Adjacent Benign Tissues Research Articles

Detection of differentially methylated CpGs between tumour and adjacent benign cells in diagnostic prostate cancer samples

Differentially methylated CpG sites (dmCpGs) that distinguish prostate tumour from adjacent benign tissue could aid in the diagnosis and prognosis of prostate cancer. Previously, the identification of such dmCpGs has only been undertaken in radical prostatectomy (RP) samples and not primary diagnostic tumour samples (needle biopsy or transurethral resection of the prostate). We interrogated an Australian dataset comprising 125 tumour and 43 adjacent histologically benign diagnostic tissue samples, including 41 paired samples, using the Infinium Human Methylation450 BeadChip. Regression analyses of paired tumour and adjacent benign samples identified 2,386 significant dmCpGs (Bonferroni p < 0.01; delta-β ≥ 40%), with LASSO regression selecting 16 dmCpGs that distinguished tumour samples in the full Australian diagnostic dataset (AUC = 0.99). Results were validated in independent North American (npaired = 19; AUC = 0.87) and The Cancer Genome Atlas (TCGA; npaired = 50; AUC = 0.94) RP datasets. Two of the 16 dmCpGs were in genes that were significantly down-regulated in Australian tumour samples (Bonferroni p < 0.01; GSTM2 and PRKCB). Ten additional dmCpGs distinguished low (n = 34) and high Gleason (n = 88) score tumours in the diagnostic Australian dataset (AUC = 0.95), but these performed poorly when applied to the RP datasets (North American: AUC = 0.66; TCGA: AUC = 0.62). The DNA methylation marks identified here could augment and improve current diagnostic tests and/or form the basis of future prognostic tests.

Immunohistochemical findings and clinicopathological features of breast cancers with pathogenic germline mutations in Non-BRCA genes

Deleterious germline mutations in multiple genes confer an increased breast cancer (BC) risk. Immunohistochemical (IHC) expression of protein products of mutated high-risk genes has not been investigated in BC. We hypothesized that pathogenic mutations may lead to an abnormal IHC expression pattern in the tumor cells. BCs with deleterious germline mutations in CHEK2, ATM, PALB2 &PTEN were identified. Immunohistochemistry was performed using Dako staining platform on formalin fixed paraffin embedded tumor tissue. Primary antibodies for PALB2 (ab202970), ATM [2C1(1A10)}, CHK2 (EPR4325), and PTEN (138G6) proteins were used for BCs with respective deleterious mutations. IHC expression was assessed in tumor and adjacent benign breast tissue. Total 27 BCs with 10 CHEK2, 9 ATM, 6 PALB2 & 2 PTEN deleterious germline mutations were identified. IHC staining was performed on 8 CHEK2, 7 ATM, 6 PALB2 & 2 PTEN cases. Abnormal CHEK2 IHC staining was identified in 7/8(88%) BCs. Three distinct CHK2 IHC patterns were noted: 1) Strong diffuse nuclear positivity (5 BC), 2) Null-pattern (2 BC), & 3) Normal breast–like staining in 1 BC Four of 5 (80%) strong CHK2 staining BC had missense CHEK2 mutations. Null-pattern was present with a missense & a frameshift mutation. Normal breast-like CHEK2 IHC staining pattern was present in 1 BC with CHEK2 frameshift mutation. Loss of nuclear/cytoplasmic PTEN IHC expression was noted in 2 in-situ carcinomas. Abnormal PTEN and CHK2 IHC were present in atypical ductal hyperplasia and flat epithelial atypia. ATM and PALB2 IHC expression patterns were similar in tumor cells and benign breast epithelium: mild to moderate intensity nuclear and cytoplasmic staining. We report abnormal CHEK2 IHC expression in 88% of BCs with pathogenic CHEK2 mutations. With PTEN and CHEK2 pathogenic mutations, abnormal IHC patterns are seen in early atypical proliferative lesions. IHC may be applied to identify CHEK2 &PTEN mutated BCs and precursor lesions.

Abstract 6825: Epigenetic dysregulation in well-differentiated pancreatic neuroendocrine tumor (PNET): An immunohistochemistry (IHC) analysis

Abstract Epigenetic dysregulation through DNA Methyltransferase 1 (DNMT1) is integral to pancreatic neuroendocrine tumorigenesis. We aim to investigate the interplay between epigenetic modifiers and pathogenetic markers in the PNET microenvironment. 12 paraffin-embedded PNET specimens and 12 benign adjacent tissue specimens, used as controls, were obtained from Cooper University Hospital Pathology Department (IRB 23-130). IHC was performed using epigenetic markers, including DNMT1, DNMT3A, DNMT3B, and 5-Hydroxymethylcytosine (5-HMC), and pathogenic markers, including Multiple Endocrine Neoplasia 1 (MEN1), Protein-Tyrosine Phosphatase (PTEN), and Ras Association Domain Family 1 (RASSF1). Image J software was utilized for IHC scoring under the guidance of a board-certified pathologist. DNMT1 was upregulated in PNET tumor specimens with a mean density score of 72.02 +/- 31 versus 16.02 +/- 17 in adjacent benign tissue (p&amp;lt;.001). Grade 2 tumors displayed increased DNMT1 expression compared to Grade 1 tumors with a mean score of 37 +/-21 versus 71 +/- 23 (p&amp;lt;.05). Decreased expression of 5-HMC was noted in tumor specimens in comparison to benign tissue with a mean score of 3.92 +/- 5.05 versus 19.37 +/- 5.05 (p&amp;lt;.05). Loss of 5-HMC was observed in higher tumor grades with a mean score of .79 +/- 2 versus 8.2+/-2 (p&amp;lt;.01). DNMT3B was upregulated in PNET tumor specimens with a mean score of 13.16 +/- 3.94 versus 7.02+/- 2.553 (p&amp;lt;.01) in adjacent benign tissue and was positively correlated to DNMT1 expression. DNMT3A was not found to have a difference in expression between tumor and benign tissue. MEN1 was downregulated in PNET, with 45% of tumor specimens displaying loss of MEN1 expression. Mean MEN1 density in tumor tissue was 4.47 +/- 5.95 versus 11.41 +/- 3.53 in adjacent benign (p&amp;lt;.01). Increased expression of DNMT1 found to negatively correlate with MEN1 expression (R2=.23). Loss of RASSF1 expression was present in 70% of tumor specimens with a mean score of 1.32 +/- 1.99 versus 9.50+/- 6.89 (p&amp;lt;.001) in adjacent benign tissue. RASSF1 expression correlated negatively with DNMT1 (R2=.21). PTEN expression showed intratumoral homogeneity to benign tissue, and its expression was not correlated with any epigenetic markers. Our work provides a framework of epigenetic regulators involved in the pathobiology of PNET, and can refine prognostic evaluation while providing therapeutic benefits for patients with PNET. Citation Format: Zena Saleh, Yazid Ghanem, Hansa Joshi, Mathew C. Moccia, Helen Toma, Yahui Li, Upsana Joneja, Tao Gao, Young Hong. Epigenetic dysregulation in well-differentiated pancreatic neuroendocrine tumor (PNET): An immunohistochemistry (IHC) analysis [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 6825.

Magnetic resonance imaging-ultrasound fusion guided focal cryoablation for men with intermediate-risk prostate cancer

IntroductionFocal therapy (FT) is a form of ablative treatment offered to men with localized, organ-confined prostate cancer (CaP). Pelvic multiparametric magnetic resonance imaging (mpMRI) and mpMRI/transrectal ultrasound fusion (MRI-US) guidance enable the precise delivery of FT with limited ablation of adjacent benign tissue or vital genitourinary structures. This article presents our findings on using MRI-US to perform FT as a primary treatment for men with intermediate-risk CaP. MethodsThirty-six men underwent MRI-US fusion-guided FT cryoablation at a single center from 2018 to 2023 as a primary treatment for intermediate-risk CaP. Following FT, quarterly prostate-specific antigen (PSA) testing and a 6 to 9 month mpMRI and combined MRI-US targeted and systematic biopsy were performed. Oncological outcomes were determined using several endpoints containing biochemical recurrence, imaging failure, and pathological failure. Functional outcomes were measured using reported erectile dysfunction/potency rates, urinary incontinence rates, and the American Urologic Association Symptom Score (AUA-SS) and Sexual Health Inventory for Men (SHIM) indices. ResultsMedian follow-up was 29.1 months, most (75%) of whom had grade group 2 CaP. Out of the 36 men, 32 (88.9%) completed the combined MRI-targeted and systematic biopsy follow-up after treatment. The study had no major complications, but 12 (33.3%) patients experienced Clavien-Dindo grade II or lower complications. For oncological outcomes, 6 (16.7%) men had biochemical recurrence, 9 (25%) showed imaging failure, and 8 (22.2%) met the criteria for positive biopsy- out-of-field vs. in-field. 88.2% of previously potent patients remained potent postoperatively at 12 months. All patients were continent at 12 months. There were no statistically significant changes in the AUA-SS and SHIM scores postoperatively. ConclusionMRI-US-guided cryoablation to target lesions in intermediate-risk CaP appears to be a safe treatment option, with functional outcomes indicating minimal short and intermediate-term morbidity and acceptable oncological outcomes. However, despite close monitoring and follow-up, there is still a limitation in accurately predicting/detecting pathological failure after FT. The long-term durability of FT for intermediate-risk, organ-confined CaP remains uncertain.

Serine/threonine kinase 36 induced epithelial-mesenchymal transition promotes docetaxel resistance in prostate cancer

To investigate the role and potential mechanism of serine/threonine kinase 36 (STK36) in docetaxel resistance-prostate cancer (PCa). The expression of STK36 in PCa and the correlation with clinicopathological characteristics of PCa patients were analyzed using the data from different databases and tissue microarrays. To investigate the role of STK36 on cell proliferation, invasion, and migration, STK36 was overexpressed and silenced in DU-145 and PC-3 cell lines. Cell counting kit-8 (CCK8) was used to test cell proliferation. Cell invasion and migration were detected by cell wound scratch assay and trans well, respectively. The expression profile of STK36, E-Cadherin, and Vimentin was analyzed by Western blot. Cell apoptosis was detected by the TUNEL assay. STK36 expression was upregulated in PCa tissue compared with adjacent benign PCa tissue; it was higher in patients with advanced stages compared with lower stages and was significantly correlated with decreased overall survival. Up-regulation of STK36 significantly promoted the proliferation, invasion, and migration of DU-145 and PC-3 cells and compensated for the suppression caused by docetaxel treatment in vitro. A striking apoptosis inhibition could be observed when dealing with docetaxel, although the apoptosis of DU-145 and PC-3 cells was not affected by the STK36 exclusive overexpression. Besides, E-Cadherin expression was restrained while the expression levels of vimentin were all enhanced. The knockdown of STK36 reversed the above process. STK36 up-regulation could accelerate the biological behavior and docetaxel resistance of PCa by epithelial-mesenchymal transition (EMT) activation. STK36 may be potentially used as a target in PCa resolvent with docetaxel.

Racial differences in serum chemokines in prostate cancer patients.

This study aimed to understand the differential levels of inflammatory chemokines in association with higher prostate cancer incidence and mortality in African American (AA) men than in Caucasians (CA). The authors used a chemokine assay to simultaneously measure 40 chemokines and cytokines levels in the serum of preoperative prostate cancer patients and healthy controls of AA and CA races. Selected chemokines (CXCL2, CXCL5, and CCL23) serum level was validated in 211 serum samples from prostate cancer patients and healthy controls. Differential expression of CXCL5 and CCL23 was analyzed using immunohistochemistry in a representative cohort of prostate tumor tissues of AA and CA races. Race-specific comparisons from 211 serum samples showed significantly higher levels of CXCL2 (control: 3104.0 pg/mL vs. cancer: 2451.0 pg/mL) and CXCL5 (control: 5189.0 pg/mL vs. cancer: 5459.0 pg/mL) in AA men than in CAs (CXCL2; control: 1155.0 pg/mL vs. cancer: 889.3 pg/mL, and CXCL5; control: 1183.0 pg/mL vs. cancer: 977.5 pg/mL). CCL23 differed significantly within and between the races with a lower level in AA cancer cases (454.5 vs. 966.6 pg/mL) than healthy controls (740.5 vs. 1263.0 pg/mL). Patient age, prostate-specific antigen, or Gleason scores were not significantly associated with these chemokines. Immunostaining for CXCL5 and CCL23 in a representative cohort of archival prostate tissues displayed significantly higher CXCL5 in prostate tumors than in adjacent benign tissues, whereas CCL23 was nondetectable in most of the analyzed tumor tissues. Lower levels of CCL23 in AA prostate cancer patient sera and tumor tissues and high CXCL2 and CXCL5 may contribute to aggressive prostate cancer, as often seen in AA men. The disproportionate levels of serum chemokines associated with race warrant further exploration to improve equitability in precision oncology to benefit prostate cancer patients.

Prediction of coexisting invasive carcinoma on ductal carcinoma in situ (DCIS) lesions by mass spectrometry imaging.

Due to limited biopsy samples, ~20% of DCIS lesions confirmed by biopsy are upgraded to invasive ductal carcinoma (IDC) upon surgical resection. Avoiding underestimation of IDC when diagnosing DCIS has become an urgent challenge in an era discouraging overtreatment of DCIS. In this study, the metabolic profiles of 284 fresh frozen breast samples, including tumor tissues and adjacent benign tissues (ABTs) and distant surrounding tissues (DSTs), were analyzed using desorption electrospray ionization-mass spectrometry (DESI-MS) imaging. Metabolomics analysis using DESI-MS data revealed significant differences in metabolite levels, including small-molecule antioxidants, long-chain polyunsaturated fatty acids (PUFAs) and phospholipids between pure DCIS and IDC. However, the metabolic profile in DCIS with invasive carcinoma components clearly shifts to be closer to adjacent IDC components. For instance, DCIS with invasive carcinoma components showed lower levels of antioxidants and higher levels of free fatty acids compared to pure DCIS. Furthermore, the accumulation of long-chain PUFAs and the phosphatidylinositols (PIs) containing PUFA residues may also be associated with the progression of DCIS. These distinctive metabolic characteristics may offer valuable indications for investigating the malignant potential of DCIS. By combining DESI-MS data with machine learning (ML) methods, various breast lesions were discriminated. Importantly, the pure DCIS components were successfully distinguished from the DCIS components in samples with invasion in postoperative specimens by a Lasso prediction model, achieving an AUC value of 0.851. In addition, pixel-level prediction based on DESI-MS data enabled automatic visualization of tissue properties across whole tissue sections. Summarily, DESI-MS imaging on histopathological sections can provide abundant metabolic information about breast lesions. By analyzing the spatial metabolic characteristics in tissue sections, this technology has the potential to facilitate accurate diagnosis and individualized treatment of DCIS by inferring the presence of IDC components surrounding DCIS lesions. © 2023 The Pathological Society of Great Britain and Ireland.

Regulation of SELENOF translation by eIF4a3: Possible role in prostate cancer progression.

The levels of the SELENOF selenoprotein are dramatically reduced in prostate cancer compared to adjacent benign tissue and reducing SELENOF in prostate epithelial cells results in the acquisition of features of the transformed phenotype. It was hypothesized that the aberrant increase in the eiF4a3 translation factor, which has an established role in RNA splicing and the regulation of selenoprotein translation, contributes to the lower levels of SELENOF. Using the available databases, eIF4a3messenger RNA (mRNA) levels are elevated in prostate cancer compared to normal tissue as is the hypomethylation of the corresponding gene. Using a prostate cancer tissue microarray, we established that eiF4a3 levels are higher in prostate cancer tissue. Ectopic expression of eIF4a3 in prostate cancer cells reduced SELENOF levels and attenuated the readthrough of the UGA codon using a specialized reporter construct designed to examine UGA decoding, with the opposite effects observed using eIF4a3 knock-down constructs. Direct binding of eIF4a3 to the regulatory regions of SELENOF mRNA was established with pull-down experiments. Lastly, we show that an eIF4a3 inhibitor, eIF4a3-IN-2, increases SELENOF levels, UGA readthrough, and reduces binding of eIF4a3 to the SELENOF mRNA 3'-UTR in exposed cells. These data establish eIF4a3 as a likely prostate cancer oncogene and a regulator of SELENOF translation.

Methylation status of various gene loci in localized prostate cancer: Novel biomarkers for diagnostics and biochemical recurrence.

Oncologic outcomes for patients with localized prostate cancer (PCa) undergoing radical prostatectomy (RP) can vary widely. Hypermethylation of tumor-associated genes has potential as a novel diagnostic tool and predictive biomarker in PCa. We investigated the methylation status of tumor-associated genes in patients who underwent RP. Patients who underwent RP during 2004 to 2008 were matched retrospectively based on post-operative D'Amico risk stratification. Quantitative pyrosequencing was used to analyze methylation status of 10 gene loci in cancerous and adjacent benign tissue from histological specimen. Follow-up was performed according to EAU guideline recommendations. Statistical analyses were performed to correlate methylation levels in cancerous and benign tissue with risk profiles and biochemical recurrence (BCR). The cohort included 71 patients: 22 low-risk, 22 intermediate-risk, and 27 high-risk. Mean follow-up time was 74 months. Methylation status differed significantly between cancerous and adjacent benign tissue for the 5 gene loci GSTP1, APC, RASSF1, TNFRFS10c, and RUNX3 (each P < 0.001). Also, the methylation level was significantly higher in high-risk than in low-risk patients for Endoglin2 and APC (P = 0.026; P = 0.032). Using ROC analysis, hypermethylation of APC in PCa tissue was associated with higher risk of BCR (P = 0.005). Methylation status of various gene loci holds diagnostic and predictive potential in PCa. Hypermethylation of APC, RASSF1, TNFRFS10c and RUNX3 were identified as novel PCa-specific biomarkers. Furthermore, increased methylation levels of APC and Endoglin2 were associated with high-risk PCa. Additionally, hypermethylation of APC was associated with increased risk of BCR after RP.

Palmitoyl acyltransferase ZDHHC7 inhibits androgen receptor and suppresses prostate cancer.

The hormonal transcription factor androgen receptor (AR) is a master regulator of prostate cancer (PCa). Protein palmitoylation, which attaches a palmitate fatty acid to a substrate protein, is mediated by a class of 23 ZDHHC (Zinc-Finger DHHC motif)-family palmitoyltransferases. Although palmitoylation has been shown to modify many proteins and regulate diverse cellular processes, little is known about ZDHHC genes in cancer. Here we examined ZDHHC family gene expression in human tissue panels and identified ZDHHC7 as a PCa-relevant member. RNA-seq analyses of PCa cells with ZDHHC7 de-regulation revealed global alterations in androgen response and cell cycle pathways. Mechanistically, ZDHHC7 inhibits AR gene transcription and therefore reduces AR protein levels and abolishes AR signaling in PCa cells. Accordingly, ZDHHC7 depletion increased the oncogenic properties of PCa cells, whereas restoring ZDHHC7 is sufficient to suppress PCa cell proliferation and invasion in vitro and mitigate xenograft tumor growth in vivo. Lastly, we demonstrated that ZDHHC7 is downregulated in human PCa compared to benign-adjacent tissues, and its loss is associated with worse clinical outcomes. In summary, our study reveals a global role of ZDHHC7 in inhibiting androgen response and suppressing PCa progression and identifies ZDHHC7 loss as a biomarker for aggressive PCa and a target for therapeutic intervention.

ATF4-mediated circTDRD3 promotes gastric cancer cell proliferation and metastasis by regulating the miR-891b/ITGA2 axis and AKT signaling pathway.

Gastric cancer (GC) is a cancer of the gastrointestinal tract that is highly malignant and has poor prognosis. Circular RNAs are a class of nonclassical RNA molecules that have been determined to be involved in GC malignancy in various ways. However, the underlying function and mechanism of circTDRD3 in gastric cancer remain largely unknown. We analyzed circTDRD3 expression in databases and verified the findings in GC cell lines and tissue specimens. A series of functional gene overexpression and knockdown assays in vivo and in vitro were carried out to investigate the role of circTDRD3 in proliferation and metastasis. Here, we revealed the role of the miR-891b/ITGA2 axis by analyzing bioinformatics datasets. Furthermore, we performed dual-luciferase, fluorescence in situ hybridization, RNA pull-down, and functional rescue experiments to examine the relationships between circTDRD3 and its interacting molecules. Western blot confirmed the positive regulatory role of circTDRD3 in the AKT signaling pathway. A promoting effect of ATF4 on circTDRD3 was determined through chromatin immunoprecipitation. CircTDRD3 was significantly overexpressed in GC tissues compared with adjacent benign tissue, and its expression level was positively correlated with tumor volume and lymph node metastasis. CircTDRD3 promoted GC cell proliferation and migration in vitro and in vivo. Mechanistically, circTDRD3 exerted a tumor-promoting effect by regulating the miR-891b/ITGA2 axis and AKT signaling pathway in a positive feedback manner mediated by the transcription factor ATF4. ATF4-mediated circTDRD3 overexpression modulates the proliferation and metastasis of GC cells through the miR-891b/ITGA2 axis in a positive feedback manner.

CCT3 as a Diagnostic and Prognostic Biomarker in Cervical Cancer.

The chaperonin-containing TCP1 complex subunit 3 (CCT3) has been reported to be involved in the development and prognosis of many tumors, including cervical cancer (CC). This study aimed to analyze the expression and prognostic value of CCT3 in CC by bioinformatics and retrospective study. CCT3 gene expression profiles and clinical information in CC were downloaded from the cancer genome atlas (TCGA) and gene expression omnibus (GEO) databases. CCT3 expression was verified by quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry (IHC). Logistic regression and chi-square testing were used to analyze the relationship between CCT3 expression and the clinical characteristics of CC. Kaplan-Meier and Cox analyses were used to evaluate whether CCT3 affects the prognosis of CC. Nomogram and calibration curves were used to test the predictive value of CCT3. The expression of CCT3 in CC tissues was significantly upregulated compared with that in adjacent benign tissues, and was related to HPV16/18 infection, grade, and positive lymph nodes. High expression of CCT3 is associated with poor prognosis of CC and can be used as an independent risk factor for CC. The prognostic model based on CCT3 and CC clinical features has good predictive ability. CCT3 is overexpressed in CC, which is related to poor prognosis and expected to become a biomarker for CC.

Protein Expression of NEK2, JMJD4, and REST in Clear Cell Renal Cell Carcinoma (ccRCC): Clinical, Pathological, and Prognostic Findings.

Cells of renal cell carcinoma (RCC) are resistant to the most currently used chemotherapeutic agents and targeted therapies; hence, we evaluated the expression of NEK2, JMJD4, and REST in cases of clear cell renal cell carcinoma (ccRCC) and benign adjacent tissues of kidney to detect associations between their expression and clinicopathological features, prognostic data, tumor recurrence, and survival rates. We collected 200 samples including tumoral and adjacent non-neoplastic tissues related to 100 ccRCC patients. All samples were evaluated for the expression of NEK2, JMJD4, and REST, and the patients were followed up for about 5 years. Tumor recurrence and survival data were documented and analyzed. NEK2 and JMJD4 expression showed increase in ccRCC tissues (P=0.002 and 0.006), while REST was downregulated (P<0.001). The elevated expression of NEK2 was positively related ro the tumor size (P=0.015), higher grades (P=0.002), higher stages (P=0.013), distant spread (P=0.004), tumor recurrence, shorter progression-free survival (PFS) rate, and overall survival (OS) rate (P<0.001). Likewise, the high expression of JMJD4 showed positive correlation with the tumor size (P=0.047), higher grades (P=0.003), higher stages (P=0.043), distant spread (P=0.001), tumor recurrence, shorter PFS rate, and OS rate (P<0.001). Conversely, low expression of REST demonstrated positive relationship with the tumor size, higher grades, higher stages, distant spread, tumor recurrence, and shorter PFS and OS rates (P<0.001). Overexpression of NEK2 and JMJD4 and downregulation of REST may be noted in malignant renal tissues compared to benign renal tissues and may be correlated with unfavorable pathological findings, poor clinical parameters, and poor patient outcomes.

Enrichment of “Cribriform” morphologies (intraductal and cribriform adenocarcinoma) and genomic alterations in radiorecurrent prostate cancer

AbstractLocally relapsed prostate cancer (PCa) after radiation therapy (RT) is associated with substantial morbidity and mortality. Morphological and molecular consequences that may contribute to RT resistance and local recurrence remain poorly understood. Locally recurrent PCa tissue from 53 patients with clinically localized PCa who failed with primary RT and subsequently underwent salvage radical prostatectomy (RP) was analyzed for tumor focality, clinicopathological, molecular, and genomic characteristics. Targeted next-generation sequencing with full exon coverage of 1,425 cancer-related genes was performed on 10 representative radiorecurrent PCas exhibiting no RT effect with matched adjacent benign prostate tissue. At RP, 37 (70%) of PCas had no RT effect with the following characteristics: grade group (GG) ≥ 3 (70%), unifocal tumor (75%), extraprostatic disease (78%), lymph node metastasis (8%), and “cribriform” morphologies (84%) [cribriform PCa (78%) or intraductal carcinoma (IDC-P) (61%)] at a median percentage of approximately 80% of tumor volume. In the setting of multifocal tumors (25%) at RP, the cribriform morphologies were restricted to index tumors. Of 32 patients with available pre-RT biopsy information, 16 had GG1 PCa, none had cribriform morphologies at baseline but 81% demonstrated cribriform morphologies at RP. Notable alterations detected in the sequenced tumors included: defects in DNA damage response and repair (DDR) genes (70%) (TP53, BRCA2, PALB2, ATR, POLQ), PTEN loss (50%), loss of 8p (80%), and gain of MYC (70%). The median tumor mutational burden was 4.18 mutations/Mb with a range of 2.16 to 31.86. Our findings suggest that most radiorecurrent PCas are enriched in cribriform morphologies with potentially targetable genomic alterations. Understanding this phenotypic and genotypic diversity of radiorecurrent PCa is critically important to facilitate optimal patient management.

High levels of unfolded protein response component CHAC1 associates with cancer progression signatures in malignant breast cancer tissues.

The aberrant mRNA expression of a UPR component Cation transport regulator homolog 1 (CHAC1) has been reported to be associated with poor survival in breast and ovarian cancer patients, however, the expression of CHAC1 at protein levels in malignant breast tissues is underreported. The following study aimed at analyzing CHAC1 protein expression in malignant breast cancer tissues. Evaluation of CHAC1 expression in invasive ductal carcinomas (IDCs) with known ER, PR, and HER2 status was carried out using immunohistochemistry (IHC) with CHAC1 specific antibody. The Human breast cancer tissue microarray (TMA, cat# BR1503f, US Biomax, Inc., Rockville, MD) was used to determine CHAC1 expression. The analysis of CHAC1 IHC was done to determine its expression in terms of molecular subtypes of breast cancer, lymph node status, and proliferation index using Qu-Path software. Survival analysis was studied with a Kaplan-Meier plotter. Immunohistochemical analysis of CHAC1 in breast cancer tissues showed significant up-regulation of CHAC1 as compared to the adjacent normal and benign tissues. Interestingly, CHAC1 immunostaining revealed high expression in tumor tissues with high proliferation and positive lymph node metastasis suggesting that CHAC1 might have an important role to play in breast cancer progression. Furthermore, high CHAC1 expression is associated with poor overall survival (OS) in large breast cancer patient cohorts. As a higher expression of CHAC1 was observed in tissue cores with high Ki67 index and positive lymph node metastasis it may be concluded that enhanced CHAC1 expression correlates with proliferation and metastasis. The further analysis of breast cancer patients' survival data through KM plot indicated that high CHAC1 expression is associated with a bad prognosis hinting that CHAC1 may have a possible prognostic significance in breast cancer.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

Abstract 3555: Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas

Abstract Head and neck squamous cell carcinomas (HNSCC) are the sixth most common malignancies worldwide. 45% of patients are diagnosed at a late tumor stage associated with poor survival. For metastatic, unresectable or recurrent (m/uR) HNSCC, immune checkpoint inhibition (ICI) was recently approved as a novel therapeutic option showing significant survival benefits compared to standard chemotherapy-based treatment. However, response to ICI is still limited to a small number of patients calling for further improvement of T cell-based immunotherapies. Peptide-based approaches, which rely on the specific immune recognition of tumor-associated human leukocyte antigen (HLA) presented peptides, represent promising and low side effect treatment options. Peptide vaccination has been shown to enhance and induce long-term anti-tumoral immune responses and even clinical responses in HNSCC patients. However, current vaccines are either monovalent, based on patient-individual tumor-specific mutations or restricted to a single HLA allotype and therefore neither widely applicable nor suitable for reliable studies and large-scale production. In this study, using mass spectrometry (MS) -based immunopeptidome analysis of a large cohort of HNSCC patient (n = 30) tumor and adjacent benign samples, we established a tumor-associated off-the-shelf peptide warehouse for broadly applicable personalized therapies. The malignant dataset, comprising 91651 HLA ligands, was compared to adjacent benign and various benign tissues (www.hla-ligand-atlas.org) to identify tumor-exclusive antigens. Further antigen selection was based on allotype-specific high frequent presentation. In total, 23 frequently presented and tumor-exclusive HNSCC-associated peptides were selected for six of the most common HLA class I allotypes (A*01, A*02, A*24, B*15, B*35, B*40) covering &amp;gt;75% of the world population, as well as five HLA class II presented peptides binding various different HLA class II allotypes. Immunogenicity was validated by IFN-γ ELISPOT screening for spontaneous preexisting T cell responses targeting the respective peptides as well as by in vitro priming experiments of naïve T cells in HNSCC patients and healthy volunteers. Furthermore, immunopeptidome analyses identified these antigens in patient plasma samples providing first evidence for “liquid biopsy” immunopeptidome analysis without the need of primary tumor tissue. A phase I study evaluating safety, immunogenicity as well as first efficacy of this warehouse-based vaccine in combination with ICI in HNSCC patients is currently being set up, with personalized peptide selection based on individual HLA-allotype and MS analysis of patient tumor/plasma sample. In conclusion, we here designed a peptide warehouse that enables a polyvalent and widely applicable but still personalized peptide vaccination in HNSCC patients. Citation Format: Sarah Schroeder, Thorben Gross, Annika Nelde, Marcel Wacker, Jens Bauer, Jonas Rieth, Marissa Dubbelaar, Lena Muehlenbruch, Yacine Maringer, Paul-Stefan Mauz, Martin Sailer, Julia Philipp, Sven Becker, Thomas Breuer, Helmut R. Salih, Hans-Georg Rammensee, Hubert Löwenheim, Juliane S. Walz. Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3555.

Abstract 6398: Functional characterization of Androgen Receptor splice variants in breast cancer invasion and metastasis

Abstract Introduction: A growing body of evidence suggests the prognostic and therapeutic value of Androgen Receptors (AR) in Breast Cancer (BrCa), particularly Triple Negative BrCa (TNBC). The splice variants of AR (ARVs); notably full-length AR (AR-fl) of ~110 kDa and a C-terminally Ligand Binding Domain (LBD) truncated AR isoform (AR-V7) of ~67 kDa are present in a variety of tissues. The ligand-independent AR-V7 is constitutively expressed and confers castration resistance in prostate cancer. With the irrefutable role of ARVs in BrCa, we evaluated their expression profile in cell lines with their prevalence and clinicopathological correlation in BrCa patients from India. Methods: Breast Cancer cell lines MDA-MB-231, MCF7, and MDA-MB-453 were procured from American Type Culture Collection. Thirty-eight de novo cases of Breast Cancer undergoing surgical intervention at a tertiary healthcare center in India were recruited. Tumor and adjacent benign tissue were collected after informed consent. The ARVs expression at transcriptomic and proteomic levels was evaluated by qRT-PCR and western blotting &amp; Immunohistochemistry (IHC) respectively. For functional annotation, ARVs were knocked down in vitro using differently targeting siRNAs facilitated by Lipofectamine 3000 based transfection. There was a strong complementarity observed in western blotting and IHC. Results: All the ARVs were significantly higher in MDA-MB-453 followed by MCF7 and MDA-MB-231 cells. The expression ratio of AR-V7 versus AR-fl is, however, elevated in highly aggressive TNBC cell line MDA-MB-231. 67% of patients recruited were immunostained positive for AR (n=38). A majority of patients were of Luminal phenotype (37%) expressing both the prominent ARVs. One-third of the TNBC fraction were AR positive. Following the TNBC cell line, a subset of patients' samples also exhibited a substantial upregulation of AR-V7 compared to AR-fl. These patients had poor prognoses and aggressive features. The siRNA knockdown of all the ARVs resulted in decreased invasion and Epithelial-mesenchymal transition (EMT). Interestingly knocking down of ARVs barring AR-V7, restored invasiveness suggesting the compensatory behavior of AR-V7. Conclusion: Our work demonstrates the presence of ARVs in BrCa with a particular focus on the AR-V7. The LBD truncated AR-V7 potentiates the growth in BrCa cell lines and patients. This study further encourages the ARVs expression screening with AR positive BrCa for a prognostic biomarker of aggressive phenotype and therapeutic resistance. Citation Format: Tryambak Pratap Srivastava, Ruby Dhar, Anurag Srivastava, SVS Deo, Rajinder Parshad, Subhradip Karmakar. Functional characterization of Androgen Receptor splice variants in breast cancer invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6398.

Abstract 1375: The intra-tumoral spatial heterogeneity of T-cell antigens in glioblastoma: An integrated multi-omics approach

Abstract Glioblastoma is the most common primary malignant neoplasm of the central nervous system in adults. Current treatment options comprise maximal surgical resection followed by radiation and/or chemotherapy with temozolomide. However, these procedures are unable to eliminate all tumor cells, which in turn lead to disease recurrence accounting for the poor prognosis. Glioblastoma is a highly infiltrative tumor in which recurrence originates from the unresectable peritumoral infiltration zone. Thus, novel treatment options specifically targeting the tumor cells in the infiltration zone are needed to prevent relapse and enable long-lasting remission. In this work, we performed multi-omics spatial analysis of the necrotic center (NEC), the gadolinium contrast-enhanced region (T1), and the infiltration zone (INF) to assess immunological relevant aspects of tumor heterogeneity. By integrating mass spectrometry-based immunopeptidome analysis with next generation sequencing methods (whole exome and RNA sequencing, DNA methylation), we aimed to identify the intra-tumoral regional heterogeneity of T cell antigens with a special focus on the peritumoral infiltration zone. For multi-omics analysis, HLA peptides and genetic material from 15 glioblastoma patients were extracted and analyzed from the three zones NEC, T1, INF and for four patients additionally from adjacent benign (BEN) brain tissue. A total of 24,699 unique HLA class I and 17,394 HLA class II peptides were identified. Comparative profiling of peptides from our study and a benign tissue database (in-house (n=429) combined with HLA ligand atlas (https://hla-ligand-atlas.org)) revealed that 6% (970/15,579), 5% (829/16,442) and 5% (725/13,891) of HLA class I peptides were exclusively presented in the INF, T1 and NEC zone, respectively. 6% (394/7,150), 7% (589/8,736) and 13% (1,725/12,908) of HLA class II peptides showed exclusive representation in the INF, T1 and NEC zone, respectively. Importantly, due to the spatial analysis of the immunopeptidome, we revealed 970 HLA class I and 394 HLA class II peptides exclusively presented in the INF zone. Interestingly, one INF-associated HLA class I peptide, which showed frequent presentation in 36% of glioblastoma immunopeptidomes, is derived from the brain and acute leukemia cytoplasmic protein (BAALC), a highly expressed cell cycle inducer (via MEK kinase-1) in several cancers, including glioblastoma. Integrated RNA/DNA sequencing enabled a greater understanding of spatial tumor antigen presentation and lead to the identification of INF-specific neoepitopes derived from tumor-specific mutations. In summary, we identified the intra-tumoral regional heterogeneity of tumor antigens, which could be used in the future for specific immunotherapy approaches targeting the infiltration zone of glioblastoma. Citation Format: Marcel Wacker, Gioele Medici, Marissa Dubbelaar, Jens Bauer, Annika Nelde, Luca Regli, Michael Weller, Helmut R. Salih, Hans-Georg Rammensee, Marian C. Neidert, Juliane S. Walz. The intra-tumoral spatial heterogeneity of T-cell antigens in glioblastoma: An integrated multi-omics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1375.

Handheld PET Probe for Pediatric Cancer Surgery.

Simple SummaryPositron emission tomography (PET)/computed tomography (CT) scans are widely used as a form of full body imaging and allow for the early detection of small, asymptomatic tumors that may represent cancer metastasis or recurrence. Tissue diagnosis is critical in determining the choice of ongoing targeted therapy for pediatric patients with solid tumors. These small tumors may be difficult to localize in the operating room, especially in a re-operative or radiated area of the body. An adjunct such as a PET probe, used to guide intra-operative dissection, is the ideal tool to assist in cases where an occult tumor requires an excisional biopsy.18F-fluorodeoxyglucose (FDG) is a glucose analog that acts as a marker for glucose uptake and metabolism. FDG PET scans are used in monitoring pediatric cancers. The handheld PET probe localization of FDG-avid lesions is an emerging modality for radio-guided surgery (RGS). We sought to assess the utility of PET probe in localizing occult FDG-avid tumors in pediatric patients. PET probe functionality was evaluated by using a PET/CT scan calibration phantom. The PET probe was able to detect FDG photon emission from simulated tumors with an expected decay of the radioisotope over time. Specificity for simulated tumor detection was lower in a model that included background FDG. In a clinical model, eight pediatric patients with FDG-avid primary, recurrent or metastatic cancer underwent a tumor excision, utilizing IV FDG and PET probe survey. Adequate tissue for diagnosis was present in 16 of 17 resected specimens, and pathology was positive for malignancy in 12 of the 17 FDG-avid lesions. PET probe gamma counts per second were higher in tumors compared with adjacent benign tissue in all operations. The median ex vivo tumor-to-background ratio (TBR) was 4.0 (range 0.9–12). The PET probe confirmed the excision of occult FDG-avid tumors in eight pediatric patients.