Formalin-fixed Paraffin-embedded Tumor Tissue Research Articles

Concordance analysis between liquid biopsy (ctDNA) and tumor DNA molecular profiles from panel-based next-generation sequencing

IntroductionAnalysis of circulating tumor DNA (ctDNA), also known as liquid biopsy, has been postulated to be a useful test in the prognostication, molecular profiling, and monitoring of cancer patients. In this series we aimed to analyze the concordance between the mutation status of formalin-fixed paraffin-embedded (FFPE) tumor samples and matched ctDNA, considering tumor molecular profiling as the gold standard technique. MethodsThis retrospective study included cancer patients with complete diagnostics and gene mutations detected in a previous FFPE tumor tissue Next-Generation Sequencing (NGS) study with a matched frozen plasma sample available for an NGS ctDNA assay. Results and discussionSixty patients were included, 24 with colorectal carcinoma (CRC) and 36 with non-small cell lung cancer (NSCLC). In 27.1% of ctDNA studies a new mutation not previously detected in the matched tumor was found. 11.9% of these ctDNA results had the potential to impact clinical management. Globally, the concordance rate between FFPE tumor samples and ctDNA was 44.4%. When tumors were stratified by stage, the concordance was 76.5%, 70%, 36.4%, and 0% in tumor stages IV, III, II, and I, respectively. ctDNA molecular profiles showed a good concordance rate in advanced stage tumors and identified undetected mutations in tumor tissues. In early tumor stages the concordance was low, casting doubt on the usefulness of ctDNA in these patients.

Loss of ARID1A expression is associated with systemic inflammation markers and has important prognostic significance in gastric cancer.

The tumor suppressor gene AT-rich interactive domain 1A (ARID1A) and systemic inflammatory response (SIR) have been reported to be related to the sensitivity to immunotherapy. This study intended to explore the relationship between ARID1A expression and SIR, and to further elucidate the prognostic value of ARID1A expression in gastric cancer (GC). The mRNA and protein expression of ARID1A were detected in 272 formalin-fixed paraffin-embedded (FFPE) tumor tissues. The data of nine systemic inflammation markers were collected 1 week before gastrectomy. Univariate and multivariate COX analysis were used to screen out independent predictors of GC. Negative expression of ARID1A protein was related to GC with deficient mismatch repair (dMMR) (p = 0.033), positive programmed cell death-ligand 1 (PD-L1) (p = 0.005) and lower albumin level (p = 0.0064). Low expression of ARID1A mRNA was common in GC with abnormal E-cadherin (p = 0.020) and a higher platelet/lymphocyte ratio (PLR) (p = 0.0391). Multivariate COX analysis showed that the expression of ARID1A protein (p = 0.023), age (p = 0.004), T stage (p = 0.009) and N stage (p = 0.009) were independent predictors of GC. The nomogram established by independent predictors can accurately evaluate the survival risk of patients with GC. The loss of ARID1A protein expression was associated with the dMMR subtype and high expression of PD-L1 in GC. Negative ARID1A protein and low expression of mRNA were associated with aberrant systemic inflammatory markers. The expression of ARID1A protein had important prognostic significance in GC.

Evaluation of NKILA lncRNA and NFκB Genes Expression in an Iranian Gastric Cancer Patient Compared with Healthy Tissue and the Relationship between Clinicopathological Features

Background & Objective: Long non-coding RNAs play critical roles in the initiation and development of gastric cancer (GC). The aim of this study was to assess the expression of NKILA and NFKB genes and the relationship between their expressions with clinical characteristics in gastric cancer. Also, relative expression of lncRNAsNKILA, compared NFKB in GC tissues was evaluated.
 Materials & Methods: This cross-sectional study was performed on 50 gastric formalin-fixed paraffin-embedded tumor tissue samples and 50 normal tissues. The RNA level of lncRNAsNKILA and NFKB genes was assessed using the quantitative real-time polymerase chain reaction. B2M was used as an internal control. The 2 -ΔΔCq method was used to determine the expression fold changes.
 Results: A significant association was observed between the levels of NKILA, in gastric tumor tissues compared with normal tissues (Mean = 2.087, p= 0.0484). The relative expression of the NFKB gene revealed no statistically significant difference between the gastric tumor tissues compared with normal tissues (P=0.3728). In addition, clinicopathologic data compared with NKILA and NFKBmRNA expression levels in gastric cancer tissues showed no significant association. Here, we found no significant association between the level of NKILA expression compared with NFKB mRNA level in gastric cancer tissues (R=0.03; P=0.2355).
 Conclusion: Our results showed that NKILA had a significant association with GC. Our findings also revealed that NKILA expression was not correlated with NFKB mRNA level in GC tissues.

Prognostic value of HER2 expression levels for upper tract urothelial carcinoma.

557 Background: HER2 expression is an adverse prognostic factor of bladder urothelial carcinoma (UBC) while the prognostic value of HER2 expression in upper tract urothelial carcinoma (UTUC) remains to be explored. This study aims to investigate the prognostic value of different HER2 expression and gene amplification levels in UTUC. Methods: The baseline clinicopathological data of 130 patients with UTUC were reviewed. The expression level of HER2 was detected by immunohistochemistry (IHC) staining ( Anti-Her2/neu, Cat.4B5, Ventana ) from formalin-fixed paraffin-embedded (FFPE) tumor tissue of patients. The HER2 gene amplification level was detected by second-generation sequencing (NGS) (HER2 gene copy number [CN]) or Fluorescence in situ hybridization (FISH) from FFPE tumor tissue of patients. HER2 negative was defined as IHC 0. HER2 low expression was defined as IHC 2+ but CN- or FISH-, while HER2 overexpression included IHC 3+, and IHC 2+ but CN+ or FISH+. The correlation between HER2 expression levels or other clinicopathological characteristics and overall survival (OS) were analyzed. The Cox proportional-hazards model was used to analyze the independent prognostic factors of UTUC. Results: All the 130 patients provided FFPE tumor slides for the IHC detection of the HER2 expression levels including: IHC 0 (n = 47), IHC 1+ (n = 28), IHC 2+ (n = 41) and IHC 3+ (n = 14), the median OS were 27.5 months, 48.7 months, 67.3 months, and not reached, respectively, with significantly difference ( P = 0.014). Among the 41 patients with HER2 IHC 2+, 15 patients received the detection of the HER2 gene amplification level by NGS or FISH while the other 26 patients whose HER2 gene amplification levels were unknown were excluded from the analysis. The patients of HER2 negative, low expression, and overexpression had a median OS of 27.5 months, 58.0 months, and 60.0 months, respectively, but the difference was not significant ( P = 0.184). Cox proportional-hazards model also verified that the HER2 IHC expression is an independent prognostic factor on the survival, while the prognostic value of the HER2 gene amplification levels was not found. Conclusions: HER2 IHC expression is an independent factor for the prognosis of patients with UTUC. However, HER2 gene amplification levels have no impact on the prognosis of UTUC patients. HER2 expression especially the HER2 gene amplification level in UTUC needs to be explored further.

Impact of different HER2 expression levels on the outcomes of second-line immunotherapy for metastatic urothelial carcinoma.

524 Background: Immunotherapy has become an option of second-line treatment for metastatic urothelial carcinoma (mUC) after the failure of platinum-containing chemotherapy. HER2 expression is an adverse prognostic factor of urothelial carcinoma (UC) while the predictive value of HER2 expression in UC treatment remains unknown. This study attempts to explore the impact of different HER2 expression and gene amplification levels on the outcomes of second-line immunotherapy in metastatic UC. Methods: The baseline clinical data and the outcomes after second-line anti-PD-1 immunotherapy from 2017 to 2021 in 79 patients with metastatic UC who failed to the first-line platinum-containing chemotherapy were reviewed. The expression level of HER2 was detected by immunohistochemistry (IHC) staining ( Anti-Her2/neu, Cat.4B5, Ventana ) from formalin-fixed paraffin-embedded (FFPE) tumor tissue of patients. The copy number amplification level of HER2 gene was detected by second-generation sequencing (NGS) from FFPE tumor tissue of patients. The correlation between HER2 expression and objective response rate (ORR), median progression-free survival (PFS) and overall survival (OS) were analyzed. Results: HER2 expression levels by IHC were performed in 77 patients and the copy number (CN) amplification level of 20 patients with HER2 IHC 2+ were detected by NGS. The median PFS of HER2 negative (IHC 0), low expression (IHC 2 +/ CN - and IHC 1 +) and overexpression (IHC 3 + and IHC 2 +/ CN +) patients were 11.0 months (95% confidence interval [CI]: 1.4-20.6 months), 3.7 months (95% CI: 1.3-6.1 months) and 1.8 months (95% CI: 0.8-2.8 months), respectively, with significant difference in the overall comparison ( P = 0.001) and in the each two comparisons. ORR had a trend to decrease gradually with the increase of HER2 expression level from negative to overexpression (42.4% vs. 31.6% vs. 0%, P = 0.08). However, no correlation between HER2 expression and OS after anti-PD-1 immunotherapy was observed. Conclusions: Different HER2 expression levels have a significant impact on the PFS of the second-line anti-PD-1 immunotherapy in patients with metastatic urothelial carcinoma. The overexpression of HER2 (IHC 3 + and IHC 2 +/ CN +) may be an adverse factor, while the HER2-negative status may be a favorable factor. HER2 expression have potential value in predicting the efficacy of second-line immunotherapy for advanced urothelial carcinoma, which needs further research.

Abstract OT2-19-03: Solti-1801. Analysis of the efficacy of CDK4/6 inhibitors in combination with hormonal treatment in luminal breast cancer in relation to the intrinsic subtype and markers of immunity (CDK-predict)

Abstract Introduction. The incorporation of cyclin-dependent kinase inhibitors 4 and 6 (CDK4/6 inhibitors) with endocrine therapy in patients with hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer has demonstrated its efficacy improving progression-free survival (PFS), overall response rate (ORR) and, more recently, overall survival (OS). However, patients eventually progress due to resistance to treatment. To date, no clinical or molecular markers defining the HR+/HER2- patient population that obtains the greatest benefit from these drugs have been found, apart from estrogen receptor positivity. However, data from multiple retrospective analysis suggest that within HR+/HER2- disease, the non-luminal intrinsic subtypes (20-30% of these patients) have a worse prognosis and may not benefit equally from every CDK4/6 inhibitor (Finn SABCS 2017, Prat. JCO 2021). Furthermore, the prognostic impact of tumor infiltrating lymphocytes (TILs) and gene expression related to the immune response in the context of HR+/HER2- advanced breast cancer have not been deeply investigated. Design. CDK-PREDICT is an observational, non-interventional, multicenter study that will include 114 patients with advanced breast cancer treated in first-line with CDK4/6 inhibitors + hormone therapy. The primary objective is to correlate the baseline intrinsic subtypes (defined by PAM50) with the efficacy (measured as PFS) of CDK4/6 inhibitors + hormone therapy. As secondary objectives, the correlation of the intrinsic subtypes with ORR and with the histopathological characteristics of the tumor will be analyzed. In addition, the expression of immune response and cell cycle genes, as well as the presence of TILs, will be correlated with the intrinsic subtypes and with PFS and ORR. Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples will be hematoxylin and eosin (H&E) stained and percentage of TILs will be determined. RNA will be purified and analyzed at the nCounter (Nanostring Technologies) using a panel of 72 genes including the PAM50 genes and immune genes such as PD1, PDL1, CD8 or CD4. Overall, we aim to develop a predictive score combining clinical, genomic, and immune expression data integrating tumor biology and microenvironment. For inclusion in the study, a metastatic sample taken within 100 days prior to CDK4/6 inhibitors treatment is required. Once this sample has been collected, registered, and assessed for quality, patients will be followed up every 6 months until disease progression, death or withdrawal from the study. As of July 2021, 80 patients have been included at 5 sites in Spain. This study is included within the Biomarker program of SOLTI. Recruitment of this study started in June 2020. Acknowledgements.This project has received a research grant from “Instituto de Salud Carlos III (ISCIII), Ministerio de Economía y Competitividad” (Spain) awarded within the National Research Program with reference PI 18/01408, co-funded with European Union ERDF funds (European Regional Development Fund). Citation Format: Pablo Tolosa, Tomás Pascual, Cristina Hernando, Sonia Servitja, María Fernández Abad, Fara Brasó-Maristany, Javier Benítez, Laura Lema, Mario Martínez, Yolanda Ruano, Lucía Parrilla, Alejandra Bernardini, Ana María Roncero, Laia Paré, Jordi Canes, Patricia Villagrasa, Fernando Salvador, Aleix Prat, Eva Ciruelos. Solti-1801. Analysis of the efficacy of CDK4/6 inhibitors in combination with hormonal treatment in luminal breast cancer in relation to the intrinsic subtype and markers of immunity (CDK-predict) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-19-03.

Abstract P3-13-06: Single nucleotide polymorphisms and mortality after docetaxel-based chemotherapy in premenopausal breast cancer: A population-based cohort study in Denmark

Abstract Background Taxane-based chemotherapy is first line treatment in various cancers, including premenopausal breast cancer, but the inter-individual effectiveness is unpredictable. Differences in expression and activity of docetaxel-metabolizing enzymes and transporters (DMETs) may modify docetaxel effectiveness. Although reported data are inconsistent, some findings suggests that variant alleles that reduce the function of 1) SLC-transporters reduce drug influx into hepatocytes; 2) CYP-450 enzymes hamper drug metabolism; and 3) ABC-transporters decrease docetaxel clearance. The net effect is hypothesized to be increased docetaxel exposure and effectiveness. In contrast, GSTP1 variants have been associated with poorer docetaxel effectiveness. We investigated whether single nucleotide polymorphisms (SNPs) in drug metabolizing enzymes and transporters were associated with mortality in premenopausal breast cancer patients. Materials and methods Using the Danish Breast Cancer Group (DBCG) clinical database, we identified data on premenopausal women aged 18–55 years, diagnosed with non–metastatic breast cancer during 2007–2011. All women were recommended epirubicin, cyclophosphamide and docetaxel-based adjuvant chemotherapy, and tamoxifen if the disease was estrogen receptor (ER) positive. From DBCG and other Danish administrative and medical registries we retrieve data on death, emigration, and tumor characteristics. We collected archived formalin-fixed paraffin-embedded primary tumor tissue from Danish pathology departments. We genotyped 17 candidate SNPs using TaqMan SNP genotyping assays and, for each SNP, categorized the women as having two normal alleles (wildtype) or at least one variant allele. We followed the women from six months after breast cancer diagnosis until death, emigration or 30th June 2019, whichever came first. Comparing variant carriers with wildtype, we computed cumulative incidence proportions (CIPs) and used Poisson regression models to calculate unadjusted incidence rate ratios (IRRs) and associated 95% confidence intervals (CIs) of all–cause mortality. We stratified by ER status to evaluate interaction. Analyses were repeated for breast cancer specific mortality. Results Our cohort included 2,262 women. During follow–up (median 9.6 years, interquartile range: 8.4–11.0), 250 women died (CIP: 14%); 219 due to breast cancer (CIP: 11%). Genotyping was successful for ≥95% of the study cohort, with exception of two SNPs, which were excluded from analyses. We detected decreased mortality in variant carriers of SLCO1B1 rs2306283 (IRR: 0.75, 95% CI 0.58–0.97), and ABCB1 rs1128503 (IRR: 0.81, 95% CI: 0.63–1.05), ABCB1 rs2032582 (IRR: 0.80, 95% CI: 0.62–1.04) and ABCC2 rs12762549 (IRR: 0.80, 95% CI: 0.62–1.04). In contrast, mortality was increased in carriers of GSTP1 rs1138272 (IRR: 1.28, 95% CI: 0.93–1.76) and, unexpectedly, in carriers of CYP3A rs10273424 (IRR: 1.36, 95% CI: 0.99–1.86) variant carriers. Sensitivity analyses yielded similar findings. ER status did not modify the associations. Conclusions In this study, mortality may be associated with SNPs in ABCB1, ABCC2, SLCO1B1, CYP3A and GSTP1 in premenopausal women with non-metastatic breast cancer receiving docetaxel. The mechanisms underlying our findings remains unclear but may be related to docetaxel pharmacokinetics. Citation Format: Cathrine F Hjorth, Per Damkier, Tore B Stage, Søren Feddersen, Bent Ejlertsen, Timothy L Lash, Stephen Hamilton-Dutoit, Thomas P Ahern, Mikael Rørth, Henrik T Sørensen, Deirdre Cronin-Fenton. Single nucleotide polymorphisms and mortality after docetaxel-based chemotherapy in premenopausal breast cancer: A population-based cohort study in Denmark [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-06.

Abstract P1-07-07: Endoresist: Prognostic and predictive gene profiles in endocrine-resistant breast cancers

Abstract Background:. Up to 75% of breast cancer patients have tumors expressing the predictive biomarker estrogen receptor alpha (ER) and are consequently offered endocrine therapy. Approximately one third eventually relapse in their disease with the majority still expressing ER but resistant to one or several types of endocrine-based therapies. Molecular studies of ER-signaling suggest several potential factors influencing endocrine response such as ER structural and functional domains, posttranslational modifications and unbalanced receptor and co-regulator activity with alterations in downstream signaling pathways. However, the mechanism behind endocrine resistance is elusive and tumor cell expression of ER is the only clinically utilized biomarker for endocrine therapeutic response. This calls for a broader understanding of the resistance process in order to develop novel tools for therapy prediction and treatment alternatives. This study aims to improve our understanding of the underlying mechanism behind endocrine resistance and to identify biomarkers for an individualized approach to cancer therapy. Materials and methods:. A cohort comprising verified endocrine-resistant early breast cancer diagnosed in 2005-2006 and 2008-2014 at the Karolinska University Hospital, Stockholm, Sweden was retrospectively collected. Patients with ER+ and human epidermal growth factor receptor 2 (HER2)- primary breast cancer who had an ER+/HER2- relapse tumor during ongoing endocrine therapy (<5 years from diagnosis) were included as cases, with both primary tumors (n=68) and relapse tumors (n=67). A control cohort was selected consisting of patients with ER+/HER2- early breast cancer diagnosed in 2005, without relapse or disease progression at 10 years of follow-up (n=78), and matched to cases (2:1 ratio) by age and tumor stage. To determine the transcriptomic and genomic profiles, RNA and DNA were extracted from archived formalin-fixed paraffin-embedded tumor tissue. RNA samples were analyzed using Affymetrix DNA Microarray with Clariom D Chip and assessed through Transcriptome Array Console and R. DNA was analyzed through panel sequencing (GMCK gene panel consisting of 370 genes and a size of 2,4 Mb intended for genetic screening of solid tumors) with following assessments in R. Results:. The preliminary transcriptomic analysis showed several differentially expressed pathways and specific genes that differentiate both the primary tumors of cases that eventually relapse during endocrine therapy compared to controls, as well as between the cases’ relapses and their corresponding primary tumors. Intrinsic analyses show upregulation in tumor-driving pathways such as PI3K signaling pathway, androgen receptor and cell adhesion pathways in primary tumors of cases versus controls. This has further been investigated by gene analysis where genes such as PIK3CA, PTEN, CDK4/6 and ESR1 were upregulated between the groups. In the analysis of relapses compared to their primary tumors, pathways involving apoptosis and response to hypoxia were downregulated as well as specific genes such as IGF1R, whereas pathways including immune response were upregulated. Conclusion:. Our study suggests that endocrine resistant tumors are associated with distinct genomic and transcriptomic profiles that potentially could be utilized as predictive and prognostic markers for endocrine resistant breast cancer. These preliminary results can guide further investigations deciphering the mechanisms of endocrine resistance in breast cancer. This trial is a non-interventional study set up as a research collaboration between Karolinska Institutet and Novartis Sweden AB. Citation Format: Caroline Schagerholm, Stephanie Robertson, Barbro Holm, Karolina Lindberg, Emmanouil Sifakis, Linnea Hases, Cecilia Williams, Johan Hartman. Endoresist: Prognostic and predictive gene profiles in endocrine-resistant breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-07.

Tissue miR-200c-3p and circulating miR-1290 as potential prognostic biomarkers for colorectal cancer

Epithelial–mesenchymal transition (EMT)-related cancers generally elicit low immune responses. EMT is regulated by several microRNAs (miRNAs) in cancers. Thus, this study aimed to evaluate the prognostic potential of EMT-related miRNAs as biomarkers in colorectal cancer (CRC). Formalin-fixed paraffin-embedded tumor and normal tissue and plasma samples were obtained from 65 patients with pathologically confirmed CRC. In addition, plasma samples were obtained from 30 healthy volunteers. Immunohistochemical staining for E-cadherin, ZEB1, PD-1, PD-L1, CD3, CD4, CD8, Foxp3, and CD68 was conducted on tissue samples. Droplet digital polymerase chain reaction (ddPCR) analysis was performed to evaluate miR-21-5p, 34a-5p, 138-5p, 200a-3p, 200b-5p, 200c-3p, 630, 1246, and 1290 expression in tissue samples and miR-630, 1246, and 1290 expression in plasma samples. miR-21-5p, 34a-5p, 630, 1246, and 1290 expression was higher in tumor tissues than in normal tissues (P < 0.05). EMT was significantly associated with reduced tumor-infiltrating T cells. Moreover, miR-21-5p, miR-34a-5p, miR-200a-3p, and miR-200c-3p expression was negatively correlated with T cell density (P < 0.05). High tissue levels of miR-200c-3p were associated with poor overall survival (OS) (P < 0.001). CRC patients with the EMT phenotype had poor OS; however, PD-L1 positivity and abundant PD-1 positive immune cells were correlated with better OS (P < 0.05). miR-1246 and miR-1290 levels were significantly higher in the plasma of patients with CRC than in the plasma of healthy controls (P < 0.05). High plasma levels of miR-1290 were correlated with advanced stage and poor OS (P < 0.05). The tissue expression of miR-200c-3p and plasma levels of miR-1290 measured by ddPCR indicate their potential as prognostic biomarkers for CRC.

Liquid BIOpsy for MiNimal RESidual DiSease Detection in Head and Neck Squamous Cell Carcinoma (LIONESS)\u2014a personalised circulating tumour DNA analysis in head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) remain a substantial burden to global health. Cell-free circulating tumour DNA (ctDNA) is an emerging biomarker but has not been studied sufficiently in HNSCC.MethodsWe conducted a single-centre prospective cohort study to investigate ctDNA in patients with p16-negative HNSCC who received curative-intent primary surgical treatment. Whole-exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumour tissue. We utilised RaDaRTM, a highly sensitive personalised assay using deep sequencing for tumour-specific variants, to analyse serial pre- and post-operative plasma samples for evidence of minimal residual disease and recurrence.ResultsIn 17 patients analysed, personalised panels were designed to detect 34 to 52 somatic variants. Data show ctDNA detection in baseline samples taken prior to surgery in 17 of 17 patients. In post-surgery samples, ctDNA could be detected at levels as low as 0.0006% variant allele frequency. In all cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times ranging from 108 to 253 days.ConclusionsThis study illustrates the potential of ctDNA as a biomarker for detecting minimal residual disease and recurrence in HNSCC and demonstrates the feasibility of personalised ctDNA assays for the detection of disease prior to clinical recurrence.

Association between tumor mutation profile and clinical outcomes among Hispanic-Latino patients with metastatic colorectal cancer.

17 Background: According to the World Health Organization GLOBOCAN database, in 2019, approximately 1.8 million new colorectal cancer cases were diagnosed, and almost 861,000 deaths were reported. In the United States, CRC is the third most frequent type of cancer and the second leading cause of cancer-related death. Although the overall incidence of CRC among the Hispanic population has been declining over the last decades, recently, a dramatic increase in CRC incidents among Hispanics younger than 50 years of age (early-onset CRC) has been reported. The increase in the incidence of early-onset CRC is markedly more significant in Hispanic-Latino (HL) patients population (45%) than in non-Hispanic Whites (NHW) (27%) and African-Americans (AA) (15%). Additionally, in contrast to NHW, Hispanics have a worse survival rate. The exact reason for these racial disparities and the biology of CRC in the HL population are not well understood. Therefore, we performed this study to better understand the biology of the disease in HL patients, which might help to identify new directions for targeted therapy. Methods: For the study, 52 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples were collected and analyzed. We compared the results with individual patient clinical histories and outcomes. Of 52 patients with mCRC, 52 (100%) were identified as HL. We identified several commonly altered genes in HL patients ( APC, TP53, KRAS, GNAS, PICK3CA, and NOTCH). Compared to those in other studies. Results: Mutation frequencies in the APC gene were significantly higher among HL patients with mCRC. Moreover, the prevalence of the APC mutation was significantly higher among male HL patients compared to female patients. The combination of mutations in the APC, NOTCH, and KRAS genes in the same tumors was associated with a higher risk of progression after the first-line of chemotherapy and worse overall survival. In addition, mutations in the combination of GNAS and AURKA genes were associated with a significantly higher risk of progression after the first-line of chemotherapy. Conclusions: The data support the notion that the molecular drivers of colon cancer might be different in HL patients compared to other racial/ethnic groups.

Association Between Tumor Mutation Profile and Clinical Outcomes Among Hispanic-Latino Patients With Metastatic Colorectal Cancer.

In the United States, CRC is the third most common type of cancer and the second leading cause of cancer-related death. Although the incidence of CRC among the Hispanic population has been declining, recently, a dramatic increase in CRC incidents among HL younger than 50 years of age has been reported. The incidence of early-onset CRC is more significant in HL population (45%) than in non-Hispanic Whites (27%) and African-Americans (15%). The reason for these racial disparities and the biology of CRC in the HL are not well understood. We performed this study to understand the biology of the disease in HL patients. We analyzed formalin-fixed paraffin-embedded tumor tissue samples from 52 HL patients with mCRC. We compared the results with individual patient clinical histories and outcomes. We identified commonly altered genes in HL patients (APC, TP53, KRAS, GNAS, and NOTCH). Importantly, mutation frequencies in the APC gene were significantly higher among HL patients. The combination of mutations in the APC, NOTCH, and KRAS genes in the same tumors was associated with a higher risk of progression after first-line of chemotherapy and overall survival. Our data support the notion that the molecular drivers of CRC might be different in HL patients.

NanoString Digital Molecular Profiling of Protein and microRNA in Rhabdomyosarcoma.

Simple SummaryNanoString digital profiling methods are novel techniques to identify biologic markers from human formalin-fixed, paraffin-embedded cancer tissue. We have applied NanoString Digital spatial profiling and microRNA profiling methods in non-alveolar rhabdomyosarcoma, a common soft tissue tumor in young adults and children with variable prognosis. Our results have highlighted aberrant miRNA expression and over-expression of several members of PI3-AKT, MAPK and apoptosis signaling pathways in fusion-negative rhabdomyosarcoma, particularly in tumors with unfavorable prognosis. INPP4B, an entry molecule in the PI3/AKT pathway, was significantly over-expressed in tumors with poor prognosis, confirmed by traditional immunohistochemistry. Several microRNAs had increased expression in association with poor patient prognosis. These results highlight the utility of NanoString digital profiling as a screening method to identify prognostic biomarkers of interest in rhabdomyosarcoma from formalin-fixed paraffin-embedded tissue.Purpose: Rhabdomyosarcoma (RMS) exhibits a complex prognostic algorithm based on histologic, biologic and clinical parameters. The embryonal (ERMS) and spindle cell-sclerosing RMS (SRMS) histologic subtypes warrant further studies due to their heterogenous genetic background and variable clinical behavior. NanoString digital profiling methods have been previously highlighted as robust novel methods to detect protein and microRNA expression in several cancers but not in RMS. Methods/Patients: To identify prognostic biomarkers, we categorized 12 ERMS and SRMS tumor cases into adverse (n = 5) or favorable (n = 7) prognosis groups and analyzed their signaling pathways and microRNA profiles. The digital spatial profiling of protein and microRNA analysis was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue using NanoString technology. Results: The detectable expression of several component members of the PI3K/AKT, MAPK and apoptosis signaling pathways was highlighted in RMS, including INPP4B, Pan-AKT, MET, Pan-RAS, EGFR, phospho-p90 RSK, p44/42 ERK1/2, BAD, BCL-XL, cleaved caspase-9, NF1, PARP and p53. Compared to cases with favorable prognosis, the adverse-prognosis tumor samples had significantly increased expression of INPP4B, which was confirmed with traditional immunohistochemistry. The analysis of microRNA profiles revealed that, out of 798 microRNAs assessed, 228 were overexpressed and 134 downregulated in the adverse prognosis group. Significant over-expression of oncogenic/tumor suppressor miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548ar-5p and miR-548y (p < 0.05) was noted in the adverse prognosis group. Conclusion: This study highlights the utility of NanoString digital profiling methods in RMS, where it can detect distinct molecular signatures with the expression of signaling pathways and microRNAs from FFPE tumor tissue that may help identify prognostic biomarkers of interest. The overexpression of INPP4B and miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548y and miR-548ar-5p may be associated with worse overall survival in ERMS and SRMS.

Mutation patterns of epidermal growth factor receptor gene in non-small cell lung cancer among Egyptian patients

ABSTRACT Epidermal growth factor receptor (EGFR) mutations have been reported to be associated with non–small cell lung cancer (NSCLC) and correlated to the responsiveness of tumors to EGFR tyrosine kinase inhibitors (TKIs). EGFR mutations in NSCLC Egyptian patients was investigated in formalin-fixed paraffin-embedded lung tumor tissues of 120 NSCLC patients and 20 control tissues from patients with benign lung tumor using ViennaLab StripAssay. Patients showed higher rates of females (87/120, 72.5%) (P=0.043) and never-smokers (82/120, 68.3%) (P=0.003), than the control group. EGFR mutations were significantly related to NSCLC adenocarcinoma, where 49 (40.8%) of patients were mutant (P=0.013) compared with the control group who have no mutations. EGFR mutations were most found in exons 1821, whereas the most mutated exons were exon 19 (55.1%) and exon 21 (26.5%). While exon 18 (10.2%) and exon 20 (8.2%) were the less mutated exons. Moreover, the most common mutations were; L858R (Leu858Arg) in exon 21 and L747-P753 in exon 19; representing 22.4% and 18.4% of all mutations; respectively. Our findings imply that, somatic EGFR mutations could be helpful for NSCLC diagnosis and can be used in combination with clinical factors to select the patients in Egypt who will respond more effectively to TKIs.

Accuracy and Clinical Relevance of Intra-Tumoral Fusobacterium nucleatum Detection in Formalin-Fixed Paraffin-Embedded (FFPE) Tissue by Droplet Digital PCR (ddPCR) in Colorectal Cancer.

The use of droplet digital PCR (ddPCR) to identify and quantify low-abundance targets is a significant advantage for accurately detecting potentially oncogenic bacteria. Fusobacterium nucleatum (Fn) is implicated in colorectal cancer (CRC) tumorigenesis and is becoming an important prognostic biomarker. We evaluated the detection accuracy and clinical relevance of Fn DNA by ddPCR in a molecularly characterized, formalin-fixed, paraffin-embedded (FFPE) CRC cohort previously analyzed by qPCR for Fn levels. Following a ddPCR assay optimization and an analytical evaluation, Fn DNA were measured in 139 CRC FFPE cases. The measures of accuracy for Fn status compared to the prior results generated by qPCR and the association with clinicopathological and molecular patients’ features were also evaluated. The ddPCR-based Fn assay was sensitive and specific to positive controls. Fn DNA were detected in 20.1% of cases and further classified as Fn-high and Fn-low/negative, according to the median amount of Fn DNA that were detected in all cases and associated with the patient’s worst prognosis. There was a low agreement between the Fn status determined by ddPCR and qPCR (Cohen’s Kappa = 0.210). Our findings show that ddPCR can detect and quantify Fn in FFPE tumor tissues and highlights its clinical relevance in Fn detection in a routine CRC setting.

Classic Hodgkin Lymphoma Refractory for ABVD Treatment Is Characterized by Pathologically Activated Signal Transduction Pathways as Revealed by Proteomic Profiling.

Simple SummaryClassic Hodgkin lymphoma (cHL) patients refractory to standard ABVD chemo-therapy are known to have a dismal prognosis. This has led to the hypothesis that ABVD treatment-sensitive and ABVD treatment-refractory tumours are biologically distinct. In this study, cHL patients refractory to standard ABVD treatment show subtle but significant differences in protein expression that enable clustering of the two response groups, thus indicating differences between ABVD sensitive and refractory patients at the molecular level, and thereby strengthening the hypothesis that ABVD sensitive and ABVD refractory tumours may be biologically distinct.In classic Hodgkin lymphoma (cHL), the tumour microenvironment (TME) is of major pathological relevance. The paucity of neoplastic cells makes it important to study the entire TME when searching for prognostic biomarkers. Cure rates in cHL have improved markedly over the last several decades, but patients with primary refractory disease still show inferior survival. We performed a proteomic comparison of pretreatment tumour tissue from ABVD treatment-refractory versus ABVD treatment-sensitive cHL patients, in order to identify biological differences correlating with treatment outcome. Formalin-fixed paraffin-embedded tumour tissues from 36 patients with cHL, 15 with treatment-refractory disease, and 21 with treatment-sensitive disease, were processed for proteomic investigation. Label-free quantification nano liquid chromatography tandem mass spectrometry was performed on the tissues. A total of 3920 proteins were detected and quantified between the refractory and sensitive groups. This comparison revealed several subtle but significant differences in protein expression which could identify subcluster characteristics of the refractory group. Bioinformatic analysis of the biological differences indicated that a number of pathologically activated signal transduction pathways are disturbed in ABVD treatment-refractory cHL.

The role of pericytes on the efficacy of bevacizumab in colorectal cancer.

Pericytes are mesenchymal cells surrounding capillary vessels and are known to play an essential role in tumor angiogenesis. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell surface proteoglycan and its release from pericytes and vascular smooth muscle cells is very important in tumor angiogenesis. Bevacizumab, which is a monoclonal antibody frequently used in the treatment of metastatic colorectal cancer, binds to the ligand of vascular endothelial growth factor A (VEGFA) and inhibits tumor angiogenesis. However, no reliable biomarker for predicting patients that will show a good response to this therapy has been established yet. In this study, we aimed to identify the significance of the presence of pericyte and VEGFA and CSPG4 expressions on the efficacy of Bevacizumab. Fifty patients with metastatic or recurrent colorectal cancer who had been treated with Bevacizumab combined chemotherapy treatment were included in the study. The expressions of VEGFA and CSPG4 genes and also human β-actin as the reference gene were examined using the quantitative real-time polymerase chain reaction method in the formalin-fixed paraffinembedded tumor tissues. For determining vascular and pericyte density in tumor tissue, immunohistochemical analysis was performed with CD31, alpha-smooth muscle actin, and CD34 antibodies. CSPG4 positive group had better objective response rate, as well as longer progression-free and overall survival than CSPG4 negative ones. Progression-free survival was significantly longer in VEGFA low group and CD31 low group. No significant correlation was found between CD34 positivity, SMA positivity, and progression-free and overall survival. Our results suggested that bevacizumab may be more effective in patients having less vascular density in the tumor tissue. But further studies are needed to support this finding.

Manual Immunofluorescence of Formalin-Fixed Paraffin-Embedded Human Tumor Tissues.

Immunofluorescence (IF) of tumor tissues has become a key tool in the study of cancer. With a wide variety of formalin-fixed paraffin-embedded (FFPE) preserved tissues available, there are possibilities to assess large cohorts using archived tissue which may have archived associated clinical outcomes. Although best practice guidelines for the assessment of tissues have been published, a standardized method for immunofluorescence of FFPE tumor tissues is elusive. Here we provide a protocol for using classical secondary fluorescent antibodies that bind directly to the primary antibody of interest. This protocol can easily be adapted to use several primary antibodies, of different species, with unique secondary fluorophores that correspond to each species of origin. It can also be adapted for cyclic amplification-based immunofluorescence of FFPE tissues. We aim to provide a beginner-friendly and highly accessible method for immunofluorescence of FFPE-embedded tissues, hoping to enable more laboratories to take on this highly informative technique and empower them to begin IF analysis in their own tissues of interest.

Heat Shock Proteins 27, 70, and 110: Expression and Prognostic Significance in Colorectal Cancer.

Simple SummaryHeat shock proteins (HSPs) are cytoprotective chaperones occurring in virtually all living organisms including various types of cancer cells to repair proteotoxic damage. Some of HSPs influence tumor prognosis and represent promising therapeutic targets. To clarify prognostic significance of HSPs 27, 70, and 110 in colorectal carcinoma, we retrospectively performed HSP immunohistochemistry on tissue microarrays from archive tumor tissue from 297 patients. Survival analysis revealed significantly shorter overall survival (OS) and borderline insignificantly shorter cancer specific survival (CSS) in patients with HSP70+ tumors. In HSP27+, both OS and CSS were insignificantly shorter. HSP110 showed no influence on survival. We found an association of HSP27 and HSP70 expression with advanced cancer stage. In multivariate analysis, only advanced stage and right sided localization were independent predictors of worse survival, whereas all three examined HSPs were insignificant. In conclusion, from all three HSPs examined in our study, only HSP70 had an impact on colorectal cancer prognosis, although it is stage-dependent.Heat shock proteins (HSPs) are evolutionarily conserved chaperones occurring in virtually all living organisms playing a key role in the maintenance of cellular homeostasis. They are constitutively expressed to prevent and repair protein damage following various physiological and environmental stressors. HSPs are overexpressed in various types of cancers to provide cytoprotective function, and they have been described to influence prognosis and response to therapy. Moreover, they have been used as a tumor marker in blood serum biochemistry and they represent a potentially promising therapeutic target. To clarify prognostic significance of two canonical HSPs (27 and 70) and less known HSP110 (previously known as HSP105) in colorectal carcinoma (CRC), we retrospectively performed HSP immunohistochemistry on tissue microarrays from formalin-fixed paraffin-embedded tumor tissue from 297 patients with known follow-up. Survival analysis (univariate Kaplan–Meier analysis with the log-rank test and multivariate Cox regression) revealed significantly shorter overall survival (OS, mean 5.54 vs. 7.07, p = 0.033) and borderline insignificantly shorter cancer specific survival (CSS, mean 6.3 vs. 7.87 years, p = 0.066) in patients with HSP70+ tumors. In the case of HSP27+ tumors, there was an insignificantly shorter OS (mean 6.36 vs. 7.13 years, p = 0.2) and CSS (mean 7.17 vs. 7.95 years, p = 0.2). HSP110 showed no significant impact on survival. Using Pearson’s chi-squared test, there was a significant association of HSP27 and HSP70 expression with advanced cancer stage. HSP27+ tumors were more frequently mismatch-repair proficient and vice versa (p = 0.014), and they occurred more often in female patients and vice versa (p = 0.015). There was an enrichment of left sided tumors with HSP110+ compared to the right sided (p = 0.022). In multivariate Cox regression adjusted on the UICC stage, grade and right/left side; both HSPs 27 and 70 were not independent survival predictors (p = 0.616 & p = 0.586). In multivariate analysis, only advanced UICC stage (p = 0) and right sided localization (p = 0.04) were independent predictors of worse CSS. In conclusion, from all three HSPs examined in our study, only HSP70 expression worsened CRC prognosis, although stage-dependent. The contribution of this article may be seen as a large survival analysis of HSPs 27 and 70 and the largest analysis of HSP110 described in CRC.

Abstract 364: The RAD51-FFPE test rapidly and reliably identifies homologous recombination deficient ovarian and endometrial carcinomas

Abstract Sensitivity to PARP inhibitors is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2-related deficiencies. Therefore, there is great interest in a robust method to identify HR-deficient (HRD) carcinomas in routine diagnostics. DNA-based assays that are developed to identify HRD are relatively complex, costly and time-consuming. As an alternative to DNA-based assays we developed the REcombination CAPacity (RECAP) test exploiting the accumulation of RAD51 protein after irradiation of fresh tumor tissue as a biomarker for HR proficiency. The RECAP test reliably identified HRD ovarian (OC) and endometrial (EC) carcinomas including those not related to BRCA1/2 deficiency. However, the implementation of the RECAP test in clinical diagnostics is hampered by dependency on ex vivo irradiation of fresh tumor tissue. We therefore explored the use of diagnostic formalin-fixed paraffin-embedded (FFPE) tumor tissue for the functional analysis of HR and aimed to determine the performance of RAD51 assessment on diagnostic FFPE blocks as compared to RECAP test. FFPE tumor tissue from tumors for which previously published RECAP scores were available (OC n = 49; EC n = 25) were subjected to a three-step quality assessment: 1) Tumor tissue quality analysis of hematoxylin and eosin stained slides by an expert pathologist, 2) Evaluation of yH2AX foci to confirm the presence of DNA damage and 3) Evaluation of the presence of a sufficient number of geminin positive (GMN+) tumor cells. When all quality criteria were met, the capacity of tumor cells to accumulate RAD51 protein at sites of endogenous DNA damage was analyzed using co-immunofluorescent staining. The RAD51-FFPE score was determined as the percentage of GMN+ cells with RAD51 foci. The performance of the RAD51-FFPE test was determined using corresponding RECAP scores. Seventy-four FFPE tumor tissues were subjected to the quality assessment. In total, 58 out of 74 (78%) met our tissue quality criteria. RAD51-FFPE scores correlated significantly with RECAP scores (p= &amp;lt;0.0001). Of the 16 tumors (including ten BRCA-related tumors) that were classified as HRD in the RECAP test (RECAP score ≤20% GMN+/RAD51+ cells), 15 showed a RAD51-FFPE score below 16%. Thirty-one out of 38 (82%) RECAP HR-proficient tumors (≥50% GMN+/RAD51+ cells) had RAD51-FFPE scores above 16%. The four tumors with RECAP scores in the HR intermediate group (20-50% GMN+/RAD51+ cells) showed RAD51-FFPE scores between 3-38%. We demonstrated that the RAD51-FFPE test on diagnostic FFPE tumor tissue is an attractive alternative to DNA-based tests to identify HRD in OC and EC. Although the optimal RAD51-FFPE threshold to identify HRD tumors needs to be established using larger cohorts with genomic and clinical trial outcome data, the RAD51-FFPE test is a promising candidate to serve as a low-cost routine diagnostic HRD test in the clinic. Citation Format: Lise M. van Wijk, Claire J. Kramer, Sylvia Vermeulen, Natalja T. ter Haar, Cor D. de Kroon, Marthe M. de Jonge, Katja N. Gaarenstroom, Harry Vrieling, Violeta Serra, Tjalling Bosse, Maaike P. Vreeswijk. The RAD51-FFPE test rapidly and reliably identifies homologous recombination deficient ovarian and endometrial carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 364.