Torben Steiniche

Background: Ultrastaging of sentinel lymph nodes (SLNs) is standard for nodal assessment in early stage cervical cancer but is limited by sampling constraints and workload. Molecular detection of human papillomavirus (HPV) may identify metastatic disease. This study evaluated next generation sequencing (NGS)-based HPV detection in SLNs as a complementary approach to SLN assessment. Patients and Methods: This prospective cohort study included 122 women with early stage cervical cancer treated with primary surgery and SLN mapping. SLNs were assessed by ultrastaging and HPV detection using an NGS-based HPV genotyping panel. A subset of patients additionally underwent polymerase chain reaction (PCR)-based HPV analysis. Results: A total of 17 patients (13.9%) had SLN metastases identified by ultrastaging, all of which were HPV-positive by NGS. HPV read counts correlated with SLN metastasis size (Spearman’s ρ = 0.73, p < 0.001). NGS additionally identified HPV in 13 patients (10.7%) with ultrastaging-negative SLNs. Compared with patients negative by both methods, these patients had significantly larger tumors ( p = 0.009) and higher International Federation of Obstetrics and Gynecology (FIGO) stage ( p = 0.02), while exhibiting significantly lower HPV read counts than patients positive by both ultrastaging and NGS ( p = 0.03). PCR detected HPV in more SLNs than NGS. Conclusions: NGS detected HPV in all ultrastaging-confirmed SLN metastases and identified an additional subgroup of patients with molecular HPV detection despite negative ultrastaging. The association between HPV quantity, metastasis size, and adverse clinicopathological characteristics supports the biological relevance of quantitative HPV detection in SLNs. These findings warrant further evaluation of NGS as a complementary molecular biomarker for SLN assessment in larger prospective studies.

Objective: Brain branks preserve extensive material relevant to neurodegenerative disease research. As these collections age, tissue becomes archival, raising the question of whether long-term fixed and stored human brain tissue remains suitable for contemporary immunohistochemical analyses. Materials and Methods: Forty-one autopsy brains collected between 1946 to 1980 were examined. For each case, midbrain and hippocampus were available both as original paraffin-embedded blocks and as tissue stored long term in fixative. New paraffin blocks were prepared from the long-term fixated tissue. Sections from original and newly prepared blocks were immunohistochemically stained for α-synuclein, hyperphosphorylated tau and amyloid-β. Immunoreactivity was assessed using semi-quantitative scoring. Results: Original blocks consistently showed good staining intensity and morphological preservation for each protein pathology. Newly prepared blocks showed slightly lower semi-quantitative scores for Lewy-related pathology, without statistically significant differences, except for astrocytic α-synuclein in the substantia nigra in cases from the 1960s. Tau pathology displayed modestly reduced labelling, particularly of the neuropil threads and neurofibrillary tangles, most evident in cases from the 1950s. Amyloid-β-positive senile plaques showed similar or slightly higher scores in newly prepared blocks, with no significant differences across regions. Conclusion: Human brain tissue preserved as paraffin-embedded blocks or stored in fixative for up to 78 years remains suitable for immunohistochemical analyses. Adequate-to-good detection of aggregated of α-synuclein, hyperphosphorylated tau and amyloid-β is achievable, indicating preserved pathological hallmarks of Lewy Body Disease and Alzheimer’s Disease in archival tissue.

Background and purpose: This study investigated prognostic biomarkers in oropharyngeal squamous cell carcinoma (OPSCC), with a focus on tumors related to human papillomavirus (HPV) infection and potential molecular effects of tobacco smoking, as smokers with HPV+ OPSCC often have poorer outcomes. Patients/material and methods: We first analyzed 56 previously untreated OPSCC patients (exploration cohort), assessing HPV status, gene expression related to hypoxia, tumor subtype, and radiosensitivity, together with next-generation sequencing (NGS) of cancer-related genes. A custom NGS panel was subsequently designed and validated in 162 patients from the DAHANCA 19 randomized controlled trial (RCT), all treated with curative (chemo-)radiotherapy. Results:In the exploration cohort (40 HPV+, 79%), the most common molecular events in HPV+ tumors were PIK3CA and ATR mutations and chromosome 3q amplification. ATR and CREBBP mutations occurred more often in heavy smokers (>10 pack-years), but these associations were not confirmed in the DAHANCA 19 cohort. No specific smoking-related mutational signature or link to TP53 mutations was observed. In the DAHANCA 19 cohort, 17 locoregional failures (LRF) occurred among 128 HPV+ patients. No unifying molecular features were identified. However, mutations in NFE2L2 and CASP8, as well as amplifications of 3q genes (BCL6, SOX2), were associated with LRF. Interpretation: In HPV+ OPSCC, only few molecular alterations appear to act as drivers or prognostic biomarkers. Importantly, no molecular features of tobacco smoking exposure were identified, and the mechanism behind the worse prognosis in smokers remains unclear.

Psoriasis, a chronic inflammatory skin disorder characterised by erythematous and scaly plaques, can be both physically and emotionally distressing for patients. Dead Sea climatotherapy (DSC), a treatment modality combining sun exposure, mineral‐rich water and mud therapy during 4 weeks at Ein Gedi, Israel, is used for a small group of patients with psoriasis. This study aimed to investigate the cellular composition of psoriatic skin lesions at relapse after complete clearance from DSC. Skin biopsies from baseline, end of treatment and relapse were collected from eight patients with plaque psoriasis who achieved complete clearance from Dead Sea climatotherapy treatment. These biopsies were subjected to immunohistochemistry, RNA sequencing and quantitative polymerase chain reaction analysis (qPCR). Our findings demonstrate that DSC effectively reduces inflammatory markers to levels comparable to baseline non‐lesional skin in the short term. The differential expression analysis identified several upregulated differentially expressed genes, including OSM, CXCL8, TREM1, CXCL1, CSF3R, BCL2A1 and CXCL2, in relapsed psoriasis skin compared with baseline lesional skin. These findings were confirmed by qPCR analysis. Pathway enrichment analysis indicated a marked upregulation of neutrophil‐associated pathways in relapse skin compared with baseline lesional skin. Immunohistochemical staining for neutrophil markers, such as CD11b, CD15, CD66b, CD207, MPO and NE, showed a non‐significant trend towards enhanced neutrophil infiltration and activation at relapse. In conclusion, while DSC provides short‐term effectiveness in managing psoriasis, the initial relapse phase is associated with neutrophil activation and migration. Thus, targeting neutrophils early in the psoriasis disease course may disturb the evolution of psoriasis, potentially preventing disease chronicity.

Introduction Immune checkpoint inhibitors (ICI) have revolutionized the treatment of metastatic malignant melanoma (MM) and improved long-term survival. Despite the impressive results, some patients still have progressive disease, and the search for biomarkers predicting response to ICI treatment is ongoing. In this search, galectin-3 (Gal-3) has been suggested as a molecule of interest, both as a marker of treatment response and as a treatment target to potentiate ICI therapy. We have previously demonstrated the binding between programmed cell death 1 (PD-1) and Gal-3, and here, we investigated the interaction between PD-1, pembrolizumab, and Gal-3 in metastatic MM patients. Methods The binding between PD-1, pembrolizumab and Gal-3 was investigated by surface plasmon resonance (SPR) and cryogenic electron microscopy (cryo-EM). The function was studied in in vitro cultures and soluble levels of both PD-1 and Gal-3 were measured in metastatic MM patients, treated with pembrolizumab. Results By SPR, we demonstrated that Gal-3 can block the binding between PD-1 and pembrolizumab, and further visualized a steric inhibition using cryo-EM. T cells cultured with Gal-3 had reduced pro-inflammatory cytokine production, which could not be rescued by pembrolizumab. In patients with metastatic MM, high levels of Gal-3 in plasma were found in patients with a longer progression-free survival in the study period, whereas high Gal-3 expression in the tumor was seen in patients with disease progression. Soluble PD-1 levels in plasma increased after treatment with pembrolizumab and correlated with disease progression. Conclusion We demonstrate that the interaction between PD-1 and Gal-3 interferes with the binding of pembrolizumab, supporting that an immune suppression induced by Gal-3 in the tumor microenvironment cannot be rescued by pembrolizumab.

Secukinumab and Dead Sea treatment result in clear skin for many psoriasis patients, through distinct mechanisms. However, recurrence in the same areas after treatments suggests the existence of a molecular scar. We aimed to compare the molecular and genetic differences in psoriasis patients who achieved complete response from secukinumab and Dead Sea climatotherapy treatments. We performed quantitative immunohistochemical and transcriptomic analysis, in addition to digital spatial profiling of skin punch biopsies. Histologically, both treatments resulted in a normalization of the lesional skin to a level resembling nonlesional skin. Interestingly, the transcriptome was not normalized by either treatments. We revealed 479 differentially expressed genes between secukinumab and Dead Sea climatotherapy at the end of treatment, with a psoriasis panel identifying SERPINB4, SERPINB13, IL36G, IL36RN, and AKR1B10 as upregulated in Dead Sea climatotherapy compared with secukinumab. Using digital spatial profiling, pan-RAS was observed to be differentially expressed in the microenvironment surrounding CD103⁺ cells, and IDO1 was differentially expressed in the dermis when comparing the two treatments. The differences observed between secukinumab and Dead Sea climatotherapy suggest the presence of a molecular scar, which may stem from mechanistically different pathways and potentially contribute to disease recurrence. This may be important for determining treatment response duration and disease memory.

Clinically, unique markers in fetal membrane cells may contribute to the search for biomarkers for preterm prelabor rupture of the fetal membranes (pPROM) in maternal blood. pPROM is associated with overwhelming inflammation and premature cellular senescence causing “biological microfractures” of the fetal membranes. We hypothesize that these pathological processes are associated with the shedding of fetal membrane cells into the maternal circulation. The aim of this study was to identify markers expressed exclusively in fetal membrane cells to facilitate their isolation, characterization, and determination of biomarker potential in maternal blood. We have (1), by their transcriptomic profile, identified markers that are upregulated in amnion and chorion tissue compared to maternal white blood cells, and (2), by immunohistochemistry, confirmed the localization of the differentially expressed proteins in fetal membranes, placenta, and the placental bed of the uterus. RNA sequencing revealed 31 transcripts in the amnion and 42 transcripts in the chorion that were upregulated. Among these, 22 proteins were evaluated by immunohistochemistry. All but two transcripts were expressed both on mRNA and protein level in at least one fetal membrane cell type. Among these remaining 20 proteins, 9 proteins were not significantly expressed in the villous and extravillous trophoblasts of the placenta.

The prognosis after primary (chemo-)radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) is affected by Human Papillomavirus (HPV) status, with a better prognosis in HPV-positive OPSCC. HPV-status is routinely assessed by p16 immunohistochemistry (IHC), but additional HPV DNA testing is debated. Also, there are numerous HPV genotypes, which prognostic role may need clarification. The purpose of this study was: (1) to test a custom-made targeted HPV next generation sequencing (NGS) panel in OPSCC, (2) to determine correlation with p16 IHC, and (3) to assess the impact of HPV DNA testing on outcome in the prospectively randomized clinical trial DAHANCA 19. We included 271 patients with OPSCC treated with primary (chemo-)radiotherapy in the DAHANCA 19 trial. Of these, 199 (73%) were p16-positive. HPV-status was determined by targeted HPV next generation sequencing (NGS), using a custom-made HPV genotyping panel. HPV was detected in 194 tumor samples. p16 IHC and NGS HPV status were concordant in 265 (98%) of 271 patients, whereas we did not detect HPV DNA in 5 p16-positive tumors. HPV16 accounted for 169 of 194 HPV-positive cases (87%). HPV genotypes 18, 31, 33, 35, and 59 were also detected.Loco-regional failure and overall survival were similar whether patients were separated by p16 IHC, or HPV DNA status (p < 0.0001 for all) and did not depend on HPV genotype (p = 0.9 and p = 0.7). In the present study, HPV DNA testing or typing in a Danish OPSCC cohort did not add additional information to p16 IHC, the most widely used and accepted prognostic indicator.