Paraffin-embedded Sections Research Articles

Novel miR-490-3p/hnRNPA1-b/PKM2 axis mediates the Warburg effect and proliferation of colon cancer cells via the PI3K/AKT pathway

BACKGROUND Heterogeneous ribonucleoprotein A1 (hnRNPA1) has been reported to enhance the Warburg effect and promote colon cancer (CC) cell proliferation, but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated. AIM To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway. METHODS Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b. The relationship between the expression values and the clinicopathological features of the patients was investigated. Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction, while differences in protein expression were analyzed using western blot. Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2’-deoxyuridine assays, and cell cycle and apoptosis were detected using flow cytometric assays. The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay. The Warburg effect was evaluated by glucose uptake and lactic acid production assays. RESULTS The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls (P < 0.05). Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC, including stage I, II-III, and IV. Furthermore, the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification. HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway, thereby promoting proliferation of HCT116 and SW620 cells. However, the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b, effectively blocking the Warburg effect. CONCLUSION These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.

Exploring the Role of EZH2 and BCL2 in Demarcating Oral Verrucous Hyperplasia and Verrucous Carcinoma

Introduction Oral verrucous hyperplasia (OVH) and verrucous carcinoma (OVC) are precursors of oral squamous cell carcinoma exhibiting overlapping histopathological picture which warrants distinction. EZH2 is an epigenetic marker possessing multifaceted function in cellular proliferation, migration, and malignant transformation, whereas BCL2 is an integral part of the antiapoptotic mechanism regulating cellular homeostasis. Aim The aim was to distinguish OVH and OVC by analysis of immunohistochemical expression of EZH2 and BCL2. Material and Methods The study sample consisted of 79 formalin-fixed paraffin-embedded tissue sections of normal oral mucosa (10), OVH (10), oral OVC (27), and oral squamous cell carcinoma (32). Immunohistochemical analysis of EZH2 and BCL2 was done and labeling indices were calculated. Additionally, six histopathological parameters were assessed in OVH and OVC. Statistical analysis was done using Kruskal–Wallis test, Tukey honest significant difference test, and Spearman's correlation. Receiver operating characteristic curve was plotted and sensitivity, specificity, and cutoff score of each marker were calculated. Result and Discussion Labeling indices of EZH2 and BCL2 depicted a gradual incline from normal mucosa to oral squamous cell carcinoma. Significant difference of EZH2 and nonsignificant difference in BCL2 expression between OVH and OVC were noted. Out of the six histopathological parameters, keratin plugging, juxtaepithelial lymphocytic response, and frank endophytic growth yielded a significant difference. EZH2 serves as a superior marker than BCL2 to differentiate OVH and OVC. Juxtaepithelial lymphocytic response can also serve as a histopathological parameter in distinguishing OVH and OVC.

Abstract PO4-16-01: EZH2 is Overexpressed in Invasive Carcinoma and DCIS in both BRCA1 and BRCA2 Mutation Carriers

Abstract Background The transcriptional repressor EZH2 (Polycomb Group Protein Enhancer of Zeste Homologue 2) is overexpressed in multiple neoplasms including breast cancer where it associates with high proliferation, aggressive features and poor prognosis and is of interest as a potential therapeutic target. EZH2 nuclear expression in breast cancer has been shown to correlate specifically with high tumor grade and basal-like histologic features but has not been well studied in cancers from BRCA carriers. Here we evaluate the expression of EZH2 protein in BRCA-associated invasive breast carcinomas and precursor lesions. Methods Eighteen cases of invasive breast cancer in consented BRCA carriers (8 BRCA1 and 10 BRCA2) treated at Dana Farber Cancer Institute from 2001-2019 were reviewed by two pathologists (TAD and RBPP). Histopathologic features (histologic subtype, nuclear grade, mitotic score) and presence of precursor lesions were recorded. Immunohistochemistry for EZH2 was performed on freshly cut formalin-fixed paraffin-embedded tissue sections (1-3 sections/case) using a monoclonal antibody against EZH2 (D2C9, Cell Signaling). Nuclear EZH2 expression was scored as negative (score=1, no staining); weak (score 2, < 25%); moderate (score=3, 25–75%); and strong (score=4, >75%), of any intensity following published criteria. EZH2 overexpression was defined as scores 3 and 4. Results The majority of the invasive carcinomas showed grade III histology (13/18, 72%), as expected. Of 8 BRCA1 cases, 6 were classified as grade III (75%), 1 as grade II, and 1 as grade I (7 cases of invasive ductal carcinoma and 1 case of invasive tubular carcinoma). Of 10 BRCA2 cases, 7 were classified as grade III (70%) and 3 as grade II (30%) (5 cases of invasive ductal carcinoma and 5 cases of invasive carcinoma with ductal and lobular features). The majority of cases (13 of 18, 72%) showed high expression of EZH2 in the invasive carcinoma (6 BRCA1 and 7 BRCA2 cases; 2 grade II and 11 grade III). Of 13 cases with EZH2 overexpression, 6 were ER+/PR+/HER2- and 7 were triple negative, with 9 invasive ductal carcinomas and 4 invasive carcinomas with ductal and lobular features. Of 5 cases with no/low EZH2 overexpression, 1 was ER+/PR+/HER2-, 3 were ER+/PR-/HER2- and 1 was triple negative, with 3 invasive ductal carcinomas, 1 invasive carcinoma with ductal and lobular features and 1 invasive tubular carcinoma. In all cases with adjacent ductal carcinoma in situ (DCIS) (n=14), the pattern of EZH2 expression in the DCIS was identical to that seen in the invasive carcinoma (11 high and 3 low expression). Overexpression of EZH2 was not observed in any other precursor lesions (LCIS, ADH, ALH) or in normal tissue. Conclusion EZH2 protein overexpression is seen in the majority of invasive carcinomas in BRCA carriers, both in BRCA1 and in BRCA2. In all cases, the associated DCIS shows the same pattern of expression as the invasive carcinoma, suggesting that overexpression occurs early in tumorigenesis, while tumor cells are still confined within the ducts. The high rate of overexpression of EZH2 in BRCA1- and BRCA2-associated tumors suggests a potential role for EZH2 as a target and/or a biomarker in these cancers. Citation Format: Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Rodrigo Fonseca Abreu, Ashka Patel, Krishan Taneja, McKenna Moore, Celina Kleer, Deborah Dillon. EZH2 is Overexpressed in Invasive Carcinoma and DCIS in both BRCA1 and BRCA2 Mutation Carriers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-16-01.

Abstract PO3-08-08: Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers

Abstract Background Single-cell studies of normal breast tissue in BRCA mutation carriers have led to the identification of a candidate cancer precursor cell, the basal-luminal (BL) cell. The localization of BL cells within normal, pre-malignant and malignant breast tissue architecture is not well characterized. Here, using cyclic immunofluorescence (CyCIF), we quantify and localize BL cells within normal and malignant breast tissues from BRCA1 and BRCA2 carriers. Methods Formalin-fixed paraffin-embedded breast tissue sections from 9 BRCA1 and 9 BRCA2 consented patients treated at Dana Farber Cancer Institute (2001-2019) were retrieved from the clinical archives and subjected to CyCIF for luminal and basal markers including CK5, CK14 and CK19. Using one section/case, cells co-expressing basal and luminal cytokeratins (CK14/CK19 and/or CK5/CK19) were visually identified in invasive carcinoma, ductal carcinoma in situ (DCIS) and in adjacent normal breast tissue. In histologically normal tissue, regions of interest (ROIs) with co-expressing cells were classified as terminal duct lobular units (TDLUs) or isolated ducts. Within each ROI, the quantity of BL cells was scored as low (1-5 cells), moderate (6-10 cells), or high ( >10 cells). Results The 9 BRCA1 tumors included 8 invasive ductal carcinomas/no special type and 1 tubular carcinoma. The 9 BRCA2 tumors included 4 invasive ductal carcinomas/no special type and 5 invasive carcinomas with ductal and lobular features. 496 ROIs were evaluated in histologically normal tissue: 297 from BRCA1 cases and 199 from BRCA2 cases. BL cells co-expressing CK14/CK19 and/or CK5/CK19 were identified in normal TDLUs and isolated ducts in both BRCA1 and BRCA2 cases. BL cells were more frequently identified in BRCA2 cases, with at least one BL cell present in 97/297 ROIs (33%) in BRCA1 cases and in 133/199 ROIs (67%) in BRCA2 cases (p< 0.01). 55% of BRCA2 ROIs had >10 BL cells each versus 42% of BRCA1 ROIs; however, this difference was not significant. Four BRCA1 cases showed a diffuse BL cell phenotype within the invasive carcinoma with either a diffuse or scattered BL cell phenotype within the DCIS. The BL cell phenotype was not seen diffusely in invasive carcinoma or DCIS in the other 14 cases; however individual tumor cells or small cell clusters with the BL cell phenotype were occasionally found. Conclusions BL cells are found in histologically normal TDLUs and isolated ducts in breast tissue from BRCA1 and BRCA2 carriers, with greater prevalence in BRCA2 carriers. The BL cell phenotype is present diffusely within tumor cells in almost half of BRCA1-associated cancers and none of the BRCA2-associated cancers in this small cohort. Further study of this interesting cell population is warranted in efforts to understand early changes in epithelium at high risk for the development of cancer. Citation Format: Rodrigo Fonseca Abreu, Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Ashka Patel, Krishan Taneja, McKenna Moore, Alicia Pollaci, Judy Garber, Nomeda Girnius, Deborah Dillon. Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-08-08.

Abstract PO3-01-05: Enhancing HER2 Evaluation: Correlation between APIS Breast Cancer Subtyping Kit and IHC/ISH for Accurate HER2 Quantification

Abstract Background: The quantification of HER2-low in breast cancer (BC) has garnered considerable interest due to emerging evidence of the efficacy of targeted therapies in this patient subgroup. Trastuzumab deruxtecan (T-Dxd), an antibody-drug conjugate targeted at HER2, has recently gained approval in the USA and Europe for treating HER2-low BC, which is currently defined as immunohistochemical (IHC) scores of 1+ or 2+ without HER2/ERBB2 in situ hybridization (ISH) amplification. HER2 quantification using IHC/ISH was primarily designed to identify tumors overexpressing HER2, rather than differentiate between HER2-low and the absence of expression. Consequently, the adequacy of these assays in accurately detecting HER2- low remains uncertain. Improving the accuracy of HER2 evaluation is crucial to prevent the incorrect selection of patients for T-Dxd treatment. There is a clear need to develop HER2 testing methods that are more precise, reliable, and capable of detecting HER2-low expression with greater sensitivity and reproducibility. This is particularly important as the minimum threshold of HER2 expression required for T-Dxd efficacy is still under investigation, with clinical data suggesting the potential activity of T-Dxd even in patients with IHC 0 HER2 score. Here, we evaluated the correlation between HER2 mRNA expression levels, detected by APIS Breast Cancer Subtyping Kit, and IHC HER2 classification. Methods: Formalin-fixed paraffin-embedded (FFPE) tumour tissue sections (n=642) obtained by core needle biopsy or resection underwent histological analysis in accordance with laboratory’s standard of care methods. Samples with a HER2 score of 2+ were referred to ISH to determine ERBB2 amplification. To evaluate the diagnostic performance, the concordance between HER2 IHC score, and APIS Breast Cancer Subtyping Kit RNA expression level was reported in terms of Overall Percent Agreement (OPA), Negative Percent Agreement (NPA), and Positive Percent Agreement (PPA), along with their corresponding 95% confidence intervals (CI). Results: HER2 expression detected by APIS Breast Cancer Subtyping Kit showed strong correlation with IHC/ISH, with an OPA of 94.2% (95% CI: 92.2-95.8), PPA of 89.2% (95% CI: 80.1-94.4) and NPA of 94.9% (95% CI: 92.8-96.4). The expression of HER2 was detected by APIS Breast Cancer Subtyping Kit in a subset of patients with 0 and 1+ IHC HER2 scores, suggesting its potential as a more sensitive and reliable method for detection of HER2 expression, and providing the opportunity to enhance HER2 stratification. Conclusions: APIS Breast Cancer Subtyping Kit can accurately detect HER2 expression and has the potential to further stratify the HER2-low patients. Further studies examining the relationship between HER2 expression and the response to anti-HER2 therapies could yield valuable insights into treatment administration and identify patients who could benefit from such therapies. Incorporating continuous quantification of HER2 could optimize patient outcomes. Citation Format: Anna Gasior, Joanna Gorniak, Sara Rollinson, Leanne Gough, Anne-Sophie Wegscheider, Axel Niendorf. Enhancing HER2 Evaluation: Correlation between APIS Breast Cancer Subtyping Kit and IHC/ISH for Accurate HER2 Quantification [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-01-05.

Exploration of identifying individual tumor tissue based on probabilistic model.

Variations in the tumor genome can result in allelic changes compared to the reference profile of its homogenous body source on genetic markers. This brings a challenge to source identification of tumor samples, such as clinically collected pathological paraffin-embedded tissue and sections. In this study, a probabilistic model was developed for calculating likelihood ratio (LR) to tackle this issue, which utilizes short tandem repeat (STR) genotyping data. The core of the model is to consider tumor tissue as a mixture of normal and tumor cells and introduce the incidence of STR variants (φ) and the percentage of normal cells (Mxn) as a priori parameters when performing calculations. The relationship between LR values and φ or Mxn was also investigated. Analysis of tumor samples and reference blood samples from 17 colorectal cancer patients showed that all samples had Log 10(LR) values greater than 1014. In the non-contributor test, 99.9% of the quartiles had Log 10(LR) values less than 0. When the defense's hypothesis took into account the possibility that the tumor samples came from the patient's relatives, LR greater than 0 was still obtained. Furthermore, this study revealed that LR values increased with decreasing φ and increasing Mxn. Finally, LR interval value was provided for each tumor sample by considering the confidence interval of Mxn. The probabilistic model proposed in this paper could deal with the possibility of tumor allele variability and offers an evaluation of the strength of evidence for determining tumor origin in clinical practice and forensic identification.

Feasibility of intraoperative pathologic examination for sentinel lymph nodes during sentinel node navigation surgery in early gastric cancer: results of pathologic protocol for SENORITA trial.

During sentinel node navigation surgery in patients with gastric cancer, intraoperative pathologic examination of sentinel nodes is crucial in determining the extent of surgery. In this study, we evaluated the feasibility and accuracy of intraoperative pathologic protocols using data from a prospective, multicenter, randomized trial. A retrospective analysis was conducted using data from the SEntinel Node ORIented Tailored Approach trials from 2013 to 2016. All sentinel lymph nodes were evaluated during surgery with hematoxylin-eosin (HE) staining using a representative section at the largest plane for lymph nodes. For permanent histologic evaluation, sentinel basin nodes were stained with HE and cytokeratin immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) sections and examined with HE for three deeper-step sections at 200-μm intervals. The failure rate of identification by frozen section and the metastasis rate in non-sentinel basins were investigated. Of the 237 patients who underwent sentinel node basin dissection, 30 had lymph node metastases on permanent pathology. Thirteen patients had macrometastasis confirmed in frozen sections as well as FFPE sections (failure rate: 0%). Patients with negative sentinel nodes in frozen sections but micrometastasis in FFPE sections had no lymph node recurrence during the follow-up period (0%, 0/6). However, in cases with tumor-positive nodes in frozen sections, metastases in non-sentinel basins were detected in the paraffin blocks (8.3%, 2/24). The single-section HE staining method is sufficient for detecting macrometastasis via intraoperative pathological examination. If a negative frozen-section result is confirmed, sentinel basin dissection can be performed safely. Otherwise, standard surgery is required.

Clinical Relevance of Pathological Diagnosis of Hirschsprung's Disease with Acetylcholine-Esterase Histochemistry or Calretinin Immunohistochemistry.

Hirschsprung disease (HD) manifests as a developmental anomaly affecting the enteric nervous system, where there is an absence of ganglion cells in the lower part of the intestine. This deficiency leads to functional blockages within the intestines. HD is usually confirmed or ruled out through rectal biopsy. The identification of any ganglion cells through hematoxylin and eosin (H&E) staining rules out HD. If ganglion cells are absent, further staining with acetylcholine-esterase (AChE) histochemistry or calretinin immunohistochemistry (IHC) forms part of the standard procedure for determining a diagnosis of HD. In 2017, our Institute of Pathology at University Hospital of Heidelberg changed our HD diagnostic procedure from AChE histochemistry to calretinin IHC. In this paper, we report the impact of the diagnostic procedure change on surgical HD therapy procedures and on the clinical outcome of HD patients. We conducted a retrospective review of the diagnostic procedures, clinical data, and postoperative progress of 29 patients who underwent surgical treatment for HD in the Department of Pediatric Surgery, University of Heidelberg, between 2012 and 2021. The patient sample was divided into two groups, each covering a treatment period of 5 years. In 2012-2016, HD diagnosis was performed exclusively using AChE histochemistry (AChE group, n = 17). In 2017-2021, HD diagnosis was performed exclusively using calretinin IHC (CR group, n = 12). There were no significant differences between the groups in sex distribution, weeks of gestation, birth weight, length of the aganglionic segment, or associated congenital anomalies. Almost half of the children in the AChE group, twice as many as in the CR group, required an enterostomy before transanal endorectal pull-through procedure (TERPT). In the AChE group, 4 patients (23.5%) required repeat bowel sampling to confirm the diagnosis. Compared to the AChE group, more children in the CR group suffered from constipation post TERPT. Elevated AChE expression is linked to hypertrophied extrinsic cholinergic nerve fibers in the aganglionic segment in the majority of patients with HD. The manifestation of increased AChE expression develops over time. Therefore, in neonatal patients with HD, especially those in the first 3 weeks of life, an increase in AChE reaction is not detected. Calretinin IHC reliably identifies the presence or absence of ganglion cells and offers multiple benefits over AChE histochemistry. These include the ability to perform the test on paraffin-embedded tissue sections, a straightforward staining pattern, a clear binary interpretation (negative or positive), cost-effectiveness, and utility regardless of patient age. The ability of calretinin IHC to diagnose HD early and time-independently prevented repeated intestinal biopsies in our patient population and allowed us to perform a one-stage TERPT in the first months of life, reducing the number of enterostomies and restoring colonic continuity early. Patients undergoing transanal pull-through under the age of 3 months require a close follow-up to detect cases with bowel movement problems.

Immunohistochemical and molecular profiles of heterogeneous components of metaplastic breast cancer: a squamous cell carcinomatous component was distinct from a spindle cell carcinomatous component

Metaplastic breast carcinoma (MBC), a category of breast cancer, includes different histological types, which are occasionally mixed and heterogeneous. Considering the heterogeneity of cancer cells in a tumour mass has become highly significant, not only from a biological aspect but also for clinical management of recurrence. This study aimed to analyse the immunohistochemical and molecular profiles of each MBC component of a tumour mass. Twenty-five MBC tumours were histologically evaluated, and the most frequent MBC component (c) was squamous cell carcinoma (SCC), followed by spindle cell carcinoma (SpCC). A total of 69 components of MBC and non-MBC in formalin-fixed paraffin-embedded sections were examined for 7 markers by immunohistochemistry. SCC(c) were significantly PTEN negative and CK14 positive, and SpCC(c) were significantly E-cadherin negative and vimentin positive. Multivariate analyses revealed that immunohistochemical profiles of normal/intraductal (IC)(c), no special type (NST)(c), and MBC(c) differed; moreover, SCC(c) and SpCC(c) were distinctly grouped. PTEN gene mutation was detected only in SCC(c) (2/7), but not in SpCC(c). Next-generation sequence analyses for 2 cases with tumours containing SCC(c) demonstrated that PTEN gene mutation increased progressively from IC(c) to NST(c) to SCC(c). In conclusion, the immunohistochemical and molecular profiles of the SCC(c) of MBC are distinct from those of the SpCC(c).

Modification of the Hi-C Technology for Molecular Genetic Analysis of Formalin-Fixed Paraffin-Embedded Sections of Tumor Tissues

Modification of the Hi-C Technology for Molecular Genetic Analysis of Formalin-Fixed Paraffin-Embedded Sections of Tumor Tissues

Using the DNA Integrity Number (DIN) to analyze DNA quality in specimens collected from liquid-based cytology after fine needle aspiration of breast tumors and lesions.

Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded (FFPE) sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA Integrity Number (DIN) score analysis. Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

Clinical impact of IDH1 mutations and MGMT methylation in adult glioblastoma

BackgroundImpact of Isocitrate dehydrogenase1 (IDH1) and O6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma (GBM) have been of great interest due to their implications in prediction of prognosis of several types of cancer. It was aimed to investigate the clinical role of IDH1 mutation and MGMT methylation pattern among GBM patients versus non-neuro-oncological diseases (NND) patients and their impact on survival criteria.MethodsFormalin-fixed paraffin-embedded (FFPE) tissue sections of 58 GBM and 20 non-neuro-oncological diseases patients were recruited and IDH1 mutation and MGMT methylation was detected using Cast-PCR technology and Methyl II quantitative PCR approach, respectively. Results were assessed with other clinicopathological criteria and survival patterns.ResultsIDH1 mutation was detected among 15 GBM cases (15/58) and it was not reported among NND (P = 0.011). Receiver operating characteristic (ROC) curve was plotted to discriminate between MGMT methylation among studied groups. Patients with MGMT methylation ≥ 66% were reported as high methylation, which was recorded significantly in 51.7% and 100% of GBM cases and NND, respectively. Both showed significant difference with performance status, while MGMT methylation was significantly related with tumor size and tumor location. IDH1 mutation and MGMT methylation reported significant increase with GB patients revealed complete response to treatment. Survival pattern was better for IDH1 mutation and MGMT high methylation as compared to IDH1 wild type or MGMT low–moderate methylation, respectively, and favorable survival was detected when both were combined than using either of them alone.ConclusionDetection of IDH1 mutation and MGMT methylation among GB patients could aid in prediction of their response to treatment and their survival patterns, and their combination is better than using any of them alone.

Abstract 5176: TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 12% 5-year survival rate (SEER) due to a lack of early detection biomarkers and resistance to standard therapeutic options (surgery, chemotherapy, radiation). Black African Americans (BAA) have 20% increased incidence of PDAC compared to those with European Ancestry (EA). 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. Recent studies have demonstrated that immune checkpoint receptor expression (PD-1 and TIGIT) on specific T cell populations correlates to worse overall survival (OS). TIGIT inhibitors are being explored in clinical trials in pancreatic cancer due to implications of its ligand (CD155) promoting immune evasion. We hypothesize that targeted anti-TIGIT therapy, in conjunction with other therapies targeting the tumor microenvironment, could reverse the immune suppression that is characteristic of PDAC. We performed RNAscope in situ hybridization (ISH) with a probe specific for human TIGIT mRNA (combined with a nuclear counterstain) on 79 tissue samples. The cohort of tissue samples included 8 biopsies (endoscopic-guided fine needle biopsies at time of diagnosis), 66 primary (from surgical resection), and 5 metastatic (liver core biopsies from patients with a primary PDAC diagnosis) formalin-fixed paraffin-embedded (FFPE) tissue sections from patients with histologically confirmed PDAC. After cells were segmented based on nuclear recognition, the presence of TIGIT probe within the cytoplasm of each cell was determined and quantified. Percent positive cell values were then exported for statistical analysis in R. De-identified clinical metadata was obtained from REDCap, a cloud-based HIPAA-compliant database used to compile patient data for research purposes. We tested for associations between %TIGIT present and clinical covariates, using linear regression for continuous outcomes, and logistic regression for binary outcomes. ScRNAseq revealed that TIGIT mRNA is enriched in, but not exclusive to the T/NK cellular compartments in PDAC. Staining analysis showed that TIGIT expression did not differ significantly between racial groups (comparing BAA to non-BAA). The mean percentage of TIGIT positive cells was 64.0%. High expression of TIGIT was associated with clinical stage, where an increase in stage was associated with increasing %TIGIT (p < 0.05).The TIGIT biomarker assay can be conducted at time of diagnosis, time of surgical resection, and time of metastatic biopsy. If patients’ samples contain high levels of TIGIT expression, these patients may be candidates for anti-TIGIT drug therapy. Considering that TIGIT expression correlates with advancing clinical stage, patients with more advanced staging at diagnosis (stage IIB, III, IV) especially may benefit from anti-TIGIT therapy. Citation Format: Madison George, Julie Clark, Kendyll Gartrelle, Georges Nassif, Kailee Hartway, Daniel Long, Daniel Salas-Escabillas, Allison Wombwell, Thais Pichardo, Hui-Ju Wen, Simone Benitz, Samuel Zwernik, Rupen Shah, Hakmin Park, Philip, Gazala Khan, Howard Crawford, David Kwon, Brian Theisen, Nina Steele. TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5176.

Abstract 5354: High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach

Abstract The immune checkpoint involving PD-1 and PD-L1 generates suppressive signals in T cells, which help to prevent autoimmunity by inducing a state of immune exhaustion. In the context of the tumor microenvironment (TME), however, cancer cells can manipulate these pathways to camouflage themselves from an immune attack. Blocking these checkpoints has thus emerged as a key immunotherapeutic tactic. Nonetheless, the success rate of immune checkpoint inhibitors (ICIs) has been mixed, even for patients who test positive for relevant diagnostic biomarkers. Patient stratification currently depends on immunohistochemical staining for checkpoint proteins like PD-L1, but these tests do not provide adequate stratification. A more comprehensive stratification, one that includes immune profiling of the TME plus an evaluation of immune checkpoint interactions, might provide better patient stratification and ICI responsiveness. Here we describe mIF that is augmented with a PD-1 and PD-L1 protein-protein interaction assay. Human formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1. Sections were then treated with oligonucleotide-modified secondary probes suitable for in situ Proximity Ligation Assay (isPLA), enabling the detection of PD1 and PD-L1 interactions. The PLA protocol utilized a fluorescent probe complementary to amplified nucleic acid to enhance the visibility of the protein interactions. The sample was then mounted in a closed microfluidic chamber and imaged on the CellScape™ platform for Precise Spatial Multiplexing. After imaging of the isPLA signal, the CellScape cyclic multiplex mIF staining approach was used to iteratively stain immune and structural markers on the tissue sample utilizing the VistaPlex™ Spatial Immune Profiling Assay Kit. This proof-of-concept study demonstrates that high-plex mIF can be augmented with an isPLA on human FFPE samples. The combined application of both methods allows visualization of PD1/PD-L1 protein-protein interactions and integrates this interaction within the spatial context of the surrounding cell populations. This approach allows for a more comprehensive insight into the interplay of different immune cell and non-immune cell populations during checkpoint activation processes in normal and neoplastic tissues. Citation Format: Arne Christians, Jannik Boog, Charles E. Jackson, Matthew H. Ingalls, J Spencer Schwarz, Sara Bodbin, Agata Zieba Wicher, Subham Basu, Oliver Braubach. High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5354.

Abstract 94: Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling

Abstract The STING (Stimulator of Interferon Genes) pathway is a pivotal player in the innate immune response, responsible for detecting cytosolic accumulation of endogenous DNA and initiating a cascade of events leading to the production of pro-inflammatory type I interferons, which promote an antitumor environment through activation of T cells, dendritic cells, and natural killer cells. Therefore, STING agonists are currently being explored as a potential therapeutic for cancer treatment, often in combination with immune checkpoint inhibitors. While the STING pathway can be stimulated in multiple cell types, activation in antigen presenting cells, such as dendritic cells (DC), is crucial for antitumor activity in the tumor microenvironment (TME). Additionally, the type 1 interferon family consists of multiple subtypes, including IFNb and IFNa, which can produce differential biological responses. Although the STING pathway is known to induce expression of multiple cytokines, combined spatial characterization of STING and type I interferons in the TME has not been performed. To characterize STING-interferon expression in the TME, we use the Integrated MultiOmyx-RNAscope platform. MultiOmyxTM (NeoGenomics Laboratories, Inc) is a proprietary multiplex immunofluorescence (mIF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section and offers high-resolution spatial and quantitative analysis of protein expression in tissue samples. RNAscopeTM (Bio-Techne) Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. The Integrated MultiOmyx-RNAscope assay allows for simultaneous detection of both protein and RNA markers in a single sample. Herein we report the design and use of a novel panel of commercially-available antibodies and ISH probes broad enough to characterize various immune subpopulations and cytokine expressing cells, including DCs and interferons, in the TME of a variety of tumor indications including melanoma and head and neck squamous cell carcinoma. Quantification and analysis will be performed using NeoLYTXTM, the proprietary MultiOmyx Analytics pipeline, to examine the spatial distribution and expression levels of key STING pathway induced cytokines to elucidate the dynamic communication of signaling molecules and their localization within specific cell populations. Understanding of the variety and phenotype of STING/cytokine expressing cells in the TME is crucial to define the populations being targeted by therapies for cancer treatment. Citation Format: Sandra Lam, Courtney Todorov, Jiong Fei, Harry Nunns, Eric Leones, Marianne Thio, Flora Sahafi, Erinn Parnell, Qingyan Au. Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 94.

Abstract 1145: Comparative spatial transcriptomic analysis of tumor microenvironment responses to conventional chemotherapy and immunotherapy in triple-negative breast cancer

Abstract Background: In triple negative breast cancer (TNBC), pembrolizumab in combination with cytotoxic chemotherapy has proven its efficacy and improved survival outcome. Previous studies demonstrated that PD-L1 expression, tumor mutational burden as well as certain neo-antigens were associated with response to pembrolizumab but there were no biomarkers predicting the response of pembrolizumab of TNBC in neoadjuvant setting. Therefore, tumor microenvironment (TME) evaluation would be warranted to understand the mechanism of action of pembrolizumab in TNBC. Methods: We employed the Xenium In Situ platform, a cutting-edge spatial transcriptomics tool, capable of mapping hundreds of transcripts within individual cells. Using a 380-plex gene panel, we analyzed formalin-fixed, paraffin-embedded TNBC sections treated with weekly paclitaxel with carboplatin followed by doxorubicin with cyclophosphamide (CTx) with/without pembrolizumab in neoadjuvant setting. We analyzed detailed cellular compositions of tumors, encompassing immune and stromal cells, across over one million cells and we attempted to identify various cell types and states, previously characterized through single-cell RNA sequencing, and assess their spatial heterogeneity within the TME with regard to treatment strategy and the response to treatment. Results: We collected 19 serial samples from 13 patients including 12 pre-treatment and seven post-treatment samples. Among seven post-treatment samples, one was collected after three weeks of the first treatment and one was after disease relapse. Of 13 patients, seven patients had received pembrolizumab with cytotoxic chemotherapy and four out of seven have achieved pathologic complete response (pCR). In six patients treated with only cytotoxic chemotherapy, only two achieved pCR. Our analysis revealed significant alterations in the spatial organization of cells in both the pembrolizumab with CTx and CTx-only groups. Notably, an increase in CD8 T cells and plasma cells following treatments was pronounced in post-treatment samples from patients in the pembrolizumab with CTx group. We also observed treatment-specific variations in ligand-receptor interactions between immune cells and cancer cells. Conclusion: This study provides an in-depth analysis of the TME in TNBCs in response to pembrolizumab with CTx. Utilizing the Xenium In Situ platform, we uncovered significant disparities in the immune cell population according to the application of pembrolizumab. Our findings underscore the complexity of TME responses and highlight the potential of spatial transcriptomics in informing tailored treatment strategies for TNBC. Citation Format: Do-Eon Gu, Jiyeon Hyeon, Eun Seop Seo, Hae Hyun Jung, Jonghan Yu, Seok Won Kim, Young-Hyuck Im, Ji-Yeon Kim, Woong-Yang Park. Comparative spatial transcriptomic analysis of tumor microenvironment responses to conventional chemotherapy and immunotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1145.

Abstract 5573: Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma

Abstract Glutathione S-transferase omega 2 (GSTO2) is known as one of important regulators for the glutathione redox balance. We recently reported that GSTO2 is exclusively expressed in airway basal cells, Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs; however, its expression was completely lost in lung squamous cell carcinoma (LSCC) (Cancer Science, 2022). In this study, we examined the expression of GSTO2 in lung adenocarcinoma (LAC), another type of lung cancer, and analyzed the relationship between GSTO2 expression and clinicopathological features. We enrolled 139 patients who were diagnosed with LAC and underwent surgery, and immunohistochemically evaluated the expression of GSTO2 using formalin-fixed, paraffin-embedded sections of surgical specimens. Fifty eight of 139 lung adenocarcinoma samples exhibited GSTO2 expression, while remaining 81 cases did not. The expression of GSTO2 in LAC samples was significantly associated with never or light (Brinkman Index < 400) smoking history (P=0.0027) and histological subtypes (lepidic 77%; acinar 44%; papillary 22%; micropapillary 16.7%; solid 9.6%; P<0.0001). There were no significant differences between the groups with respect to pN, pM, EGFR mutation, ALK fusion, and ROS-1 fusion. However, GSTO2 expression in LAC significantly associated with pT1 (P<0.0001), early stage (P<0.0001), and absence of PD-L1 expression (P=0.0027). Moreover, patients with GSTO2-negative LAC had a significantly lower disease-free survival rate than those with GSTO2-positve LAC (P=0.028). To examine whether GSTO2 expression affects PD-L1 expression, we prepared GSTO2-transfected and mock-transfected lung adenocarcinoma cell lines (A549 and PC-9). In both cell lines, mRNA level of PD-L1 was significantly reduced in GSTO2-transfectants. Because Jang RH et al reported that vimentin, reflecting to acquire mesenchymal traits in epithelial-mesenchymal transition (EMT), regulates the PD-L1 expression, we next examined whether GSTO2 overexpression affects the expression of EMT related molecules. There was no difference in a mRNA level of E-cadherin, while the mRNA expression of vimentin and SLUG, a transcriptional inducer of EMT, were significantly suppressed in GSTO2-transfected PC-9. In both cell lines, protein expression of vimentin was also significantly reduced in GSTO2-transfectants. The present study shed light on a novel function of GSTO2 as a PD-L1 regulator, which may contribute to prevention of tumor immune escape. Citation Format: Ryusuke Sumiya, Hiroto Hatano, Teruki Hagiwara, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Hiromu Suzuki, Yuki I. Kawamura. Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5573.

Impact of Alcian blue and periodic acid Schiff expression on the prognosis of gastric signet ring cell carcinoma.

The Alcian blue (AB) and periodic acid Schiff (PAS) stains are representative mucus markers in gastric signet ring cell carcinoma (SRCC). They are low-cost special staining methods used to detect acidic mucus and neutral mucus, respectively. However, the clinical importance of the special combined AB and PAS stain is unclear. To investigate AB expression, PAS expression and the AB-to-PAS (A/P) ratio in gastric SRCC patients and to assess patient prognosis. Paraffin-embedded sections from 83 patients with gastric SRCC were stained with AB and PAS, and signet ring cell positivity was assessed quantitatively. Immunohistochemical staining for Ki67, protein 53 (P53) and human epidermal growth factor receptor 2 (HER2) was performed simultaneously. The cancer-specific survival (CSS) rate was estimated via Kaplan-Meier analysis. Cox proportional hazards models were used for univariate and multivariate survival analyses. Kaplan-Meier survival analysis revealed that the 3-year CSS rate was significantly greater in the high-PAS-expression subgroup than in the low-PAS-expression subgroup (P < 0.001). The 3-year CSS rate in the A/P ≤ 0.5 group was significantly greater than that in the A/P > 0.5 group (P = 0.042). Univariate Cox regression analysis revealed that the factors affecting prognosis included tumor diameter, lymph node metastasis, vessel carcinoma embolus, tumor stage, the A/P ratio and the expression of Ki67, P53 and the PAS. Cox multivariate regression analysis confirmed that low PAS expression [hazard ratio (HR) = 3.809, 95% confidence interval (CI): 1.563-9.283, P = 0.003] and large tumor diameter (HR = 2.761, 95%CI: 1.086-7.020, P = 0.033) were independent risk factors for poor prognosis. A/P > 0.5 is potentially a risk factor for prognosis, and low PAS expression is an independent risk factor in the prognosis of gastric SRCC. PAS expression and the A/P ratio could help in predicting the clinical prognosis of patients with SRCC.

Abcg2a is the functional homolog of human ABCG2 expressed at the zebrafish blood–brain barrier

BackgroundA principal protective component of the mammalian blood–brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs.MethodsTo determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish.ResultsWe found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature.ConclusionsThese results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for studying the role of ABCG2 at the BBB.

[18F]FDG PET/CT Signal Correlates with Neoangiogenesis Markers in Patients with Fibrotic Interstitial Lung Disease Who Underwent Lung Biopsy: Implication for the Use of PET/CT in Diffuse Lung Diseases.

The use of [18F]FDG PET/CT as a biomarker in diffuse lung diseases is increasingly recognized. We investigated the correlation between [18F]FDG uptake with histologic markers on lung biopsy of patients with fibrotic interstitial lung disease (fILD). Methods: We recruited 18 patients with fILD awaiting lung biopsy for [18F]FDG PET/CT. We derived a target-to-background ratio (TBR) of maximum pulmonary uptake of [18F]FDG (SUVmax) divided by the lung background (SUVmin). Consecutive paraffin-embedded lung biopsy sections were immunostained for alveolar and interstitial macrophages (CD68), microvessel density (MVD) (CD31 and CD105/endoglin), and glucose transporter 1. MVD was expressed as vessel area percentage per high-power field (Va%/hpf). Differences in imaging and angiogenesis markers between histologic usual interstitial pneumonia (UIP) and non-UIP were assessed using a nonparametric Mann-Whitney test. Correlation of imaging with angiogenesis markers was assessed using the nonparametric Spearman rank correlation. Univariate Kaplan-Meier survival analysis assessed the difference in the survival curves for each of the angiogenesis markers (separated by their respective optimal cutoff) using the log-rank test. Statistical analysis was performed using SPSS. Results: In total, 18 patients were followed for an average of 41.36 mo (range, 5.69-132.46 mo; median, 30.07 mo). Only CD105 MVD showed a significantly positive correlation with [18F]FDG TBR (Spearman rank correlation, 0.556; P < 0.05, n = 13). There was no correlation between [18F]FDG uptake and macrophage expression of glucose transporter 1. CD105 and CD31 were higher for UIP than for non-UIP, with CD105 reaching statistical significance (P = 0.011). In all patients, MVD assessed with either CD105 or CD31 quantification on biopsy predicted overall survival. Patients with CD105 MVD of less than 12 Va%/hpf or CD31 MVD of less than 35 Va%/hpf had a significantly better prognosis (no deaths during follow-up in the case of CD105) than did patients with higher scores of CD105 MVD (median survival, 35 mo; P = 0.041, n = 13) or CD31 MVD (median survival, 28 mo; P = 0.014, n = 13). Conclusion: Previous work has used [18F]FDG uptake in PET/CT as a biomarker in fILD. Here, we highlight a correlation between angiogenesis and [18F]FDG TBR. We show that MVD is higher for UIP than for non-UIP and is associated with mortality in patients with fILD. These data set the scene to investigate the potential role of vasculature and angiogenesis in fibrosis.