Formalin-fixed Paraffin-embedded Tissue Research Articles

Prevalence of NTRK gene fusions in solid tumors in northeastern Mexico.

e15145 Background: Gene fusions (GF) are among the intricate hallmarks of cancer that lead to abnormal cell proliferation and survival. Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are frequent in rare solid tumors but less frequent in common ones. Scarce literature has evaluated the prevalence of this aberration estimated to be from 0.5 to 1% globally. For these particular type of tumors, multikinase inhibitors is an option aside from chemotherapy with good responses. Objective: To determine the prevalence of NTRK gene fusions in a Mexican population. Methods: Sociodemographic, clinical, and histopathological data were collected in a single center from databases of Pathology and Oncology Departments of Hospital Universitario Dr. José E. González in Northeastern Mexico. Patients of all ages with solid tumors were included if tested positive for Pan-TRK by immunohistochemistry (IHC). Confirmation for gene fusion was made using Next Generation Sequencing (NGS) technology in formalin-fixed paraffin-embedded tissue. Results: A total of 156 patients from 2021 to 2023 were included for analysis. 67 (42.9%) did test positive for IHC. Of these, 52.2% were male and 20.8% were pediatric. Adult and pediatric median age of 57 and 13 years, respectively. Most frequent tumors locations were central nervous system & extremities. Gene fusion anomaly was confirmed in 6 patients. Among these, 83.3% were male and 16.7% pediatric population. Only NTRK1 and NTRK3 were found in gene fusions. Four translocations were prevalent TPM3:NTRK1 and ETV6:NTRK3 with 2 cases (33.3%), respectively. Followed by LMNA:NTRK1 and EML4:NTRK3 each with one patient. For location, arranged in descending order, head and neck (33.3%), extremities (33.3%), central nervous system and occult primary tumors both 16.6%. The only non-adult tumor reported was infantile fibrosarcoma. Prevalence was estimated to be 3.8% in our population. Conclusions: Understanding the molecular biology of tumor cells enables the development of targeted treatments other than chemotherapy. In our population, NTRK gene fusions were a little more prevalent (~4%) than global estimation. Unfortunately, in Mexico there's a lack of access to multikinase inhibitors. Further studies with large samples are required in Mexico and all over the world to afford access to these tumor-agnostic therapies.

Multinational, Multicenter Evaluation of Prostate Cancer Tissue in Sub-Saharan Africa: Challenges and Opportunities.

Prostate cancer disproportionately affects men of African descent, yet their representation in tissue-based studies is limited. This multinational, multicenter pilot study aims to establish the groundwork for collaborative research on prostate cancer in sub-Saharan Africa. The Men of African Descent and Carcinoma of the Prostate network formed a pathologist working group representing eight institutions in five African countries. Formalin-fixed paraffin-embedded prostate tissue specimens were collected from Senegal, Nigeria, and Ghana. Histology slides were produced and digitally scanned. A central genitourinary pathologist (P.L.) and eight African general pathologists reviewed anonymized digital whole-slide images for International Society of Urological Pathology grade groups and other pathologic parameters. Discrepancies were re-evaluated, and consensus grading was assigned. A virtual training seminar on prostate cancer grading was followed by a second assessment on a subcohort of the same tissue set. Of 134 tissue blocks, 133 had evaluable tissue; 13 lacked cancer evidence, and four were of insufficient quality. Post-training, interobserver agreement for grade groups improved to 56%, with a median Cohen's quadratic weighted kappa of 0.83 (mean, 0.74), compared with an initial 46% agreement and a quadratic weighted kappa of 0.77. Interobserver agreement between African pathologist groups was 40%, with a quadratic weighted kappa of 0.66 (95% CI, 0.51 to 0.76). African pathologists tended to overgrade (36%) more frequently than undergrade (18%) compared with the reference genitourinary pathologist. Interobserver variability tended to worsen with a decrease in tissue quality. Tissue-based studies on prostate cancer in men of African descent are essential for a better understanding of this common disease. Standardized tissue handling protocols are crucial to ensure good tissue quality and data. The use of digital slide imaging can enhance collaboration among pathologists in multinational, multicenter studies.

A Fast, Affordable, and Minimally Invasive Diagnostic Test for Cancer of Unknown Primary Using DNA Methylation Profiling

Currently, we cannot provide a conclusive diagnosis for 3% to 5% of people who are confronted with cancer. These patients have cancer of unknown primary (CUP), ie, a metastasized cancer for which the tissue of origin cannot be determined. Studies have shown that the DNA methylation profile is a unique “fingerprint” that can be used to classify tumors. Here we used cell-free reduced representation bisulfite sequencing (cfRRBS), a technique that allows us to identify the methylation profile starting from minimal amounts of highly fragmented DNA, for CUP diagnosis on formalin-fixed paraffin-embedded (FFPE) tissue and liquid biopsies. We collected 80 primary tumor FFPE samples covering 16 tumor entities together with 15 healthy plasma samples to use as a custom cfRRBS reference data set. Entity-specific methylation regions are defined for each entity to build a classifier based on nonnegative least squares deconvolution. This classification framework was tested on 30 FFPE, 19 plasma, and 40 pleural and peritoneal effusion samples of both known metastatic tumors and clinical CUPs for which pathological investigation finally resulted in a cancer diagnosis. Using this framework, 27 of 30 FFPE (all CUPs) and 16 of 19 plasma samples (10/13 CUPs) obtained an accurate diagnosis, with a minimal DNA input of 400 pg. Diagnosis of the 40 pleural and peritoneal effusion samples is possible in 9 of 27 samples with negative/inconclusive cytology (6/13 CUPs), showing that cell-free DNA (cfDNA) methylation profiling could complement routine cytologic analysis. However, a low “cfDNA – high-molecular weight DNA ratio” has a considerable impact on the prediction accuracy. Moreover, the accuracy improves significantly if the predicted tumor percentage is >7%. This proof-of-concept study shows the feasibility of using DNA methylation profiling on FFPE and liquid biopsy samples such as blood, ascites, and pleural effusions in a fast and affordable way. Our novel RRBS-based technique requires minimal DNA input, can be performed in <1 week, and is highly adaptable to specific diagnostic problems as we only use 5 FFPE references per tumor entity. We believe that cfRRBS methylation profiling could be a valuable addition to the pathologist’s toolbox in the diagnosis of CUPs.

Concordance of PD-L1 combined positive score (CPS) analysis between primary gastric cancer and matched peritoneal metastasis.

e16068 Background: Peritoneum represents one of the most common metastatic sites of gastroesophageal adenocarcinoma (GEA) and is associated with poor prognosis. Programmed death-ligand 1 (PD-L1) expression is a recognized predictive biomarker of response to immune checkpoint inhibitors (ICIs) in the first-line setting. However, data from pivotal trials showed poorer clinical efficacy of ICIs in the sub-set of patients with peritoneal involvement, regardless of PD-L1 score magnitude. Here, we investigated the temporal and spatial heterogeneity of PD-L1 expression between primary tumor and matched peritoneal metastasis. Methods: PD-L1 expression according to CPS was determined by immunohistochemistry using VENTANA PD-L1 (SP263) assays on formalin-fixed paraffin-embedded (FFPE) tumor tissues derived from 22 patients treated at our institution for advanced GEA from September 2015 to August 2023. Concordance rate of PD-L1 expression between primary tumor and matched peritoneal metastasis using PD-L1 CPS cut-off of 1 and 5 was reported (spatial heterogeneity); additionally, concordance between PD-L1 value before and after the administration of systemic treatments was analyzed (temporal heterogeneity). Results: 12(54.5%)/5(22.7%) primary tumor samples and 12(54.5%)/1(4.5%) peritoneal metastases showed PD-L1 CPS ≥ 1/ ≥ 5, respectively. Concordance of PD-L1 CPS between primary and peritoneal specimens was 54.5% and 72.7% according to CPS cuts-off of 1 and 5, respectively, highlighting marked spatial heterogeneity. High concordance rate (94%) was reported between negative PD-L1 CPS primary tumor samples and matched peritoneal metastases, against the cut-off of CPS ≥ 5; in contrast, none of the positive primary tumor samples retained PD-L1 expression in the peritoneum. Evident temporal heterogeneity of PD-L1 expression was also observed, with 56% concordance against CPS ≥ 5 in the pre- versus post-treatment comparison. Conclusions: PD-L1 expression in GEA is characterized by spatial and temporal heterogeneity which is more prominent for lower CPS cut-offs values. This evidence could hamper its role as predictive biomarker for ICIs. The high intra-patient discordance rate of PD-L1 CPS expression between positive primary tumor samples and matched peritoneal metastases suggests that peritoneal specimens should not be recommended as the preferred source for PD-L1 assessment.

Longitudinal tissue-based evaluation of TIGIT expression in patients with pancreatic cancer: Effect of expression with advancing clinical stages and across racial groups.

e16314 Background: While ground has been gained, pancreatic ductal adenocarcinoma (PDAC) continues to have a low 5-year survival of 13%. This is owed partially to a lack of early detection biomarkers and resistance to standard therapeutic options. TIGIT, an immune checkpoint receptor, is a marker of T-cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. TIGIT inhibitors are being explored in clinical trials in PDAC. Here we evaluate TIGIT expression in a cohort of PDAC patients and correlate level and intensity of expression with clinical parameters. We also examine changes in expression as the disease progresses from primary to metastatic disease. Methods: We performed RNAscope in situ hybridization (ISH) with a probe specific for human TIGIT mRNA on 82 formalin-fixed paraffin-embedded (FFPE) tissue samples. The cohort of tissue samples included 9 biopsies, 67 primary resections and 6 metastatic lesions. We evaluated the total TIGIT expression (%) as well as the intensity of TIGIT expression (% of cells with 3+ punctae, signifying putative immune cells), and compared these values between samples. Utilizing linear regression for continuous outcomes and logistic regression for binary outcomes, we tested for associations between TIGIT expression and clinical covariates. Results: Staining analysis showed that TIGIT expression did not differ significantly between racial groups. The mean percentage of TIGIT positive cells was 64.0%. High expression of TIGIT was associated with more advanced clinical stage (p &lt; 0.05). Evaluation of three longitudinal samples from the same patient revealed decreased TIGIT expression from the initial biopsy (41.6%) to resection (33.4%) and metastasis (2.8%). In these specimens, 3+ TIGIT expression also declined (2.1%, 1.2% and 0.02%, respectively). Conclusions: Anti-TIGIT therapy has potential to reverse immune suppression and, with other therapeutic modalities, may provide survival benefit. Here we demonstrate that increased TIGIT expression correlates with more advanced stage at diagnosis and also present data demonstrating that overall TIGIT expression, as detected by RNAscope ISH, may decrease as PDAC progresses to metastasis. As such, anti-TIGIT therapy may have important implications for evading an important mechanism of cancer progression.

Clinical and genomic characterization of sporadic medullary thyroid carcinoma in Chinese patients.

6094 Background: Medullary thyroid carcinoma (MTC) is a C-cell-derived epithelial neuroendocrine neoplasm. MTC has a low prevalence and a relatively worse prognosis compared with differentiated thyroid carcinoma. About 75% of all MTCs are sporadic. The clinicogenomic landscape of sporadic medullary thyroid carcinoma (sMTC) in the Chinese population was rarely described. Methods: Formalin-fixed paraffin-embedded (FFPE) samples and/or tumor tissues from a total of 121 sMTC patients from 22 centers were retrospectively collected and tested using a targeted 28-gene next-generation sequencing (NGS) panel (May 2022 to November 2023). Clinicopathological data, including age, gender, histological diagnoses, site, tumor size, and status of the lymph nodes and distant metastasis were reviewed. Regression analyses were performed to examine the association between the clinicogenomic characterization and pathologic features. Results: A total of 105 out of 121 sMTC were NGS-positive. The mutant genes included RET(66.9%), RAS(20.7%), BRAF(1.6%), TSHR(0.8%). The top 5 frequent mutations were RET M918T (24.8%, 30/121), HRAS Q61R (8.3%, 10/121), RET C634R (7.4%, 9/121), RET V804M (4.1%, 5/121), HRAS Q61K (4.1%, 5/121). Of the 13 co-mutations, 12 were RET mutations or co-occurrence of RET with other genetic alterations. Besides, the higher the age, the lower the probability of lymph node metastasis (OR=0.95, p=0.005). Also, patients with common RET mutations (including M918T, C634, C630, C611, C618, C620, A883F, S891 and V804) tended to have lymph node metastases (OR=5.67, p=0.016). We observed a significant positive linear relationship (Beta=2.0) between other gene mutations (non-RET mutations and non-RAS mutations) and tumor size. However, when the tumor diameter was analyzed according to whether it was greater than 1cm, we found that clinical factors such as age, sex, and gene mutation were not related to tumor size. Moreover, we did not find any association between this cutoff value of tumor size and lymph node metastases. It indicated that sMTC clinical diagnosis and treatment may consider pathologic and genomic features, not just whether the tumor size is &gt;1cm. Conclusions: Our study first provided the molecular characteristic of Chinese patients with large-scale sMTC. Analysis revealed that common RET mutations may be potential predictors of lymph node metastases. These conclusions remain to be validated in prospective large-scale cohorts.

Molecular characterization of growing teratoma syndrome in patients with testicular germ cell tumor.

e17017 Background: Teratomas are unique neoplastic growths that encompass a diverse array of tissue types derived from multiple germ layers, often exhibiting varying degrees of differentiation. Among the phenomena associated with teratomas, the growing teratoma syndrome (GTS) stands out as a rare yet clinically significant occurrence. GTS, characterized by the persistence or regrowth of teratomatous elements despite initial treatment, poses challenges for both diagnosis and management. In this abstract, we present the results of our expression array analysis of teratomas, aiming to uncover novel insights into their molecular composition, heterogeneity, and potential implications for clinical management. Methods: This study included 53 patients with retroperitoneal teratoma (7 chemotherapy naïve and 46 with postchemotherapy teratoma) and 20 patients with viable postchemotherapy germ cell tumor from retroperitoneum that had surgery at National Cancer Institute in Slovakia and for whom formalin-fixed paraffin-embedded (FFPE) tumor tissue was available. Study also includes samples from 5 patients with normal testis as control group. Ten patients (18.9%) in this cohort had growing teratoma syndrome. Analysis was performed by HTG EdgeSeq Oncology Biomarker panel (HTG Molecular Diagnostics, Inc., Tucson, USA). Identified candidate genes associated with GTS were further validated by immunohistochemistry on FFPE. Results: Molecular profiling indicates no differences in expression profile in retroperitoneal teratomas regardless of previous chemotherapy. Expression analysis indicates 1586 genes being differently expressed with high significance ( p &lt; 0.05) in teratomas compared to normal testis and 1571 genes differentially expressed compared postchemotherapy viable tumors. We identified 14 genes being differently expressed in growing teratoma syndrome compared to teratoma without growth (CYP4A22, IGFBP1, CXCL6, LTF, CYP2C8, CFTR, CCL20, PFKFB3, SLC2A3, LIPA, HMOX1, GPNMB, CCL18, DLK1. Pathway analysis revealed MAPK and PI3K−Akt signaling pathways are mostly dysregulated in GTS. Conclusions: This molecular profiling identify genes differentially expressed in teratomas that are potential biomarkers for prediction of teratoma element in postchemotherapy residues in testicular germ cell tumor patients. Moreover, inhibition of identified signaling pathways associated with GTS could represent potentially novel therapeutic targets in case of unresectable disease.

Association of overexpression of TNF receptor superfamily members (CD40, BAFFR, LTβR and RANK) with favorable prognosis in patients with colorectal adenocarcinoma.

e15516 Background: Numerous studies by our and other groups have highlighted the implication of NF-κB in colorectal cancer (CRC) through the deterioration of classical and alternative pathway signaling. The alternative pathway of NF-κB can be activated by members of TNF superfamily, such as lymphotoxin β (LTβ), tumor necrosis factor ligand superfamily member 5 (CD40L), B-cell activation factor (BAFF) and receptor activator of NF-κB ligand (RANKL). We have previously shown that alternative pathway of NF-κΒ is deteriorated in CRC. In the present study, we investigated the clinical significance of the aforementioned receptors in patients with CRC as well as their relation with effector transcription factors NF-κB2 and RELB. Methods: Gene expression of CD40, BAFFR, LT β R, RANK, NF- κ B2 and RELB, quantified by using real-time qRT-PCT and specific primers and probes, was assessed in 97 cancerous and 61 paired non-neoplastic tumor-adjacent formalin-fixed paraffin-embedded (FFPE) tissue specimens from CRC patients who were surgically managed in the University Hospital of Patras, Greece. Concurrently, we assessed protein expression of the same molecules by immunohistochemistry in the same cohort of patients. Both, mRNA and protein levels of the receptors were analyzed in association with clinicopathological parameters and clinical outcome of the patients in terms of time to disease progression. Results: Cytoplasmic expression of all molecules was higher in cancer compared to tumor-adjacent epithelial cells ( p= 0.047 for CD40, p= 0.007 for BAFFR, p&lt; 0.001 for LTβR and p= 0.003 for RANK) and in stromal cells ( p&lt; 0.001 for all molecules), while the reverse pattern (lower in neoplastic epithelial cells) was observed for nuclear expression ( p&lt; 0.001 for all molecules). Similarly, mRNA levels of LTβR and RANK were higher in cancer compared to adjacent non-neoplastic tissues ( p= 0.019 and p= 0.037, respectively). Notably, patients with high mRNA levels of all studied receptors had significantly longer time to disease progression ( p= 0.026 for CD40, p= 0.023 for BAFFR, p= 0.027 for LTβR and p= 0.038 for RANK). In addition, mRNA levels of BAFFR, LTβR and RANK levels were higher in ulcerative tumors compared to polypoid tumors ( p= 0.002, p= 0.005 and p= 0.011, respectively). RELB mRNA levels were positively correlated with mRNA levels of CD40, BAFFR and LTβR in cancerous ( p&lt; 0.001 for all molecules) and non-cancerous tissues (p &lt; 0.001 for all molecules). Surprisingly, NF-κB2 mRNA levels were positively correlated with BAFFR and LTβR mRNA levels in malignant tissues, while in non-malignant tissues were positively correlated with all receptors’ mRNA levels ( p&lt; 0.001 for all molecules). Conclusions: This study suggests a prognostic value for CD40, BAFFR, LTβR and RANK expression in patients with CRC and further supports the role of the alternative pathway of NF-κB in CRC.

Extracellular (EC) Mucin5AC (MUC5AC) detection after neoadjuvant therapy (NAT) and the risk of recurrence in resected pancreatic ductal adenocarcinoma (R-PDA).

4171 Background: The clinical significance of MUC5AC glycoforms is unclear to date. Our earlier study showed the association of lower mature (MM) and immature (IM) MUC5AC in R-PDA with pathological treatment response and EC detection of MM (EC-MM). Here, we have the significance of EC-MM detection in the tissue of R-PDA post-NAT. Methods: Formalin-fixed paraffin-embedded tissue blocks of R-PDA from January 2010 to June 2021 were obtained from the Ohio State University biorepository. Using monoclonal antibodies for 45M1 (MM) and CLH2 (IM), immunohistochemistry was performed to study the cellular expression of MM and IM, and H-scores were calculated. Also, the presence or absence of EC expression of MM is noted. progression-free survival (PFS) and overall survival (OS) were the primary endpoints. Univariate and multivariate Cox regression models were used to model PFS and OS, and clinical and pathological variables were adjusted in the multivariate models. We performed univariate logistic regression analysis to examine the impact of MUC5AC expression on pathological features in resected specimens. Results: Among 100 R-PDA patients, 43 received NAT and 57 had upfront surgery (UpS)). In NAT-group (36-FOLFIRINOX, 2-FOLFOX, and 5-gemcitabine/nab-paclitaxel), EC-MM detection in the resected specimens was associated with worse PFS on multivariate analysis (MVA) (Hazard Ratio (HR) 0.2, 95 % CI: 0.059-0.766, p = 0.01). In the FOLFIRINOX-treated group, the EC-MM detection had a similar impact (HR 0.2, 95 % CI: 0.052-0.847, p = 0.02) on PFS. Cellular MM and IM expression were significant (p &lt; 0.05) on MVA for OS in both groups, but the HRs were not clinically significant (NAT-group, MM - 0.9 &amp; IM - 1.04, FOLFIRNIOX-group, MM - 0.8 &amp; IM -1.02). Expression levels of cellular MM and IM in FOLFIRINOX group and MM alone in NAT-group significantly (p &lt; 0.05) correlated with incomplete resection (R1/R2 vs R0) and perineural invasion. In the UpS group, MUC5AC glycoforms did not significantly correlate with survival or pathological features. Conclusions: Post NAT EC-MM detection and cellular MUC5AC (MM and IM) expression levels are associated with worse PFS and pathological features. Their role in the treatment of naïve patients is still unclear. MUC5AC can be a clinically useful predictive biomarker. Future research should focus examining its importance in advanced tumors and manipulating it for therapeutic purposes.

Evaluation of an E6/E7 PCR-capillary electrophoresis fragment analysis in the genotyping of human papillomavirus in archival FFPE samples of oropharyngeal cancer.

Accumulating evidence has demonstrated that high-risk human papillomaviruses (HR-HPVs) are involved in the etiology of a subset of oropharyngeal squamous cell carcinoma (OPSCC). In this regard, the International Agency for Research on Cancer (IARC) has recommended direct molecular HPV testing. So far, there is no agreement on the most appropriate method for HPV detection on OPSCC formalin-fixed paraffin-embedded (FFPE) materials. In this study, we aimed to evaluate the performance of the high-sensitive SureX HPV assay in OPSCC FFPE tissues compared with LiPA-25 and p16ink4a immunostaining. A retrospective series of FFPE primary OPSCC cases were diagnosed between 2008 and 2019 and provided by the Henan Cancer Hospital, China. The level of agreement of two assays was determined using Cohen's Kappa (κ) statistics. A total of 230 FFPE OPSCC samples from tumor resections (n = 160) and diagnostic biopsies (n = 70) were detected. Sixty-six (28.7%) and 70 (30.4%) samples were identified as HPV-DNA-positive by LiPA-25 and SureX, respectively, of which HPV16 was largely the most common type (95.5% vs 94.3%). We found a perfect concordance between LiPA-25 and SureX for HPV-DNA status (κ = 0.906, 95% CI: 0.875-0.937) and for HPV16 (κ = 0.925, 95% CI: 0.897-0.953). In addition, SureX and p16ink4a immunostaining had a perfect concordance (κ = 0.917, 95% CI: 0.888-0.946). Moreover, the HPV-driven fraction, based on double positivity for HPV-DNA and p16ink4a, was similar between SureX (63 of 230, 27.4%) and LiPA-25 (60 of 230, 26.1%). Similar results were found in samples from resections and biopsies. SureX and LiPA-25 are comparable. SureX could be used for routine HPV-DNA detection and genotyping on archival OPSCC FFPE tissues.

Age-Related Clusters and Favorable Immune Phenotypes in Young Breast Cancer Patients

Breast cancer (BC) patients aged <40 years at diagnosis experience aggressive disease and poorer survival compared with women diagnosed with BC at 40 to 49 years, but the age-related biology is described to little extent. Here, we explored transcriptional alterations in BC to gain better understanding of age-related tumor biology. We studied a subset of the Bergen in-house cohort (n = 127; age range, 26-49 years) and used the NanoString Breast Cancer 360 expression panel on formalin-fixed paraffin-embedded BC tissue, and publicly available global BC messenger RNA expression data (n = 204; age range, 22-49 years), to explore differentially expressed genes between the young (age <40 years) and older (age 40-49 years) patients. Unsupervised hierarchical clustering was applied to identify gene expression–based patient clusters. We applied established computational approaches to define the PAM50 subtypes, risk of recurrence scores (ROR), and risk groups and to infer the proportions of 22 immune cell types from bulk gene expression profiles of patients aged <50 years at BC diagnosis. Differentially expressed genes and gene sets were investigated using OncoEnrichR and g:Profiler to describe functional profiles and pathway enrichment. We identified 4 age-related patient clusters presenting distinct characteristics of PAM50 subtypes and ROR profiles, which demonstrated independent prognostic value when adjusted for traditional clinicopathologic variables and the known molecular subtypes. Our findings showed better survival than expected in the basal-enriched cluster 2 and in triple-negative and basal-like BC. Deconvolution analyses of immunophenotypes indicated higher levels of M0 and M1 macrophages than M2 macrophages in subsets of young BC. Our approach identifies age-based patient clusters with distinct clinicopathologic profiles, to a large extent overlapping with the PAM50 subtypes, although with independent prognostic values in multivariate survival analyses. The patient clusters provided new insight in the immune cell distribution across tumor subtypes, potentially contributing to survival differences between the clusters and the molecular subtypes and indicating age-related mechanisms improving outcome. Our study confirms the applicability of ROR as a valid prognosticator also in a young BC cohort.

The diagnostic potentiality of the RNA aptamer against progesterone receptor isolated by crush and soak (CRUSOAK)-SELEX.

Aptamers are a class of molecular recognition elements that exhibit high binding affinity and specificity against their respective targets. In view of the many advantages aptamers harbor over their counterpart antibodies, we were impelled to isolate an RNA aptamer against progesterone receptor, particularly its DNA binding domain. A total of eight SELEX cycles were executed against the recombinant Progesterone Receptor DNA-binding domain (PR DBD). The RNA-protein complex in the gel shift assay was subjected to crush and soak method to elute the binders prior to conventional sequencing, the step of which was based upon to coin the term CRUSOAK-SELEX. The sequencing revealed three different classes of sequences, with one class termed, PRapt-3, showing the strongest binding against PR DBD. The dissociation constant of PRapt-3 RNA aptamer was estimated at 380nM ± 35nM. PRapt-3 was successfully used to develop aptamer-based diagnostic assays such as ELASA, aptamer-based dot blot, and aptamer-based western blot. The prominent highlight is the performance of the aptamer in aptacytostaining, which was unachievable with antibodies. Compared to its counterpart antibodies, PRapt-3 has a better penetration capacity in aptahistostaining using the formalin-fixed paraffin-embedded (FFPE) breast cancer cells and tissue blocks. This study represents the first ever demonstration of an aptamer against progesterone receptor and its diagnostic capacity.

Combining RNAscope, Immunohistochemistry (IHC) and Digital Image Analysis to Assess Podoplanin (PDPN) Protein and PDPN_mRNA Expression on Formalin-Fixed Paraffin-Embedded Normal Human Placenta Tissues.

The expression and function of podoplanin (PDPN) in the normal human placenta has been debated in placental evaluation. This study emphasizes the importance of a multimodal approach of PDPN expression in normal human placentas. A complete examination is performed using immunohistochemistry, RNAscope and automated Digital Image examination (DIA) interpretation. QuPath DIA-based analysis automatically generated the stromal and histological scores of PDPN expression for immunohistochemistry and RNAscope stains. The umbilical cord's isolated fibroblasts and luminal structures expressed PDPN protein and PDPN_mRNA. RNAscope detected PDPN_mRNA upregulation in syncytial placental knots trophoblastic cells, but immunohistochemistry did not certify this at the protein level. The study found a significant correlation between the IHC and RNAscope H-Score (p = 0.033) and Allred Score (p = 0.05). A successful multimodal strategy for PDPN assessment in human placentas confirmed PDPN expression heterogeneity in the full-term human normal placenta and umbilical cord at the protein and mRNA level. In placental syncytial knots trophoblastic cells, PDPN showed mRNA overexpression, suggesting a potential role in placenta maturation.

Expression of the Folate Receptor Proteins FOLR1 and FOLR2 in Correlation With Clinicopathological Variables of Gastric Cancer.

Gastric cancer (GC) remains a leading cause of cancer-related mortality worldwide, owing to its aggressive nature and poor prognosis. The role of folate receptors, particularly folate receptor 1 (FOLR1) and folate receptor 2 (FOLR2), in cancer has been increasingly recognized due to their overexpression in various malignancies including gastric cancer, and its potential implications in cancer progression, treatment resistance and as therapeutic targets. To evaluate the expression patterns of FOLR1 and FOLR2 in GC patients' tissue and blood specimens and to correlate these patterns with clinicopathological variables. A total of 58 gastric cancer patients were enrolled at the Regional Cancer Centre (RCC) from March 2017 to March 2020. Immunohistochemical analysis was performed to examine the expression of FOLR1 and FOLR2 in formalin-fixed paraffin-embedded (FFPE) tissue samples. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyze FOLR1 and FOLR2 expression in blood samples. Statistical analyses were conducted using chi-square tests, independent T-tests, and Kaplan-Meier survival analysis. FOLR1 and FOLR2 were overexpressed in 82.76% and 70.69% of gastric cancer tissues, respectively. High expression levels of FOLR1 were significantly associated with the diffuse type of gastric cancer (p<0.005). qRT-PCR showed significant overexpression of FOLR1 in gastric cancer blood samples compared to control samples, with a median fold change of approximately 14.18 times. Conversely, FOLR2 was significantly underexpressed in gastric cancer samples, with a fold change of 0.30. However, no significant correlation was found between FOLR2 expression and the clinicopathological features. The overall survival analysis did not show a significant difference in survival rates based on the expression levels of FOLR1 and FOLR2. This study highlights the differential expression patterns of FOLR1 and FOLR2 in gastric cancer and underscores the complexity of their roles in cancer biology. While FOLR1 shows potential as a biomarker for gastric cancer due to its overexpression, further studies are needed to fully elucidate the therapeutic and prognostic implications of folate receptors in gastric cancer.

Multiplexed in situ RNA imaging by combFISH.

Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.

Abstract A007: Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is encoded by the ERBB2 gene, and frequently mutated, amplified, and/or overexpressed in urothelial cancer (UC). Promising antibody-drug conjugates have led to new interest in HER2 as a UC clinical target. Patient selection for HER2-targeted therapy typically relies on immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of tumor tissue, however DNA sequencing can identify ERBB2 genomic alterations. Accurate biomarker-driven patient selection will be critical to optimize the clinical benefit of HER2-targeted therapy. Therefore, we evaluated plasma circulating tumor DNA (ctDNA) for ERBB2/HER2 status determination in UC, as compared to IHC and FISH of metachronous tissue. 411 plasma samples from 236 metastatic UC patients were profiled with targeted sequencing across &amp;gt;50 genes frequently altered in UC, including dense coverage flanking the ERBB2 locus. Using an established bioinformatics workflow, we determined the presence of ERBB2 alterations (mutations, amplifications, and/or structural variants) in the 181 patients with evidence of ctDNA in ≥1 sample. Clinical records were reviewed for the availability of archival tumor tissue; in total, 81 formalin-fixed paraffin-embedded tissue specimens were retrieved from 23 patients with ctDNA ERBB2 alterations and 20 ERBB2-wildtype controls. HER2 IHC was performed with a polyclonal rabbit anti-human Her2 antibody (Dako) and scored according to gastric cancer guidelines. HER2 FISH was performed with the PathVysion HER-2 DNA Probe Kit. Protein-altering ERBB2 mutations were identified in 14% of evaluable patients, with two-thirds at known oncogenic hotspots. ERBB2 copy gain was detected in 8% of patients overall, 9% when excluding low tumor fraction samples. IHC results were assessable for 82 tissue samples from 43 patients, and 33 patients had at least one sample with positive HER2 staining (2+/3+). IHC scores varied in 16/23 patients with ≥2 tissue samples, with the variation leading to a change in classification (HER2-negative [0/1+] versus positive [2+/3+]) in half (8/16). In two patients with mixed variant histology, the urothelial component showed IHC 3+ while the scores were 2+ (plasmacytoid) and 0 (squamous) in the variant regions. Frequent focal staining patterns were observed with both IHC and FISH. ERBB2 alterations in ctDNA were correlated with IHC positivity – when considering the most recent tissue sample per patient, 79% of ctDNA ERBB2-altered patient’s tumors were also positive by IHC. Conversely, 55% of ctDNA ERBB2-wildtype cases were positive by IHC. FISH results were available from 24 samples (20 positive, 4 negative), all from patients with ctDNA ERBB2 amplification. 18/20 samples with FISH positivity were HER2 IHC 2+/3+; the remaining two samples were IHC 1+. Our results demonstrate significant spatial and/or temporal heterogeneity in ERBB2/HER2 status, with implications for the rational implementation of biomarker-directed (HER2-targeted) therapy in UC. Citation Format: Gillian Vandekerkhove, Andrew J. Murtha, David C. Müller, Kimia Rostin, Carlos Vasquez-Rios, Jussi Nikkola, Maria Stephenson, Emily Fung, Jaskirat Atwal, Karan Parekh, Cecily Q. Bernales, Gráinne Donnellan, Gang Wang, Tilman Todenhöfer, Piet Ost, Peter C. Black, Kim N. Chi, Bernhard J. Eigl, Alexander W. Wyatt. Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A007.

Differential Epigenetic Regulation in Uninfected and Tuberculosis-Human Immunodeficiency Virus Co-Infected Patients.

This study aimed to compare the degree of epigenetic modifications between a TB-HIV co-infected cohort and uninfected subjects. Formalin-fixed paraffin-embedded (FFPE) tissues were retrieved from 45 TB-HIV co-infected and 45 control individuals. Real-time PCR was applied to compare the level of expression of genes involved in epigenetic regulation. The protein multiplex assay was used to assess the degree of protein modification. DNA sequencing was used to determine the evolutionary relationships between the infecting HIV and Mtb strains. Our results indicated a significant increase in the expression of the five candidate genes in the patients with TB-HIV relative to the control cohort. A sharp increase in the degree of histone methylation, acetylation and phosphorylation was observed in TB-HIV co-infected patients. The phylogenetic analysis classified the strains into three distinct HIV clusters and five Mtb clusters. The disparities in the expression profiles of our candidate genes between the TB-HIV cohort and non-TB-HIV group highlights the important role played by various TB and HIV strains in regulating the host gene expression landscape.

Correlation of High-Grade Osteosarcoma Response to Chemotherapy with Enhanced Tissue Immunological Response: Analysis of CD95R, IFN-γ, Catalase, Hsp70, and VEGF

High-grade osteosarcoma, a primary malignant bone tumour, is experiencing a global increase in reported incidence with varied prevalence. Despite advances in management, which include surgery and neoadjuvant chemotherapy often an unsatisfactory outcome is found due to poor or heterogeneous response to chemotherapy. Our study delved into chemotherapy responses in osteosarcoma patients and associated molecular expressions, focusing on CD95 receptor (CD95R), interferon (IFN)-γ, catalase, heat-shock protein (Hsp)70, and vascular endothelial growth factor (VEGF). Employing immunohistochemistry and Huvos grading of post-chemo specimens, we analysed formalin-fixed paraffin-embedded (FFPE) osteosarcoma tissue of resected post-chemotherapy specimens from Dr. Soetomo General Academic Hospital in Surabaya, Indonesia (DSGAH), spanning from 2016 to 2020. Results revealed varied responses (poor 40.38%, moderate 48.08%, good 11.54%) and distinct patterns in CD95R, IFN-γ, catalase, Hsp70, and VEGF expression. Significant differences among response groups were observed in CD95R and IFN-γ expression in tumour-infiltrating lymphocytes. The trend of diminishing CD95R expression from poor to good responses, accompanied by an increase in IFN-γ, implied a reduction in the count of viable osteosarcoma cells with the progression of Huvos grading. Catalase expression in osteosarcoma cells was consistently elevated in the poor response group, while Hsp70 expression was highest. VEGF expression in macrophages was significantly higher in the good response group. In conclusion, this study enhances our understanding of immune-chemotherapy interactions in osteosarcoma and identifies potential biomarkers for targeted interventions.

Investigating somatic variants and pathways in mismatch repair-deficient (dMMR) colorectal carcinoma in South Africa

AimsColorectal carcinoma (CRC) is a common cause of morbidity and mortality worldwide, and an emerging public health problem in sub-Saharan Africa. Several authors have described an increased frequency of mismatch...

The Expression of Ki-67 and E-cadherin in Triple Negative Breast Cancer: A Hospital Based Study

Introduction: Triple negative breast cancer (TNBC) is a subtype of breast cancer with aggressive nature as it does not respond to targeted chemotherapeutics. Expression of biomarkers is considered as an important factor for selecting treatment strategies and assessing prognosis. Objective: The aim of this study was to assess the expression of Ki-67 and E–cadherin in TNBC tissue. Methods: Immunohistochemical staining was done to assess the expression of proliferative marker Ki-67 and junctional complex marker E-cadherin in the Formalin Fixed Paraffin Embedded tissue (FFPE) of 80 TNBC patients. Ki-67 positive cells were identified on the basis of brown staining nuclei and E-cadherin expressed cells were considered positive on the basis of brown staining cell membrane. Ki-67 positive TNBCs were divided into low and high Ki-67 groups. Results: Ki-67 was expressed in 65% of the TNBC, the median of Ki-67 index was 30.25%. The difference between the medians of Ki-67 expression of low and high groups (7.5% and 53.25% respectively) was highly significant (p = 0.000). E-cadherin was negative in 67.5% of TNBC patients. The mean (±SD) age at diagnosis was 43.79 (±9.10) years. Most of the cancers were invasive ductal cell carcinoma (95%) and of grade II (70%). Lymph node metastasis was present in 73.8% patients and in 51% cases the cancer was in the left breast. Ki-67 expression was significantly associated with lymph node metastasis and E-cadherin expression was more negative in the left sided cancer. Conclusion: The study signifies that the TNBC has an early age at onset and Ki-67 expression is significantly high and associated with lymph node metastasis pointing the importance of this biomarker in TNBC patients. Negative expression of E-cadherin suggests close monitoring of the left sided TNBC.