Paraffin-embedded Biopsy Samples Research Articles

Evaluation of molecular and bacteriological detection methods performed on the formalin-fixed paraffin-embedded biopsy samples collected from endometrial and lymph node tuberculosis suspected patients

BackgroundEndometrial Tuberculosis is one of the most common gynecological problems known to have serious implications for the quality of life like infertility. The commonly practiced histopathology solely relies on the suggestive feature of Tuberculosis (TB) with low specificity. Regarding the alternative bacteriological and molecular detection tools, little evidence was generated on their utility in the diagnosis of endometrial tuberculosis in Ethiopia. Therefore, we aim to investigate the detection rate of molecular and bacteriological detection methods on formalin-fixed paraffin-embedded biopsy samples for the diagnosis of endometrial and lymph node TB.MethodsA retrospective cross-sectional study was conducted on 90 formalin fixed paraffin embedded biopsy samples from patients with gynecologic and lymph problems collected between 2018 and 2022 at St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia. SPSS version 26 was used for statistical analysis. The diagnostic performance was calculated using the histopathology method as the reference standard. Cohen’s Kappa value was used to measure the level of agreement. A test with a P-value of < 0.05 was considered statistically significant.ResultsA total of 90 samples were analyzed in the current study. Auramine O, GeneXpert MTB/RIF assay, and Real-Time PCR tests have shown a detection rate of 32/90 (36%), 43/90 (47.8%), and 54/90 (60%) respectively (P ≤ 0.01). The sensitivity and specificity of AO were 38.1% and 95% respectively. RT PCR showed superior sensitivity followed by GeneXpert MTB/RIF assay, 70% and 58.6%. AO and molecular methods have shown a similarly low level of agreement with histopathology (Kappa value = 0.2).ConclusionsIn a resource-limited setting, the selection of diagnostic tools needs careful attention. Putting the patients on anti-TB treatments based solely on histopathological findings may lead to undesired and adverse complications. Therefore, applying molecular and bacteriological detection methods along with histopathology, could help minimize inappropriate antimicrobial use.

Tumor microenvironment biomarkers predicting pathological response to neoadjuvant chemoimmunotherapy in locally advanced esophageal squamous cell carcinoma: post-hoc analysis of a single center, phase 2 study

BackgroundNeoadjuvant chemoimmunotherapy has a promising effect on locally advanced esophageal squamous cell carcinoma (ESCC). However, reliable biomarkers robustly predicting therapeutic response are still lacking.MethodsFormalin-fixed and paraffin-embedded pre-neoadjuvant chemoimmunotherapy biopsy samples...

Expression of cytokeratin 7/20 and Ki67 in Helicobacter pylori-associated gastritis and intestinal metaplasia.

Cytokeratins (CKs) have been associated with precancerous and cancerous gastric lesions in patients with Helicobacter pylori-associated chronic gastritis, making them useful for diagnosing epithelial tumors. A retrospective study was conducted utilizing 200 formalin-fixed paraffin-embedded gastric biopsy samples collected from the lesser curvature of the stomach. Samples from the control group, patients with H. pylori infection, and patients with H. pylori-associated gastritis, with complete and incomplete intestinal metaplasia (IM) were immunostained. Monoclonal antibodies were utilized to determine the expression of CK7, CK20, and Ki-67. Patients infected with H. pylori had strong CK20 expression on the surface, and weak CK7 expression on the surface and deep glands; while non-specific chronic gastritis patients had weak focal CK7 expression and strong CK20 expression. The normal gastric mucosa of patients in the control group had relatively weak CK7 expression, restricted to a few cells in the neck and deep glands. CK20 showed diffuse strong reactivity on the surface. On the other hand, patients with complete IM showed a CK7 staining pattern that was either negative or weakly focal on the surface and crypts associated with diffuse surface CK20 and focal crypt staining corresponding to gastric type IM. The Ki67 proliferating index was low (≤ 15%) in H. pylori infected patients, high (> 30%) in patients with incomplete IM, and intermediate (16-30%) in patients with complete IM. These results indicate a significant link between the expressions of CK7/CK20 and Ki67 in patients afflicted with H. pylori and IM.

A case of crystalglobulin-induced nephropathy wherein M protein was identified by mass spectrometry and immunoglobulin G subclass staining.

Crystalglobulin-induced nephropathy is a rare disease that causes the deposition of crystallized monoclonal immunoglobulins into the glomerular capillary and arteriole spaces. Here, we report the case of a patient who presented with skin ulcers, urinary protein, and renal dysfunction. The patient underwent renal and skin biopsies, and the biopsy tissue samples were subjected to mass spectrometry. The patient was diagnosed with crystalglobulin-induced nephropathy. A literature review suggested that pathological examinations using electron microscopy, mass spectrometry, and immunofluorescent staining of paraffin-embedded biopsy samples treated with pronase may be useful for the diagnosis of this condition.

Emerging need of molecular profiling in hepatobiliary cancer

: Gallbladder cancer is a rare malignancy but represents almost 50% of all biliary tract cancer. Biliary cancers are highly fatal malignancies with a 5-year survival rate of approximately 20%. The prognosis of gallbladder cancer is poor due to the aggressive tumor biology, late presentation, complicated anatomic position, and advanced stage at diagnosis. Locally advanced and metastatic disease treatment is with palliative chemotherapy. Alarming sign of gallbladder cancer is overall decreased in incidence in older patients but increased in the younger population. So many mutations have been reported for the gall bladder cancer till date. : A prospective observational study was conducted over a period of 1 year at Asian Institute of Medical Sciences Faridabad which includes hepatobiliary carcinoma patients who are at stage III and stage IV of cancer. After getting the consent formalin fixed paraffin embedded biopsy samples, and 5 ml serum sample was collected in serum separator tube (SST). A whole genome sequencing was performed using Illumina HiSEQ, Illumina (NGS) technology, allows for high-throughput sequencing of DNA and RNA. Illumina's NGS is based on "sequencing by Synthesis" to detect the mutations.: Most common mutation found was in the P53 gene. TP53 (p.Arg175His), TP53 (p.Arg306Ter), TP53 (p.Cys238Tyr), TP53 (p.Leu43Ter), TP53 (p.Glu339Ter), TP53 (p.Pro190Leu). Mutations in the TP53 gene are a common feature of carcinoma of the gallbladder, and are associated with a more aggressive tumor phenotype, resistance to chemotherapy, and poorer overall survival.

Intrahepatic Transcriptomics Differentiate Advanced Fibrosis and Clinical Outcomes in Adults With Fontan Circulation

BackgroundThe molecular mechanisms underlying Fontan-associated liver disease (FALD) remain largely unknown. ObjectivesThis study aimed to assess intrahepatic transcriptomic differences among patients with FALD according to the degree of liver fibrosis and clinical outcomes. MethodsThis retrospective cohort study included adults with the Fontan circulation. Baseline clinical, laboratory, imaging, and hemodynamic data as well as a composite clinical outcome (CCO) were extracted from medical records. Patients were classified into early or advanced fibrosis. RNA was isolated from formalin-fixed paraffin-embedded liver biopsy samples; RNA libraries were constructed with the use of an rRNA depletion method and sequenced on an Illumina Novaseq 6000. Differential gene expression and gene ontology analyses were performed with the use of DESeq2 and Metascape. ResultsA total of 106 patients (48% male, median age 31 years [IQR: 11.3 years]) were included. Those with advanced fibrosis had higher B-type natriuretic peptide levels and Fontan, mean pulmonary artery, and capillary wedge pressures. The CCO was present in 23 patients (22%) and was not predicted by advanced liver fibrosis, right ventricular morphology, presence of aortopulmonary collaterals, or Fontan pressures on multivariable analysis. Samples with advanced fibrosis had 228 upregulated genes compared with early fibrosis. Samples with the CCO had 894 upregulated genes compared with those without the CCO. A total of 136 upregulated genes were identified in both comparisons and were enriched in cellular response to cytokine stimulus or oxidative stress, VEGFA-VEGFR2 signaling pathway, TGF-β signaling pathway, and vasculature development. ConclusionsPatients with FALD and advanced fibrosis or the CCO exhibited upregulated genes related to inflammation, congestion, and angiogenesis.

Abstract A007: Expression and activity of Rac, Cdc42, and their downstream effector PAK in Puerto Rican pancreatic cancer patients

Abstract Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed after the localized tumor has metastasized, which decreases its survival rate to a grim 3%. Mortality from PDAC is also increasing in Puerto Rico with lower survival rates than in the US. Therefore, it is pivotal to identify viable targets for the late stages of PDAC. The small GTPase Ras is the most mutated oncogene in PDAC. Oncogenic Ras, as well as multiple oncogenic cell surface receptors, activate the related GTPases Rac and Cdc42, which in turn activate p21-activated kinase (PAK) by autophosphorylation (p-PAK), to drive cancer cell invasion, and thus metastasis. Rac and Cdc42 are overexpressed in 21% of PDAC patients and contribute to poor prognosis. To determine the feasibility of treating PDAC with Rac/Cdc42 inhibitors that we are developing, this study aims to identify the p-PAK levels and Rac/Cdc42 expression in PDAC tissue from Puerto Rican patients. The hypothesis is that p-PAK levels, as well as Rac/Cdc42 expression, are selectively elevated in high-grade invasive PDAC. Immunohistochemistry for p-PAK and immunofluorescence for Rac/Cdc42 were conducted in fixed, paraffin-embedded PDAC biopsy samples of Puerto Rican patients. Preliminary results demonstrate that higher p-PAK levels are correlated with stage 3 and stage 4 tissue, and Rac and Cdc42 are expressed in ductal regions of PDAC tissue. Taken together, p-PAK has potential as a diagnostic biomarker for high-grade PDAC and will aid patient stratification in the planned clinical trials for our lead Rac/Cdc42 inhibitor, MBQ-167. Citation Format: Anamaris Torres-Sanchez, Jahdiel Berrios-Rodriguez, Jerry Cruz-Rodriguez, Jasmin Contreras-Santini, Victor Carlo, Suranganie Dharmawardhane. Expression and activity of Rac, Cdc42, and their downstream effector PAK in Puerto Rican pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A007.

EBF1, MYO6 and CALR expression levels predict therapeutic response in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is a hematological malignancy representing one-third of non-Hodgkin's lymphoma cases. Notwithstanding immunotherapy in combination with chemotherapy (R-CHOP) is an effective therapeutic approach for DLBCL, a subset of patients encounters treatment resistance, leading to low survival rates. Thus, there is an urgent need to identify predictive biomarkers for DLBCL including the elderly population, which represents the fastest-growing segment of the population in Western countries. Gene expression profiles of n=414 DLBCL biopsies were retrieved from the public dataset GSE10846. Differentially expressed genes (DEGs) (fold change >1.4, p-value <0.05, n=387) have been clustered in responder and non-responder patient cohorts. An enrichment analysis has been performed on the top 30 up-regulated genes of responder and non-responder patients to identify the signatures involved in gene ontology (MSigDB). The more significantly up-regulated DEGs have been validated in our independent collection of formalin-fixed paraffin-embedded (FFPE) biopsy samples of elderly DLBCL patients, treated with R-CHOP as first-line therapy. From the analysis of two independent cohorts of DLBCL patients emerged a gene signature able to predict the response to R-CHOP therapy. In detail, expression levels of EBF1, MYO6, CALR are associated with a significant worse overall survival. These results pave the way for a novel characterization of DLBCL biomarkers, aiding the stratification of responder versus non-responder patients.

Tissue miRNA Profile Is Associated with Acute Tubular Necrosis, Rejection Phenotypes and BK Polyomavirus-Associated Nephropathy in Human Kidney Allografts

Introduction: MicroRNAs (miRNAs), short noncoding RNAs, are involved in the modulation of gene expression, mainly by inhibiting the translation of mRNAs. Under physiological conditions, miRNAs are involved in viral infections and immune responses, among others; aberrant miRNA expression has been associated with kidney transplant pathologies, but a comprehensive comparison of later, particularly in tissue sections, is still pending. Methods: We used the genome-wide screening of miRNAs to identify those potentially involved in the disease processes after kidney transplantation. RNA was isolated from formalin-fixed paraffin-embedded kidney biopsy samples. Study included 8 patients with acute tubular necrosis (ATN), 8 patients with antibody-mediated rejection (ABMR), 10 patients with T-cell-mediated rejection (TCMR), 10 patients with BK polyomavirus-associated nephropathy (BKPyVAN), and 12 surveillance biopsies from patients with stable allograft function and no major abnormalities (normal allografts, CTRL). Results: We found 136 miRNAs differentially expressed in diseased kidney transplant tissue compared with normal allografts; of these, 74 miRNAs were differentially expressed in ABMR, 65 in ATN, 62 in BKPyVAN, 69 in TCMR, and 16 miRNAs were not associated with a specific disease phenotype. In addition, 29 miRNAs were differently expressed between ABMR and ATN, 39 between BKPyVAN and TCMR, and 20 between BKPyVAN and ABMR, and 38 between ABMR and TCMR. Conclusion: Our findings show that miRNA derived from kidney allograft biopsy samples represent an additional diagnostic tool to distinguish different disease phenotypes. This finding has the potential to assist clinicians in therapeutic decision-making and to translate to noninvasive monitoring of patients, e.g., blood samples.

Confirmation of canine acanthomatous ameloblastoma using RAS Q61R immunohistochemical staining of formalin-fixed paraffin-embedded tissues.

Differentiating canine acanthomatous ameloblastoma (CAA) from oral squamous cell carcinoma (OSCC) based on routine histopathology can be challenging. We have previously shown that more than 95% of CAAs harbor an HRAS p.Q61R somatic mutation, while OSCCs carry either wild-type alleles or other MAPK pathway activating mutations (e.g., HRAS p.Q61L, BRAF p.V595E). Given that HRAS p.Q61R mutations are highly prevalent in CAA, we hypothesized that a RAS Q61R-specific rabbit monoclonal antibody may be a useful tool for confirmation of CAA by immunohistochemical (IHC) staining. In the present study, we assessed IHC staining of archived formalin-fixed and paraffin-embedded biopsy samples with a diagnosis of CAA (n = 23), using a RAS Q61R-specific rabbit monoclonal antibody (SP174) and an automated IHC stainer. Negative control samples consisted of HRAS p.Q61R mutation-negative OSCC tumors with either a known HRAS p.Q61L mutation (n = 1), BRAF p.V595E mutation (n = 4), or wild-type corresponding alleles (n = 3). We found that all 23 CAAs showed diffuse and strong membranous RAS Q61R immunoreactivity (100% sensitivity), while none of the 8 OSCCs showed immunoreactivity (100% specificity). The data supports the use of RAS Q61R-specific rabbit monoclonal antibody for diagnostic IHC confirmation of CAA and ruling out OSCC in dogs.

Use of high-plex data provides novel insights into the temporal artery processes of giant cell arteritis.

To identify the key coding genes underlying the biomarkers and pathways associated with giant cell arteritis (GCA), we performed an in situ spatial profiling of molecules involved in the temporal arteries of GCA patients and controls. Furthermore, we performed pharmacogenomic network analysis to identify potential treatment targets. Using human formalin-fixed paraffin-embedded temporal artery biopsy samples (GCA, n = 9; controls, n = 7), we performed a whole transcriptome analysis using the NanoString GeoMx Digital Spatial Profiler. In total, 59 regions of interest were selected in the intima, media, adventitia, and perivascular adipose tissue (PVAT). Differentially expressed genes (DEGs) (fold-change > 2 or < -2, p-adjusted < 0.01) were compared across each layer to build a spatial and pharmacogenomic network and to explore the pathophysiological mechanisms of GCA. Most of the transcriptome (12,076 genes) was upregulated in GCA arteries, compared to control arteries. Among the screened genes, 282, 227, 40, and 5 DEGs were identified in the intima, media, adventitia, and PVAT, respectively. Genes involved in the immune process and vascular remodeling were upregulated within GCA temporal arteries but differed across the arterial layers. The immune-related functions and vascular remodeling were limited to the intima and media. This study is the first to perform an in situ spatial profiling characterization of the molecules involved in GCA. The pharmacogenomic network analysis identified potential target genes for approved and novel immunotherapies.

P-444 A comparison of immunohistochemistry and laser microdissection tandem mass spectrometry to identify the amyloid fibril protein from formalin-fixed paraffin embedded biopsy samples

P-444 A comparison of immunohistochemistry and laser microdissection tandem mass spectrometry to identify the amyloid fibril protein from formalin-fixed paraffin embedded biopsy samples

Cancer Stem Cells' Biomarker ALDH1&2 Increased Expression in Erosive Oral Lichen Planus Compared to Oral Leukoplakia.

ALDH1&2 has been considered an oral cancer stem cell (CSC) marker. Oral carcinogenesis is a process that usually passes through oral potentially malignant disorders (OPMD). Oral lichen planus (OLP) consists of immune-related chronic disorders that have been included in the OPMDs due to their possible transformation into oral cancer. The aim of this study was to investigate the early presence of ALDH1&2 in OLP compared to early oral leukoplakias (OL), especially mildly and non-dysplastic OL. The study type is experimental, and the study design is characterized as semiquantitative research which belongs to the branch of experimental research. The study sample consisted of paraffin-embedded OLP biopsy samples from the archives of the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece, during the period 2009-2019. The study sample contained 24 cases of OLP (14 erosive and 10 reticular) and 30 cases of OL (16 cases of moderately and severely dysplastic OL and 14 cases of mildly and non-dysplastic OL). The CSC-related biomarker ALDH1&2 was examined using semiquantitative immunohistochemistry (monoclonal antibody sc-166362, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:100). ALDH1&2 expression was evaluated through a scale of 1 to 3 depending on the percentage of positive epithelial cells and was compared to normal epithelium as well as cases of OL (the most prominent OPMD). The statistical analysis was performed with the Pearson chi-square test and the significance level was set at p≤0.05. The cytoplasmic staining of ALDH1&2 was observed mostly in the epithelial cells of the basal layer of the epithelium of OLP. Overall, this expression was significantly increased compared to normal epithelium. In addition, statistically significantly higher expression of ALDH1&2 was observed in the erosive form of OLP. Interestingly, this OLP positivity was higher compared to mild and non-dysplastic leukoplakias (p<0.001). ALDH1&2 is a confirmed CSC marker that was found to be clearly increased in OLP and characteristically in erosive OLP epithelium for the first time. Noteworthy, it was more prominent in erosive OLP rather than in mildly and non-dysplastic OL. Whether this pattern of expression raises the red flag of an early epithelial "CSC" phenotype in OLP or that ALDH1&2 expression indicates a response to the OLP inflammatory process requires further investigation.

Development and validation of Ran as a prognostic marker in stage I and stage II primary breast cancer

PurposeExisting prognostic biomarkers are inadequate for stratifying breast cancer patients with the highest risk of tumor progression at the time of diagnosis. Here, we demonstrate that the small GTPase Ran has predictive value for breast cancer (BC) patients as a whole, and for specific BC subtypes. Patients and methodsRan expression was quantified by immunohistochemistry in 263 patients with primary breast cancer diagnosed at the Breast Unit, Royal Liverpool Hospital. Additionally as an independent validation, we also analyzed the mRNA expressions of Ran, ER, PR, and Cerb-2, the triple-negative endocrine receptors, and their associations with patient survival in a combined patient cohorts of multiple public datasets (n = 1079). We analyzed the data with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. ResultsRan nuclear, cytoplasmic, and total staining are substantially associated with poor survival, independent of conventional prognostic markers such as estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and lymph node status. According to the datasets, Ran was significantly correlated with distant metastasis-free survival (DMFS) and relapse-free survival (RFS). ConclusionWe found that Ran expression is a unique predictive biomarker for patient survival, metastasis, and tumor relapse. This biomarker could be used for diagnostic purposes, using formalin-fixed, paraffin-embedded tumor biopsy samples from breast cancer patients in the early stages.

Morphologic and molecular analysis of liver injury after SARS-CoV-2 vaccination reveals distinct characteristics

Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n= 6) and from patients with an initial diagnosis of AIH (n= 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.

The changes of HER3 expression in head and neck cancer patients treated with induction chemotherapy.

e18004 Background: Erb-b2 receptor tyrosine kinase 3 (HER3) is broadly expressed in various types of cancer and is associated with poor prognosis. Recent evidence supports HER3 plays a role in acquired resistance to chemotherapy. The change of HER3 expression by chemotherapy in head and neck squamous cell carcinoma (HNSCC) remains unclear. Methods: We evaluated 95 patients with locally advanced HNSCC who had received induction chemotherapy (ICT) between 2009 and 2019. Immunohistochemical staining for HER3 was performed using formalin-fixed paraffin-embedded surgical specimens and biopsy samples. H-score of membrane staining was evaluated. Results: Median age of patients was 64 years, and 85% were male. Primary locations included oropharynx (54.8%), hypopharynx (21.5%), oral cavity (17.2%), and salivary gland (6.5%). Mean H-score of HER3 in tumor tissues was 77.6 ± 7.9. H-score of HER3 in HPV-positive tumors was higher than HPV-negative tumors (p = 0.019), which was more pronounced in oropharynx and hypopharynx tumors. While, H-score of HER3 in adjacent normal tissues of these tumors did not significantly differ. Compared with the non-smokers, tumor tissues of current or ex-smokers demonstrated higher H-score of HER3 (p = 0.033), independent of tumor location. In contrast, in adjacent normal tissues, no significant differences of H-score of HER3 were observed depending on the smoking status. The H-score higher than 100 was defined as HER3-high group. Among oropharynx and hypopharynx tumors with positive correlation between H-score of HER3 and HPV status (n = 56), HER3-high group was significantly correlated with HPV positivity (p = 0.009) and good response to ICT (p = 0.035). Good clinical response was also strongly related to HPV-positivity (p = 0.001). In addition, there was a tendency for longer overall survival in HER3-high group than low HER3 expression group (93 vs. 70 months, p = 0.053). The paired tumor samples collected at pre- and post-ICT (n = 21) were analyzed. HER3 expressions in adjacent normal tissue were higher at post-ICT compared to pre-ICT (p = 0.003), and these results were more pronounced in the non-smoking group, but no differences were found according to HPV-status. Conclusions: Taken together, our results showed that H-score of HER3 was significantly higher in both HPV-positive and smoking group. Interestingly, high HER3 expression in oropharynx and hypopharynx tumors was associated with HPV positivity and good clinical response to ICT. Additional analysis of tumor immune microenvironment in tumor tissues are underway.

Hepatic myofibroblasts exert immunosuppressive effects independent of the immune checkpoint regulator PD-L1 in liver metastasis of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) represents the 4th most common cause of cancer-related deaths in Western countries. Most patients are diagnosed at advanced stages, often already with metastases. The main site of metastasis is the liver and hepatic myofibroblasts (HMF) play a pivotal role in metastatic outgrowth. Immune checkpoint inhibitors (ICI) targeting programmed death ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) improved treatment of several cancers but not of PDAC. Therefore, this study aimed to better understand the impact of HMF on PD-L1 expression and immune evasion of PDAC cells during liver metastasis. Formalin-fixed and paraffin embedded biopsy samples or diagnostic resection specimens from liver metastases of 15 PDAC patients were used for immunohistochemical analyses. Serial sections were stained with antibodies directed against Pan-Cytokeratin, αSMA, CD8, and PD-L1. To investigate whether the PD-1/PD-L1 axis and HMF contribute to immune escape of PDAC liver metastases, a stroma enriched 3D spheroid coculture model was established in vitro, using two different PDAC cell lines, HMF, and CD8+ T cells. Here, functional and flow cytometry analyses were conducted. Immunohistochemical analysis of liver tissue sections of PDAC patients revealed that HMF represent an abundant stroma population in liver metastases, with clear differences in the spatial distribution in small (1500 µm) and large (> 1500 μm) metastases. In the latter, PD-L1 expression was mainly located at the invasion front or evenly distributed, while small metastases either lacked PD-L1 expression or showed mostly weak expression in the center. Double stainings revealed that PD-L1 is predominantly expressed by stromal cells, especially HMF. Small liver metastases with no or low PD-L1 expression comprised more CD8+ T cells in the tumor center, while large metastases exhibiting stronger PD-L1 expression comprised less CD8+ T cells being mostly located at the invasion front. HMF-enriched spheroid cocultures with different ratios of PDAC cells and HMF well mimicking conditions of hepatic metastases in situ. Here, HMF impaired the release of effector molecules by CD8+ T cells and the induction of PDAC cell death, an effect that was dependent on the amount of HMF but also of PDAC cells. ICI treatment led to elevated secretion of distinct CD8+ T cell effector molecules but did not increase PDAC cell death under either spheroid condition. Our findings indicate a spatial reorganization of HMF, CD8+ T cells, and PD-L1 expression during progression of PDAC liver metastases. Furthermore, HMF potently impair the effector phenotype of CD8+ T cells but the PD-L1/PD-1 axis apparently plays a minor role in this scenario suggesting that immune evasion of PDAC liver metastases relies on other immunosuppressive mechanisms.

Genetic and Microenvironment Features Do Not Distinguish Follicular Lymphoma Patients Requiring Immediate or Deferred Treatment.

Genetic and Microenvironment Features Do Not Distinguish Follicular Lymphoma Patients Requiring Immediate or Deferred Treatment.

Long non-coding RNA lnc-CHAF1B-3 promotes renal interstitial fibrosis by regulating EMT-related genes in renal proximal tubular cells

Renal interstitial fibrosis (RIF) is a common pathological manifestation of chronic kidney diseases. Epithelial-mesenchymal transition (EMT) of tubular epithelial cells is considered a major cause of RIF. Although long non-coding RNAs (lncRNAs) are reportedly involved in various pathophysiological processes, the roles and underlying molecular mechanisms of lncRNAs in the progression of RIF are poorly understood. In this study, we investigated the function of lncRNAs in RIF. Microarray assays showed that expression of the lncRNA lnc-CHAF1B-3 (also called claudin 14 antisense RNA 1) was significantly upregulated in human renal proximal tubular cells by both transforming growth factor-β1 (TGF-β1) and hypoxic stimulation, accompanied with increased expression of EMT-related genes. Knockdown of lnc-CHAF1B-3 significantly suppressed TGF-β1-induced upregulated expression of collagen type I alpha 1, cadherin-2, plasminogen activator inhibitor-1, snail family transcriptional repressor I (SNAI1) and SNAI2. Quantitative reverse transcriptase PCR analyses of paraffin-embedded kidney biopsy samples from IgA nephropathy patients revealed lnc-CHAF1B-3 expression was correlated positively with urinary protein levels and correlated negatively with estimated glomerular filtration rate. In situ hybridization demonstrated that lnc-CHAF1B-3 is expressed only in proximal tubules. These findings suggest lnc-CHAF1B-3 affects the progression of RIF by regulating EMT-related signaling. Thus, lnc-CHAF1B-3 is a potential target in the treatment of RIF.

A postulated model for photoimmunopathogenesis of chronic actinic dermatitis around adaptive immunity, including Th17 cells, Tregs, TRMs, cytotoxic T cells, and/or common-γ chain receptor+ cells.

The pathogenesis of chronic actinic dermatitis (CAD) is more complicated than other photodermatoses. However, the relationship between the clinical severity of CAD and the offending photocontact or contact allergens or both, and the correlations of CAD immunopathogenesis with the immunoregulatory molecules involved in adaptive immunity are yet to be investigated. We performed phototesting with broad-spectrum ultraviolet (UV) B, UVA, and visible light to establish the presence of photosensitivity in 121 patients with CAD, together with photopatch and contact patch testing. Nine patients with CAD were selected according to their clinical severity score for CAD (CSS-CAD), and triple direct immunofluorescence analysis was performed with paraffin-embedded skin biopsy samples. As CSS-CAD was closely correlated with the multiplicity of photo(contact) allergens, particularly photoallergens, three or more photoallergens were detected in the severe CAD group (52.5%); less in the moderate group (32.8%); and only one in the mild group (14.8%; P=.025). In the groups showing greater severity of disease, the absolute numbers of IFN-γ+ , IL-17+ , CD4+, CD8+, common-γ chain receptor (common-γCR)+ , and CD69+ tissue-resident memory cells increased on average; there was also an increase in the CD4+/CD8+ cell ratio, with the more severely affected groups. However, the levels of TNF-α+ and FoxP3+ regulatory T (Treg) cells and the mean IL-17/IFN-γ cell ratio decreased in the more severely affected CSS-CAD subgroups. Based on the clinical analysis and immunopathogenic results, avoidance of excessive sun exposure, and topical and systemic blocking agents for photo(contact) allergens are recommended. Additionally, conventional immunomodulators and emerging agents including JAK-STAT inhibitors may be administered for CAD treatment in the future.