconnections between progesterone, HLA-F and immune cell infiltration. Glandular sHLA-G expression was also positively correlated with uNK cell abundance in the RIF subgroup with no female infertility but negatively correlated in the RIF subgroup with a female infertility diagnosis., Large Scale Data: Immunohistochemistry analyses of endometrial biopsies and DNA sequencing of HLA genes. Data will be shared upon reasonable request to the corresponding author., Limitations, Reasons for Caution: The control group of women attending their first IVF treatment had an anticipated good prognosis but was not proven fertile. A significant age difference between the RIF group and the IVF group reflects the longer treatment period for women with a history of RIF. The standardization of hormonal endometrial preparation, which allowed consistent timing of endometrial and blood sampling, might be a strength because a more uniform hormonal background may more clearly show an influence on the immune marker profile and HLA class Ib levels in the endometrium by other factors, for example genetic polymorphisms. However, the immune marker profile might be different during a normal cycle., Wider Implications of the Findings: The findings further highlight the importance of HLA-F and HLA-G at the implantation site and in early pregnancy for pregnancy success. Diagnostic measures and modulation of the complex interactions between HLA class Ib molecules, maternal immune cells and hormonal factors may have potential to improve fertility treatment., Study Funding/competing Interest(s): This work was supported by the Region Zealand Health Sciences Research Foundation and the Zealand University Hospital through the ReproHealth Research Consortium ZUH. The authors declared there are no conflicts of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The immune system recognizes and destroys cancer cells. However, cancer cells develop mechanisms to avoid detection by expressing cell surface proteins. Specific tumour cell surface proteins (e.g. HLA-G, PD-L1, CDX2) either alone or in combination with the relative presence of immune cells (CD3 and CD8 positive T-cells) in the tumour tissue may describe the cancer cells' ability to escape eradication by the immune system. The aim was to investigate the prognostic value of immunohistochemical markers in patients with colon cancer., Methods: We conducted a retrospective study including patients diagnosed with pT3 and pT4 colon cancers. Immunohistochemical staining with HLA-G, PD-L1, CDX2, CD3, and CD8 was performed on tissue samples with representation of the invasive margin. PD-L1 expression in tumour cells and immune cells was reported conjointly. The expression of CD3 and CD8 was reported as a merged score based on the expression of both markers in the invasive margin and the tumour centre. Subsequently, a combined marker score was established based on all of the markers. Each marker added one point to the score when unfavourable immunohistochemical features was present, and the score was categorized as low, intermediate or high depending on the number of unfavourable stains. Hazard ratios for recurrence, disease-free survival and mortality were calculated., Results: We included 188 patients undergoing colon cancer resections in 2011-2012. The median follow-up was 41.7 months, during which 41 (21.8%) patients had recurrence and 74 (39.4%) died. In multivariable regression analysis positive HLA-G expression (HR = 3.37, 95%CI [1.64-6.93]) was associated with higher recurrence rates, while a preserved CDX2 expression (HR = 0.23, 95%CI [0.06-0.85]) was associated with a lower risk of recurrence. An intermediate or high combined marker score was associated with increased recurrence rates (HR = 20.53, 95%CI [2.68-157.32] and HR = 7.56, 95%CI [1.06-54.16], respectively). Neither high expression of PD-L1 nor high CD3-CD8 score was significantly associated with recurrence rates. Patients with a high CD3-CD8 score had a significantly longer DFS and OS., Conclusions: In tumour cells, expression of HLA-G and loss of CDX2 expression were associated with cancer recurrence. In addition, a combination of certain tumour tissue biomarkers was associated with colorectal cancer recurrence., (© 2022. The Author(s).)

Background: Colonic stent is recommended as a bridge to elective surgery for malignant obstruction to improve short-term clinical outcomes for patients with colorectal cancer. However, since the oncological outcomes remain controversial, this study aimed to investigate the impact of self-expandable metallic stent (SEMS) on the tumor microenvironment., Methods: Patients treated with colonic stent as a bridge to surgery from 2010 to 2015 were identified from hospital records. Tumor biopsies and resected tumor samples of the eligible patients were retrieved retrospectively. Gene expression analysis was performed using the NanoString nCounter PanCancer IO 360 gene expression panel., Results: Of the 164 patients identified, this study included 21 who underwent colonic stent placement as a bridge to elective surgery. Gene expression analysis revealed 82 differentially expressed genes between pre- and post-intervention specimens, of which 72 were upregulated and 10 downregulated. Among the significantly upregulated genes, 46 are known to have protumor functions, of which 26 are specifically known to induce tumorigenic mechanisms such as proliferation, migration, invasion, angiogenesis, and inflammation. In addition, ten differentially expressed genes were identified that are known to promote antitumor functions., Conclusion: SEMS induces gene expressional changes in the tumor microenvironment that are associated with tumor progression in colorectal cancer and may potentiate a more aggressive phenotype. Future studies are warranted to establish optimal timing of surgery after SEMS insertion in patients with obstructive colorectal cancer., (© 2021. Society of Surgical Oncology.)

Background: The prognostic value of tumour-infiltrating lymphocytes (TILs) in breast cancer is well-established. However, the investigation of specific T-cell subsets exclusively in BRCA-associated breast cancer is sparse., Methods: Tumour tissues from 414 BRCA-mutated breast cancer patients were analysed by immunohistochemistry and digital image analysis for expression of CD4, CD8 and FOXP3 immune markers. Distribution of CD4-, CD8- and FOXP3-positive cells and clinicopathological characteristics were assessed according to groups of low or high expression. The prognostic value was evaluated as continuous variables in univariate and multivariate analyses of overall survival and disease-free survival., Results: Both CD4 and CD8 expression are associated with histological diagnosis, tumour grade and oestrogen and progesterone receptor expression status. CD4 expression is associated with BRCA gene status. A high percentage of tumour-infiltrating CD4-, CD8- or FOXP3-positive cells is significantly associated with lower mortality in BRCA1- and BRCA2-associated breast cancer and CD8-positive cells are associated with disease-free survival. No heterogeneity according to BRCA gene status was found for the prognostic value of the immune markers., Conclusions: The results support a prognostic role of specific T-cell subsets in BRCA-associated breast cancer and the promising potential of targeting the immune system in the treatment of these patients., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

In the search for a reliable biomarker able to diagnose immunological causes of infertility, uterine immune cells have been widely investigated. As a result, heterogeneous methods and cutoff values of what constitutes an aberrant number of immune cells have been reported, and a standardized method for quantification is needed. The objective of this study was to compare methods for quantification of immune cells visualized with immunohistochemistry in the endometrium of women in fertility treatment. Evaluation of the density of CD56 + , CD16 + and CD163 + cells by conventional microscopy on a semiquantitative scale (low, medium and high) was compared to a continuous count using digital image analysis (DIA) reported as percentage positive cells out of the total number of stromal cells and number of positive cells per mm 2 , respectively. We previously reported the CD56/CD16 ratio as a possible prognostic marker, and therefore the ratios of CD56/CD16 were compared using two different methods for selecting fields for counting with DIA: one method using principles of systematic random sampling, where glands were excluded, and one method analyzing large parts of the tissue including glands. A significant association between conventional microscopy and DIA was found when the semiquantitative scale was compared to medians of positive cells in CD56, CD16 and CD163, respectively, p < 0.001. A systematic significant difference in the ratios of CD56/CD16 was found when comparing the two methods for field selection, p < 0.001. To determine the possible use of these methods, more knowledge of the correlation to clinical outcome is warranted., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Reepithelialisation is fundamental to wound healing, but our current understanding largely relies on cellular and animal studies. The aim of the present randomised double-blind three-arm controlled trial was to correlate genuine epidermal wound healing with key proteins and topical zinc treatment in humans. Sixty wounds were produced using deroofed suction blisters in 30 healthy volunteers and randomised to topical zinc sulphate (n = 20), placebo (n = 20), or control (n = 20) treatment for 4 days. All wounds with perilesional skin were processed for automatic immunostaining of paraffin tissue sections with monoclonal antibodies against Ki-67, metallothionein (MT) and matrix metalloproteinase (MMP)-1. Protein expression was quantified by automated digital image analysis. Epidermal Ki-67 and MT labelling indices were increased in keratinocytes in the neoepidermis (∼1.1 mm) and at the wound edge (0.5 mm) compared to normal skin. Increased MMP-1 immunostaining was restricted to the neoepidermis. MT was robustly upregulated in the upper dermis of the wounds. Zinc treatment enhanced MMP-1 expression beneath the neoepidermis via paracrine mechanisms and MT under the neoepidermis and in the nonepithelialised wound bed via direct actions of zinc as indicated by the induction of MT2A mRNA but not MMP-1 mRNA in cultured normal human dermal fibroblasts by zinc sulphate. The present human study demonstrates that quantitative immunohistochemistry can identify proteins involved in reepithelialisation and actions of external compounds. Increased dermal MT expression may contribute to the anti-inflammatory activities of zinc and increased MMP-1 levels to promote keratinocyte migration., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Mycosis fungoides is the most common type of cutaneous T-cell lymphoma. The inflammatory micro-environment in mycosis fungoides is complex. There is accumulating evidence that the neoplastic T-cells take control of the microenvironment and thereby promote their own expansion by suppressing cellular immunity. B-cells have proved to be upregulated in large-cell transformed mycosis fungoides, and could potentially play a role in disease progression. To investigate the presence of B-cells in mycosis fungoides compared with controls, this study analysed 85 formalin-fixed and paraffin-embedded mycosis fungoides biopsies. MS4A1 gene expression was significantly upregulated in mycosis fungoides compared with controls (p < 0.0001) and further upregulated in disease progression, (p = 0.001). Digital quantification of PAX5+/CD20+ cells confirmed the increased presence of B-cells in mycosis fungoides compared with controls. No co-labelling of CD3/CD20 was observed in the neoplastic T-cells. This study found a significantly increased presence of B-cells in the tumour-associated microenvironment in mycosis fungoides. These findings could potentially lead to new treatment strategies for mycosis fungoides.

Neuroinflammation is an essential part of neurodegeneration. Yet, the current understanding of neuroinflammation-associated molecular events in distinct brain regions of prion disease patients is insufficient to lay the ground for effective treatment strategies targeting this complex neuropathological process. To address this problem, we analyzed the expression of 800 neuroinflammation-associated genes to create a profile of biological processes taking place in the frontal cortex and cerebellum of patients who suffered from sporadic Creutzfeldt-Jakob disease. The analysis was performed using NanoString nCounter technology with human neuroinflammation panel+. The observed gene expression patterns were regionally and sub-regionally distinct, suggesting a variable neuroinflammatory response. Interestingly, the observed differences could not be explained by the molecular subtypes of sporadic Creutzfeldt-Jakob disease. Furthermore, analyses of canonical pathways and upstream regulators based on differentially expressed genes indicated an overlap between biological processes taking place in different brain regions. This suggests that even smaller-scale spatial data reflecting subtle changes in brain cells' functional heterogeneity and their immediate pathologic microenvironments are needed to explain the observed differential gene expression in a greater detail.

Problem: To what extent do endocrine, immunological, gene expression and histological markers of endometrial receptivity correlate?, Method of Study: Between November 2017 and September 2019, 121 women referred to a University Hospitals Fertility Clinic consented to inclusion in this cohort study. The women underwent timed endometrial biopsy followed by blood samples in a hormone-substituted cycle. Of these, 37 women had just started IVF treatment, and the remaining 84 had experienced recurrent implantation failure following IVF/ICSI. The hormone-substituted cycle consisted of initiation with oral oestradiol followed by addition of vaginal progesterone treatment for five full days. Endometrial biopsies were subject to histological examination, immune cell markers by immunohistochemistry (CD56 + , CD16 + , CD163 + , FoxP3) and gene expression microarray analyses with the endometrial receptivity array (ERA ® ) test (Igenomix). Plasma progesterone and oestradiol were measured on the day of biopsy., Results: CD56 + uterine natural killer (uNK) cell counts correlate with transcriptional markers of endometrial receptivity assessed by the ERA test. Endometrial maturation, receptivity and immunological markers were not correlated with mid-luteal blood plasma progesterone level. Mid-luteal serum oestradiol level correlated with markers of endometrial maturation and receptivity. The tests were carried out during a standard hormone substitution cycle, and the findings may not apply in the natural cycle., Conclusion: CD56 + uNK cell counts and endometrial receptivity assessed by the ERA test appear to be linked. Mid-luteal progesterone levels were not correlated to the tested markers of endometrial receptivity. In contrast, mid-luteal oestradiol level was inversely related to markers of endometrial receptivity and maturation., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Research Question: What is the prevalence of disrupted markers of endometrial function among women experiencing recurrent implantation failure (RIF), and does the prevalence differ from a control cohort?, Design: Prospective controlled cohort study. In total, 86 women with a history of RIF and 37 women starting their first fertility treatment were recruited for this study. Endometrial and blood profiling were carried out in a hormone-substituted cycle using oestradiol and progesterone. Endometrial biopsies were analysed by histology, immune cell profiling, and the endometrial receptivity array (ERA®) test (Igenomix, Valencia, Spain). The vaginal microbiome was analysed using a NGS-based technology (ArtPRED, Amsterdam, the Netherlands). Blood tests included oestradiol, progesterone, prolactin, thyroid-stimulating hormone, vitamin D and anti-phospholipid antibody levels., Results: Patients who had experienced RIF produced a range of test abnormalities. Compared with controls, women with RIF had a higher prevalence of chronic endometritis (24% versus 6%), a lower vitamin D level and a borderline lower progesterone level. Women who had experienced RIF had a more favourable vaginal microbiome compared with controls. Although the RIF cohort was older than the controls (mean age 33.8 years versus 30.2 years), no differences between the groups were observed in immune cell profiling and the ERA test., Conclusion: These data demonstrate that a single test or treatment for the endometrial factor in RIF is unlikely to be clinically effective. Diagnosing the endometrium in women with RIF permits targeted rather than blind interventions. Relative vitamin D deficiency, lower mid-luteal progesterone and chronic endometritis are ready targets for treatment. Understanding the role and treatment of an unfavourable vaginal microbiome in RIF needs further investigation., (Copyright © 2020 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: Estrogen receptor positive (ER+) breast cancer constitutes almost 85% of all breast cancer patients and are a genetically highly heterogenic group. Data on the association of somatic alterations to outcome and prognosis are however sparse. In this neoadjuvant endocrine phase II trial including postmenopausal breast cancer patients with ER+, HER2 normal breast cancer, we investigated the rate of pathogenic mutations before and after treatment as well as the association with treatment response and survival., Methods: Pretreatment and posttreatment tumour samples from 109 patients treated with neoadjuvant letrozole were collected and analysed with Next Generation Sequencing utilizing a panel of 12 genes (ALK, BRAF, EGFR, ERBB2, ERBB3, ESR1, KIT, KRAS, NRAS, PDGFRA, PIK3CA, and RAF1). Residual disease was assessed by a modified Miller Payne scale and the Residual Cancer Burden index. Survival data were collected prospectively., Results: Among the 109 patients, 52 had at least one pathogenic mutation in the pretreatment sample and 60 in the posttreatment sample. The most frequently mutated gene was PIK3CA, followed by EGFR and KRAS. Twelve different pathogenic PIK3CA mutations were identified, primarily in exon 20 and exon 9. An altered PIK3CA mutation profile from the pre- to the posttreatment specimen was significantly associated to improved pathological outcome. Overall and Disease-Free Survival benefits in PIK3CA mutated patients was observed., Conclusion: Considerable heterogeneity was identified both among patients and between pre- and posttreatment samples. PIK3CA has the potential to be a predictive biomarker. To further assess the implications of a treatment related altered PIK3CA mutation profile, more data are needed.

Purpose: The Prosigna-PAM50 risk of recurrence (ROR) score has documented clinical utility for the prediction of 10-year distant recurrence (DR). The present study investigated the value of Prosigna-PAM50 for predicting 10-year DR and overall survival after 5 years of endocrine treatment for postmenopausal patients with invasive lobular carcinoma., Patients and Methods: Using the Danish Breast Cancer Group database, we identified patients with a diagnosis from 2000 to 2003 of estrogen receptor-positive, human epidermal growth factor receptor 2-negative invasive ductal (n = 1570) or lobular (n = 341) cancer > 20 mm or 1 to 3 positive lymph nodes and applied multivariate Cox models., Results: The median follow-up for DR was 9.3 years and for overall survival 15.2 years. Of the 341 lobular and 1570 ductal cases, 140 (41%) and 349 (22%) were classified as low ROR, with a 10-year DR rate of 7.7% (95% confidence interval [CI], 3.7%-13.6%) and 3.5% (95% CI, 1.8%-6.2%), respectively. The 10-year DR rate for the intermediate ROR group for those with lobular cancer was 18% (95% CI, 10.1%-27.9%) compared with 9.7% (95% CI, 6.7%-13.4%) for those with ductal cancer. Luminal B tumors had a significantly worse outcome than luminal A tumors in both lobular (hazard ratio, 1.89; 95% CI, 1.03%-3.45%; P = .04) and ductal (hazard ratio, 3.18; 95% CI, 2.29%-4.43%; P < .0001) cancer., Conclusion: Prosigna PAM-50 provides significant prognostic information beyond the clinicopathologic factors in patients with invasive lobular breast cancer. Those with lobular cancer had worse 10-year DR rates compared with those with ductal cancer in the same ROR category. Our results could have an effect on the treatment decisions regarding the addition of chemotherapy for those in the intermediate ROR group., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by effector memory T cells (TEM) and plays an important role in their activation and proliferation. Mycosis fungoides (MF), the most common subtype of cutaneous T-cell lymphoma (CTCL), was recently proposed to be a malignancy of skin-resident TEM. However, the expression of Kv1.3 in CTCL has not been investigated., Objectives: This study aims to examine the expression of Kv1.3 in situ and in vitro in CTCL., Methods: The expression of Kv1.3 was examined by immunohistochemistry in skin lesions from 38 patients with MF, 4 patients with Sézary syndrome (SS), and 27 patients with benign dermatosis. In 4 malignant T-cell lines of CTCL (Myla2059, PB2B, SeAx, and Mac2a) and a non-malignant T-cell line (MyLa1850), the expression of Kv1.3 was determined by flow cytometry. The proliferation of those cell lines treated with various concentrations of Kv1.3 inhibitor ShK was measured by 3H-thymdine incorporation., Results: Half of the MF patients (19/38) displayed partial Kv1.3 expression including 1 patient with moderate Kv1.3 positivity, while the other half (19/38) exhibited Kv1.3 negativity. An almost identical distribution was observed in patients with benign conditions, that is, 44.4% (12/27) were partially positive for Kv1.3 including 1 patient with moderate Kv1.3 positivity, while 55.6% (15/27) were Kv1.3 negative. In contrast, 3 in 4 SS patients displayed partial Kv1.3 positivity including 2 patients with weak staining and 1 with moderate staining, while 1 in 4 SS patients was Kv1.3 negative. In addition, all malignant T-cell lines, and a non-malignant T-cell line, displayed low Kv1.3 surface expression with a similar pattern. Whereas 2 cell lines (PB2B and Mac2a) were sensitive to Kv1.3 blockade, the other 2 (Myla2059 and SeAx) were completely resistant., Conclusions: We provide the first evidence of a heterogeneous Kv1.3 expression in situ in CTCL lesions., (© 2019 S. Karger AG, Basel.)

Diagnosis of mycosis fungoides and Sézary syndrome can be very challenging. Clinical and histopathological data for patients with mycosis fungoides and Sézary syndrome in Denmark are limited. A retrospective study was performed in Region Zealand, Denmark from 1990 to 2016. A total of 43 patients with mycosis fungoides or Sézary syndrome were identified during the period. At the time of diagnosis the patients' mean age was 64.3 years and 74.5% had early-stage (≤IIA) disease. The mean time from onset of skin disease to diagnosis was 4.4 years. Surprisingly, 43% progressed to a higher disease stage, and risk of disease progression was higher for stage IB than IA (p = 0.01). All cases displayed some degree of epidermotropism and the infiltrates consisted of pleomorphic lymphocytes with a T-helper (CD4+/CD8-) phenotype. This study describes, for the first time, all aspects of clinical and histopathological findings in patients with mycosis fungoides and Sézary syndrome in a well-characterized Danish cohort.

It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.

Background: The role of bacterial biofilm in hidradenitis suppurativa (HS) is highly debated. Less biofilm is found in clinically unaffected axillary perilesional skin of HS patients compared with healthy controls., Objective: To study the correlation between biofilm and the phenotypical characterization of the preclinical inflammatory infiltrate., Materials and Methods: An exploratory comparative study of punch biopsies from unaffected axillary HS skin compared to similarly biopsies from healthy controls underwent standard staining procedures for CD4, CD8, CD25, FoxP3 and IL17. Standard-sized inflammatory histological hotspots were identified manually. Slides were scanned into Leica Biosystems' Digital Image Hub. Number of stained cells per slide and hotspot was found using an algorithm., Results: 12.5% of HS had biofilm compared to 85% of controls. For full slides, HS patients had more CD4+ cells than controls; HS patients with biofilm had higher CD4+ cell number than controls with or without biofilm and HS patients without biofilm. For hotspots, HS patients with biofilm had higher number of CD4+FoxP3+ cells than HS patients without biofilm and controls with biofilm., Conclusion: The association between biofilm and the number of regulatory T cells in HS patients supports the concept of dysbiosis as a factor in the preclinical HS lesions., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown., Material and Methods: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (<10%), intermediate (10-59%) and high (≥60%). Prognostic associations of sTils were evaluated as a continuous variable in univariate and multivariate models. Only follow-up time beyond date of BRCA mutation test was included., Results: A large proportion had high sTils (27% in the full cohort, 36% in BRCA1-mutated, and 44% in ER negative breast cancers). Higher sTils were associated with BRCA1, ER negative breast cancer, high histological grade and medullary histology. In combined analysis for BRCA1 and BRCA2-mutated breast cancers, increasing sTils in 10% intervals were significantly associated with OS (HR 0.92, 95% CI 0.84-1.00, p = .05). For each 10% increment of sTils in BRCA1 breast cancers, a 10% reduction of mortality (adjusted HR 0.90 95% CI 0.81-0.99, p = .03) and a 13% reduction in risk of DFS-event (HR 0.87 95% CI 0.76-1.00, p = .05) was observed even after adjustment for ER status. No significant association with survival was of observed in the BRCA2 subgroup. Test for interaction of sTils and BRCA status was not statistically significant (p = .3)., Conclusions: Breast cancer patients with a germline BRCA mutation had higher sTils than previously reported in sporadic breast cancers, and sTils were associated with favorable survival among BRCA carriers.

Background: The PAM50-based (Prosigna) risk of recurrence (ROR) score and intrinsic subtypes are prognostic for women with high-risk breast cancer. We investigate the predictive ability of Prosigna regarding the effectiveness of cyclophosphamide-based adjuvant chemotherapy in premenopausal patients with high-risk breast cancer., Methods: Prosigna assays were performed on the NanoString platform in tumors from participants in Danish Breast Cancer Group (DBCG) 77B, a four-arm trial that randomized premenopausal women with high-risk early breast cancer to no systemic treatment, levamisole, oral cyclophosphamide (C) or cyclophosphamide, methotrexate and fluorouracil (CMF)., Results: In total, this retrospective analysis included 460 women (40% of the 1146 randomized patients). The continuous Prosigna ROR score was prognostic in the no systemic treatment group (unadjusted P < 0.001 for disease-free survival (DFS), P = 0.001 for overall survival (OS)). No statistically significant interaction of continuous ROR score and treatment on DFS and OS was found. A highly significant association was observed between intrinsic subtypes and C/CMF treatment for DFS (P interaction  = 0.003 unadjusted, P = 0.001 adjusted) and OS (P interaction  = 0.04). In the adjusted analysis treatment with C/CMF was associated with a reduced risk of DFS events in patients with basal-like (hazard ratio (HR) 0.14; 95% CI 0.06; 0.32) and luminal B (HR 0.48; 95% CI 0.27; 0.84) subtypes but not in patients with Human epidermal growth factor receptor-enriched (HR 1.05; 95% CI 0.56; 1.95) or luminal A (HR 0.61; 95% CI 0.32; 1.16) subtypes., Conclusion: The Prosigna ROR score and intrinsic subtypes were prognostic in high-risk premenopausal patients with breast cancer, and intrinsic subtypes identify high-risk patients with or without major benefit from adjuvant C/CMF treatment.

Purpose The PAM50-based Prosigna risk of recurrence (ROR) score has been validated in randomized clinical trials to predict 10-year distant recurrence (DR). The value of Prosigna for predicting DR was examined in a comprehensive nationwide Danish cohort consisting of postmenopausal women with hormone receptor-positive early breast cancer treated with 5 years of endocrine therapy alone. Patients and Methods Using the population-based Danish Breast Cancer Cooperative Group database, follow-up data were collected on all patients diagnosed from 2000 through 2003 who, by nationwide guidelines, were treated with endocrine therapy for 5 years. Primary tumor blocks from 2,740 patients were tested with Prosigna and, after determination of human epidermal growth factor receptor 2 (HER2) status, data from 2,558 hormone receptor-positive/HER2-negative samples were analyzed, including 1,395 node-positive patients. Fine and Gray models were applied to determine the prognostic value of ROR for DR. Results Median follow-up for recurrence was 9.2 years. Twenty-six percent of the node-positive patients were classified as low ROR (n = 359) with a DR risk of 3.5% (95% confidence interval [CI], 1.9% to 6.1%) versus a DR risk of 22.1% (95% CI, 18.6% to 25.8%) at 10 years for patients classified as high ROR (n = 648). Node-negative patients classified as low and high ROR had a risk of DR of 5.0% (95% CI, 2.9% to 8.0%) and 17.8% (95% CI, 14.0% to 22.0%), respectively. Luminal B tumors (n = 947; DR risk, 18.4% [95% CI: 15.7% to 21.3%]) had a significantly worse outcome than luminal A tumors (n = 1,474,;DR risk, 7.6% [95% CI: 6.1% to 9.2%]; P < .001). Conclusion Prosigna ROR score improved the prediction of outcome in this nationwide Danish population. In a real-world setting, Prosigna can reliably identify node-negative patients and a significant proportion of patients with one to three positive nodes who can be spared treatment with adjuvant chemotherapy.

Introduction: The Prosigna-PAM50 risk of recurrence (ROR) score has been validated in randomized clinical trials to predict 10-year distant recurrence (DR) in hormone receptor-positive breast cancer. Here, we examine the ability of Prosigna for predicting DR at 10 years in a subgroup of postmenopausal breast cancer patients with special histological subtypes., Methods: Using the population based Danish Breast Cancer Group database, follow-up data were collected on all patients diagnosed from 2000 to 2003 with estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2) normal breast cancer who by nationwide guidelines were treated with 5 year of endocrine therapy (N = 2558). Among patients with 1 to 3 positive lymph nodes or a tumor size >20 mm, we identified 1570 with invasive ductal carcinoma (IDC) and 89 with special histological subtypes (apocrine, medullary, mucinous, papillary, secretory, tubular, neuroendocrine) who were tested with Prosigna. Fine and Gray models were applied to determine the prognostic value of the Prosigna-PAM50 ROR score for DR special subtypes as compared to IDC., Results: Median follow-up for DR was 9.2 year and for OS 15.2 year. The 10-year DR rate for the special subtypes was 9.2% (95% CI: 4.0% to 17.2%) as compared to 13.7% (95% CI: 11.9% to 15.7%) for IDC. The 10-year OS was 74.2% (95% CI: 63.7% to 82.0%) for the special subtypes and 75.4% (95% CI: 73.2% to 77.4%) for IDC. Prosigna showed a statistical significant association of the continuous ROR score with risk of DR for both IDC and the special subtypes (IDC: p < .0001; special subtypes: p = .01)., Conclusion: In the present study, we demonstrated that Prosigna-PAM50 continuous ROR score added significant prognostic information for 10-year DR in postmenopausal patients with special subtypes (tumor size >20 mm or 1 to 3 positive lymph nodes) and ER-positive, HER2-normal early breast cancer.

Background: In breast cancer (BC) patients a cancer predisposing BRCA1/2 mutation is associated with adverse tumor characteristics, risk assessment and treatment allocation. We aimed to estimate overall- (OS) and disease-free survival (DFS) according to tumor characteristics and treatment among women who within two years of definitive surgery for primary BC were shown to carry a mutation in BRCA1/2 ., Material and Methods: From the clinical database of the Danish Breast Cancer Group we included 141 BRCA1 and 96 BRCA2 BC patients. Estrogen receptor and HER2 status were centrally reviewed on paraffin-embedded tumor tissue. Information on risk reducing surgery was obtained from the Danish Pathology and Patient Registries and included as time-dependent variables in Cox proportional hazard models., Results: Ten-year OS and DFS for BRCA1 BC patients were 78% (95% CI 69-85) and 74% (95% CI 64-81). Ten-year OS and DFS for BRCA2 BC were 88% (95% CI 78-94) and 84% (95% CI 74-91). BRCA1 BC patients as compared to BRCA2 BC patients had a higher risk of BC relapse or non-breast cancer within ten years of follow-up, independent of ER status (adjusted HR 2.78 95% CI 1.28-6.05, p = .01), but BRCA mutation was not associated with OS (adjusted HR 1.98, 95% CI 0.87-4.52, p = .10). In multivariate analysis, including both BRCA1 and BRCA2 carriers, no chemotherapy was associated with a higher risk of death (adjusted OS HR 3.58, 95% CI 1.29-9.97, p = .01) and risk reducing contralateral mastectomy (RRCM) was associated with a significantly reduced risk of death (adjusted OS HR 0.42, 95% CI =0.21-0.84, p = .01)., Conclusion: Difference in OS between BRCA1 and BRCA2 BC patients could be ascribed to tumor-biology. BRCA1 BC patients may have a shorter ten-year DFS than BRCA2 BC patients. Chemotherapy and risk reducing contralateral mastectomy reduce mortality for both BRCA1 and BRCA2 BC patients.

HLA class Ia (HLA-ABC) and HLA class Ib (HLA-E, -F and -G) molecules and FOXP3+ tumor-infiltrating lymphocytes (TILs) are often reported as relevant factors of tumor immune regulation. We investigated their expression as prognostic factors in 200 patients with primary cutaneous melanoma (PCM). In our cohort, patients with tumors showing upregulation of HLA-ABC molecules had significantly thicker tumors (32% vs 7%, P<0.001), frequent ulceration (20% vs 6%, P=0.007) and frequent nodular melanomas (20% vs 4%, P=0.001). Additionally, high expression of HLA-G in the tumor was a sign of bad prognosis for the patients, being associated with thick tumors (30% vs 12%, P=0.017), ulceration (24% vs 5%, P<0.001) and positive sentinel node (13% vs 6%, P=0.015). HLA-E, HLA-F and FOXP3+ TILs were not indicative of the prognosis in PCM. High HLA-ABC and HLA-G were associated with tumor aggressiveness and could be relevant predictive markers for effective immunotherapy of melanoma tumors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Problem: Elucidating immune mechanisms in the endometrium, which lead to the success of implantation and pregnancy, is important in reproductive medicine. Studies of immune cell abundance have shown conflicting results, and the expression and importance of HLA class Ib proteins in pre-implantation endometrium have not yet been investigated., Method of Study: The study population consisted of four subgroups: a hydrosalpinx, a salpingectomy, an unexplained infertility, and a fertile control group. Endometrial samples were collected during the implantation window. Immune markers (CD56 + and CD16 + cells, FoxP3 + Tregs, HLA-G, HLA-F) were quantified in the samples. The outcome of the subsequent IVF treatment was recorded., Results: Increased CD56 + uNK cells and high HLA-G expression served as predictor for successful pregnancy outcome. HLA-F expression was positively correlated with uNK cells, being indirectly predictive for achieving pregnancy., Conclusion: Endometrial uNK cell abundance in the pre-implantation endometrium seems to be important for normal fertility and pregnancy success, and they may be used as clinical markers to predict implantation success in IVF., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)