Archival Paraffin-embedded Tissue Research Articles

Abstract PO016: Spatial transcriptomic profiling to characterize the tumor-vascular interactome of hepatocellular carcinoma

Abstract BACKGROUND: The hepatocellular carcinoma (HCC) tumor microenvironment (TME) is composed of a complex ecosystem dominated by cancer cells and the endothelial cells that line tumor blood vessels. Although many genomic drivers have been identified and at least three transcriptional subsets have been proposed, these efforts have not yet led to novel therapies or otherwise significantly impacted management options. Single cell transcriptional profiling has generated deep insights into the multiple heterogeneous cell types within tissues, but the spatial context of these data is lost during single cell processing. Spatial transcriptomic approaches aim to bridge the gap between dissociative single cell technologies and in situ histopathological characterization.METHODS: To gain insight into potential in situ cancer-endothelial crosstalk interactions, we utilized the Nanostring GeoMx spatial transcriptomics platform with the Cancer Transcriptome Atlas ~1800 gene oligonucleotide probe panel to generate tumor (Arginase+) and blood vessel (CD31+) areas of interest (AOI) gene expression profiles from formalin-fixed, paraffin-embedded archival tissue specimens obtained from HCC resection specimens. Oligonucleotides released from each microscopic AOI were then captured, processed by DNA sequencing, and analyzed using custom computational pipelines.RESULTS: Using the 119 ROI containing data from both tumor and vessels that passed quality control filters, we performed unbiased hierarchical clustering of both the tumor and vessel areas of interest (AOI) within each ROI using the most highly variable genes for each AOI set and identified at least 3 clusters within each AOI type (tumor and vessel). Based on gene ontology analysis of the tumor AOIs, the two subsets were distinguished by unique immune and inflammatory-related genes. Analogous ontology-based characterization of the vessel AOIs demonstrated two groups: 1) an interferon-activated, inflamed progenitor, and immune checkpoint-associated cluster; and 2) a TGF-beta and oxidative stress-associated cluster. Notably, both vessel clusters also contained significant numbers of leukocyte genes, concordant with the intimate relationship of the vasculature and immune system. Canonical correlation analysis (CCA) utilizing both the most variable genes within each AOI set showed significant correlated gene sets within tumor AOIs and vessel AOIs, implying biologically significant interactions in multiple signaling pathways.CONCLUSIONS: Spatial transcriptomic profiling enables an understanding of cell-cell interactions in situ that can uncover biologically distinct tumor and blood vessel niches within the HCC microenvironment. Subsequent efforts will be focused on functionally assessing the spatially linked cancer and endothelial cell phenotypes with the goals of developing improved prognostic and predictive biomarkers and generating novel drug targets. Citation Format: Joseph W Franses, Michael J Raabe, Amaya Pankaj, Bidish Patel, Avril Coley, Irun Bhan, Martin Aryee, David T Ting. Spatial transcriptomic profiling to characterize the tumor-vascular interactome of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO016.

Possible role of high risk HPV infection and RPD3 overexpression in Iraqi patients with colorectal cancer

Aims: the aim of this study is RPD3 expression in clinicopathological characteristics in CRC patients and to study the existence of a relationship between CRC and HPV in samples of Iraqi patients. Material and methods: 30 samples of formalin-fixed, paraffin-embedded archival tissues from CRC patients were obtained, together with 10 samples of normal tissue (free cancer), for immunohistochemical analysis of RPD3 and HPV expression. Results: Immunohistochemical (IHC) staining revealed only four cases (15%) with HPV infections while PRD3 was expressed in 19 CRC samples (63.3 percent). The PRD3 expression ratio. It was found in only four (15%) of the CRC samples. At ≤ P 0. 05, there is no significant difference. Conclusion: The results of this study support RPD3 as a supplementary marker to identified histologic screening components; it could possibly be used for the prognosis and targeted therapy. In addition, we discovered a relation with both HPV infection and CRC as oncovirus and tumorigenic factor.

Comparative evaluation of immunohistochemical expression of MCM2 and Ki67 in oral epithelial dysplasia and oral squamous cell carcinoma.

The aim and objective of the study were to evaluate the immunohistochemical expression of proliferative markers, Ki67, and MCM2 in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), to compare the relationship of their staining patterns, and to look for correlation between them, if any. Thirty archival paraffin-embedded tissue blocks of previously diagnosed cases of OED, OSCC each, and 10 normal oral mucosa were used in the study. Immunohistochemical staining for MCM2 and Ki67 markers was done and the slides were individually evaluated for MCM2 and Ki67 expression, with immunopositivity determined on the basis of dark brown staining of the nucleus. The number of positively stained nuclei was counted in 10 representative areas and the data were charted and statistically analyzed. The overall mean expression of both the proteins increased progressively from normal mucosa to OED to OSCC. In normal mucosa, all positively stained nuclei were seen in the basal compartment of the epithelium, while in dysplastic cases, expression was seen toward the surface of squamous epithelium. In OSCC, the frequency of expression of MCM2 and Ki-67 proteins showed an inverse correlation with the degree of tumor differentiation. In well-differentiated cases, the positivity of either marker was restricted to the outermost layer of the tumor cells. In moderately differentiated cases, an expression of Ki-67 was more diffuse in inner layers, whereas the MCM2 antigen was found to be more intense and diffuse in both the inner and outer layers. Whereas in poorly differentiated SCC, positive expression was seen in most of the tumor cells, the mean expression of MCM2 was found to be higher than that of Ki67 in all cases. MCM2, as a proliferation marker, is superior to Ki67 as it indicates the capacity of proliferation and the ability of DNA replication of a cell.

Abstract SY25-02: Double-edged “soil”: Stromal microenvironment in breast cancer development

Abstract SY25-02: Double-edged “soil”: Stromal microenvironment in breast cancer development

Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma.

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol–chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol–chloroform DNA isolation protocol.

Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1.

DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5′/3′-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.

Unveiling the arcanum of formalin-fixed paraffin-embedded archival tissue blocks: A valuable resource for genomic DNA extraction.

Background:Formalin-fixed paraffin-embedded (FFPE) tissue blocks are routinely preserved after pathological diagnosis and possess tremendous potential for biomarker discovery. These archival samples are prone to degradation on prolonged storage due to the formalin cross-linking.Aims:This study aimed to evaluate whether the storage period of the formalin-fixed paraffin-embedded tumor blocks had a significant impact on the yield and purity of the isolated DNA archived for 11 years.Settings and Design:A retrospective study was carried out in the Department of Oral Pathology and Microbiology in accordance with the Institutional Ethics Committee.Materials and Methods:Genomic DNA extraction was performed using TaKaRa DEXPAT Easy DNA kit from 40 FFPE tissue blocks of oral squamous cell carcinoma archived for 11 years (2006–2017). NanoDrop spectrophotometer was used to determine the DNA yield (A260) and purity (A260/A280 ratio). The quality of DNA fragments was validated using agarose gel electrophoresis.Statistical Analysis Used:Statistical analysis was obtained by SPSS 22, MS Excel and analyzed using the analysis of variance (ANOVA) test. P < 0.05 was set for statistical significance.Results:There was no statistically significant difference observed both in terms of DNA yield (P = 0.996) and purity (P = 0.997) of FFPE tumor blocks archived for 11 years among the study groups.Conclusions:It was concluded that, irrespective of years of storage of the FFPE, it is possible to extract genomic DNA and use it for molecular studies.

Development of a method for isolation of melanin from archival FFPE tissues of human melanoma for structural studies by pyrolysis-gas chromatography-tandem mass spectrometry

Abstract Introduction There is some evidence that pheomelanin produced in skin melanocytes may be involved in the development of cutaneous melanoma, particularly in individuals with a light skin/red hair phenotype. However, nothing is known about possible correlation between the type and/or clinical stage of melanoma and the content of pheomelanin in the tumor tissue. We believe that archival formalin-fixed and paraffin-embedded (FFPE) melanoma tissues could be a good source of melanin pigment for future large-scale research on that issue. Aim The aim of this work was to develop a method for isolation and purification of melanin from FFPE samples of human melanoma. To test the suitability of the isolation protocol for planned structural studies, the obtained melanin was analyzed for pheomelanin content by the method based on pyrolysis (Py) coupled with gas chromatography and tandem mass spectrometry (GC/MS/MS). Material and methods For melanin isolation, microtome sections of FFPE tissue of primary lesion and lymph node metastases were subjected to a multistep procedure of paraffin removal, tissue rehydration, homogenization, and digestion with the set of proteolytic enzymes. The pigment samples were then pyrolyzed at 500 °C, and the GC-separated thermal degradation products were identified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode. Results GC/MS/MS analysis of the pyrolysis products revealed the presence of pheomelanin markers, which allowed quantitation of a pheomelanin component of each of the isolated pigments. Melanin from the FFPE primary melanoma was found to contain 6.6%, and the pigment from FFPE metastatic lymph node 7.5% of pheomelanin. Conclusions The developed protocol allows for the isolation of melanin from FFPE melanoma specimens. The pigment can be successfully studied for pheomelanin content by Py-GC/MS/MS method. The results of our study indicate that archival FFPE tumor tissues can be used as a good source of melanin for future structural studies aimed at shedding more light on the role of pheomelanin in the pathomechanism of cutaneous melanoma.

Immunohistochemical Evaluation of CD10 Expression in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma: A Cross-sectional Study

bjectives: The objective of the study is to evaluate the expression of CD10 in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC) and to determine its role as a potential biomarker in OSCC. Methods: Immunohistochemical evaluation of 40 archival paraffin-embedded tissue specimens grouped into oral epithelial dysplasia and early invasive, well-differentiated, and moderately differentiated OSCC was conducted to determine and compare the CD10 expression among the groups. Stromal positivity of the CD10 expression was calculated and statistically analyzed at p<0.05. Results: The results of the study showed that there was a statistically significant increase in the mean CD10 expression among the four groups. Intergroup comparison showed a statistically significant difference between oral epithelial dysplasia and OSCC while statistical significance was not observed in the various grades of OSCC. Conclusion: CD10 expression could be used as an important biomarker for determining the progression of oral epithelial dysplasia and the overall prognosis of OSCC. Keywords: Biomarker, CD10, oral epithelial dysplasia, oral cancer, immunohistochemistry

Cutaneous epithelioid haemangiomas show somatic mutations in the mitogen‐activated protein kinase pathway

Epithelioid haemangioma (EH) arising from the skin is a benign vascular tumour with marked inflammatory cell infiltration, which exhibits a high tendency to persist and frequently recurs after resection. So far, the underlying pathogenesis is largely elusive. To identify genetic alterations by next-generation sequencing and/or droplet digital polymerase chain reaction (ddPCR) in cutaneous EH. DNA and RNA from an EH lesion of an index patient were subjected to whole-genome and RNA sequencing. Multiplex PCR-based panel sequencing of genomic DNA isolated from archival formalin-fixed paraffin-embedded tissue of 18 patients with cutaneous EH was performed. ddPCR was used to confirm mutations. We identified somatic mutations in genes of the mitogen-activated protein kinase (MAPK) pathway (MAP2K1 and KRAS) in cutaneous EH biopsies. By ddPCR we could confirm the recurrent presence of activating, low-frequency mutations affecting MAP2K1. In total, nine out of 18 patients analysed showed activating MAPK pathway mutations, which were mutually exclusive. Comparative analysis of tissue areas enriched for lymphatic infiltrate or aberrant endothelial cells, respectively, revealed an association of these mutations with the presence of endothelial cells. Taken together, our data suggest that EH shows somatic mutations in genes of the MAPK pathway which might contribute to the formation of this benign tumour.

Interplay between copy number alterations and immune profiles in the early breast cancer Scandinavian Breast Group 2004-1 randomized phase II trial: results from a feasibility study

Emerging data indicate that genomic alterations can shape immune cell composition in early breast cancer. However, there is a need for complementary imaging and sequencing methods for the quantitative assessment of combined somatic copy number alteration (SCNA) and immune profiling in pathological samples. Here, we tested the feasibility of three approaches—CUTseq, for high-throughput low-input SCNA profiling, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) for quantitative immuno-profiling- in archival formalin-fixed paraffin-embedded (FFPE) tissue samples from patients enrolled in the randomized SBG-2004-1 phase II trial. CUTseq was able to reproducibly identify amplification and deletion events with a resolution of 100 kb using only 6 ng of DNA extracted from FFPE tissue and pooling together 77 samples into the same sequencing library. In the same samples, mfIHC revealed that CD4 + T-cells and CD68 + macrophages were the most abundant immune cells and they mostly expressed PD-L1 and PD-1. Combined analysis showed that the SCNA burden was inversely associated with lymphocytic infiltration. Our results set the basis for further applications of CUTseq, mfIHC and DIA to larger cohorts of early breast cancer patients.

Role of special stains in identification of fungi in eumycetoma among Sudanese patients in Soba University Hospital

Background: Mycetoma is a unique neglected tropical disease caused by a substantial number of microorganisms of fungal or bacterial origins. Identification of the causative organism and the disease extension are the first steps in the management of the affected patients and predicting disease treatment outcome and prognosis. Objectives: To determine the role of special stain in identification of fungi in eumycetoma among Sudanese patients at Soba University Hospital. Method: This descriptive cross- sectional study was conducted of Fifty-nine archival formalin fixed paraffin- embedded tissue blocks, four identical sections from each archival paraffin-embedded tissue block were obtained and stained with routine HE also mycetoma is most common in young adults (16–40 years old) and is uncommon in children. No significant relationship was demonstrated between diagnosis and variables (gender, age). Type I and II was the commonest type of the host tissue reaction encountered in this study. Conclusion: Results showed that GMS and PAS had the ability to stain both fungal hyphae and yeast in compare to Gridley’s stain which had some limitations to stain fungal structures.

MP66-14 DEVELOPMENT &amp; VALIDATION OF A METHOD FOR DETECTION OF CIRCULATING TUMOUR DNA IN MUSCLE-INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III (MP66)1 Sep 2021MP66-14 DEVELOPMENT & VALIDATION OF A METHOD FOR DETECTION OF CIRCULATING TUMOUR DNA IN MUSCLE-INVASIVE BLADDER CANCER Ruchira Nandurkar, Pavel Sluka, Hady Wardan, Ian Davis, and Shomik Sengupta Ruchira NandurkarRuchira Nandurkar More articles by this author , Pavel SlukaPavel Sluka More articles by this author , Hady WardanHady Wardan More articles by this author , Ian DavisIan Davis More articles by this author , and Shomik SenguptaShomik Sengupta More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002106.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Survival from muscle-invasive Bladder Cancer (MIBC) remains variable despite optimal therapy. Current clinical and pathological variables are limited in their prognostic and predictive utility. Identifying the presence and amount of circulating tumour DNA (ctDNA) in the bloodstream is of great interest as an additional tool. Currently, this relies on next-generation sequencing of tumour samples, which is expensive and can take a long time for results, thus precluding responsive clinical decision-making. This study aimed to develop a quick, sensitive, inexpensive and reliable method for detection of ctDNA in MIBC using droplet digital PCR (ddPCR). METHODS: We selected a combination of 10 mutations found in MIBC that were not only common, but when sequentially added to a gene panel, each would increase the panel’s capacity to detect a wider cohort of MIBC patients, thereby maximising the detection rate whilst maintaining assay feasibility. Mutations were selected based upon data published by The Cancer Genome Atlas (TCGA). Our 10-gene panel was tested against archival MIBC specimens. DNA extracted from archival paraffin-embedded tissue blocks was run against the gene panel using ddPCR to assess for presence and fractional abundance of the mutation in each sample. RESULTS: To date, 66 MIBC samples have been processed, of which 65 possessed at least one of the mutations within our gene panel, conferring a 98.5% detection rate, with a median of five panel mutations detected per sample (range 0-9). Our use of ddPCR may be accountable for our higher detection rate compared to the 40% predicted from the TCGA, which used Next Generation Sequencing technology. Further analyses are being undertaken to compare samples before and after neo-adjuvant chemotherapy. CONCLUSIONS: This technique utilising a 10 gene panel appears to be sensitive enough to detect mutations in the majority of patients with MIBC. Prospective validation is underway to determine the potential clinical utility of this technique. Source of Funding: Robert Bulley Research and Innovation Grant, Eastern Health Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1134-e1135 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ruchira Nandurkar More articles by this author Pavel Sluka More articles by this author Hady Wardan More articles by this author Ian Davis More articles by this author Shomik Sengupta More articles by this author Expand All Advertisement Loading ...

RNAscope compatibility with image analysis platforms for the quantification of tissue-based colorectal cancer biomarkers in archival formalin-fixed paraffin-embedded tissue

RNAscope®, has emerged as an important in-situ hybridisation method to validate mRNA expression within single cells whilst preserving tissue morphology in histological samples. The aim of this research was to compare the utility of various open-source and commercial image analysis methods, to quantify mRNA transcripts identified by RNAscope within formalin fixed paraffin embedded (FFPE) histological samples and cell monolayer preparations. Examination of MLH1 expression from 10 histological FFPE colorectal cancer specimens using four image analysis tools (Colour Deconvolution, SpotStudio, WEKA and the LEICA RNA-ISH algorithm) showed the WEKA tool as having the greatest level of agreement with manual quantification. Comparing image analysis methods to qRT-PCR for quantifying MLH1, GFI1 and TNFRSF11A expression within two colorectal cell lines results suggest that these image analysis methods perform at a similar level to qRT-PCR. Furthermore, we describe the strengths and limitations for each image analysis method when used in combination with RNAscope assays. Our study concludes that there are several freely available and commercial image analysis tools that enable reliable RNA in situ expression analysis, however operators need to consider factors, such as expected expression levels of target genes, software usability and functionality.

Molecular alterations in oral cancer using high-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue.

Loss of cell differentiation is a hallmark for the progression of oral squamous cell carcinoma (OSCC). Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues constitute a valuable resource for studying the differentiation of OSCC and can offer valuable insights into the process of tumor progression. In the current study, we performed LC-MS/MS-based quantitative proteomics of FFPE specimens from pathologically-confirmed well-differentiated, moderately-differentiated, and poorly-differentiated OSCC cases. The data were analyzed in four technical replicates, resulting in the identification of 2376 proteins. Of these, 141 and 109 were differentially expressed in moderately-differentiated and poorly differentiated OSCC cases, respectively, compared to well-differentiated OSCC. The data revealed significant metabolic reprogramming with respect to lipid metabolism and glycolysis with proteins belonging to both these processes downregulated in moderately-differentiated OSCC when compared to well-differentiated OSCC. Signaling pathway analysis indicated the alteration of extracellular matrix organization, muscle contraction, and glucose metabolism pathways across tumor grades. The extracellular matrix organization pathway was upregulated in moderately-differentiated OSCC and downregulated in poorly differentiated OSCC, compared to well-differentiated OSCC. PADI4, an epigenetic enzyme transcriptional regulator, and its transcriptional target HIST1H1B were both found to be upregulated in moderately differentiated and poorly differentiated OSCC, indicating epigenetic events underlying tumor differentiation. In conclusion, the findings support the advantage of using high-resolution mass spectrometry-based FFPE archival blocks for clinical and translational research. The candidate signaling pathways identified in the study could be used to develop potential therapeutic targets for OSCC.

Abstract PS5-06: Prospective testing for PIK3CA/AKT1/PTEN alterations in tumor tissue from 1440 patients with advanced hormone receptor-positive HER2-negative breast cancer (HR+/HER2- BC) or triple-negative breast cancer (TNBC) screened for the IPATunity130 randomized phase 3 trial

Abstract Background The PI3K/AKT signaling pathway plays a significant role in both HR+ BC and TNBC. IPATunity130 is a double-blind, placebo-controlled, randomized phase 3 trial of ipatasertib in combination with paclitaxel in patients with PI3K/AKT1/PTEN-altered HR+ or TNBC. A next-generation sequencing (NGS)-based assay from Foundation Medicine Inc. (FMI) was used to select patients prospectively for enrollment in this trial.Patients and methods An investigational clinical trial assay (CTA) of a composite 3-gene biomarker signature [Kim, Lancet Oncol 2017] based on the FoundationOne® CDx assay was used to identify patients with PI3K/AKT pathway-activated tumors as eligible for enrollment in IPATunity130 (NCT03337724). Qualifying alterations for the 3-gene signature (referred to as ‘biomarker-positive’) comprised activating mutations in PIK3CA and/or AKT1, and/or loss of function (LOF) alterations in PTEN represented by homozygous or heterozygous deletions, dominant-negative mutations, or inactivating mutations under loss of heterozygosity. Study sites were required to submit formalin-fixed paraffin-embedded archival or fresh biopsy tissue derived from primary or metastatic tumors for patient screening. IPATunity130 includes three independent cohorts: Cohort A (biomarker-positive TNBC); Cohort B (biomarker-positive HR+/HER2– BC); and Cohort C (biomarker-negative TNBC). Results In total, 1736 patients were screened, from whom 1690 samples were tested by FMI. Of these, 1475 (87%) produced a valid NGS result. The remaining 215 (13%) failed quality control for reasons including insufficient tissue, DNA yield, lab error, and computational failure. Alteration status for PIK3CA and/or AKT1 and/or PTEN was positive in 703 (49%) of 1440 CTA-evaluable samples. In the HR+/HER2– cohort, the breakdown of CTA-positive samples was 301/356 (85%) PIK3CA/AKT1 mutations and 86/356 (24%) PTEN LOF alterations. In TNBC, 183/347 (53%) had PIK3CA/AKT1 mutations and 193/347 (56%) had PTEN LOF alterations. CTA results according to baseline characteristics are shown overall and by subtype below. The most common mutations detected outside the 3-gene biomarker signature in the screened population were in the TP53, BRCA1, RB1, BRCA2, and NF1 genes in the TNBC cohort and the TP53, GATA3, CDH1, MAP3K1, and ESR1 genes in the HR+/HER2– cohort. In the HR+/HER2– cohort, 30 (14%) of 213 metastatic tumors had ESR1 mutations compared with 10/414 (2%) primary tumors. Tumor BRCA1 mutations were more common in patients aged ≤50 years whereas MAP3K1 mutations were more common in those aged &amp;gt;50 years.Conclusions IPATunity130 is the first reported pivotal trial to utilize the 3-gene biomarker CTA in HR+/HER2– BC and TNBC to screen for patients with PI3K/AKT pathway-activated tumors. The 3-gene biomarker CTA detected alterations in 49% of screened patients, with a higher prevalence of PIK3CA/AKT1/PTEN alterations in HR+/HER2– BC than TNBC. Additional analyses are planned to assess correlations between clinical outcomes and tumor characteristics. SubgroupPIK3CA/AKT1/PTEN alteration, n/N (%)OverallHR+/HER2–TNBCAll patient samples703/1440 (49)356/647 (55)347/793 (44)Age, years≤50219/475 (46)99/195 (51)120/280 (43)&amp;gt;50484/965 (50)257/452 (57)227/513 (44)Sample sourcePrimary456/941 (48)232/414 (56)224/527 (43)Metastatic222/448 (50)112/213 (53)110/235 (47)Geographic regionNorth America60/102 (59)33/49 (67)27/53 (51)Asia-Pacific172/330 (52)81/145 (56)91/185 (49)Europe301/633 (48)171/310 (55)130/323 (40)Rest of world170/375 (45)71/143 (50)99/232 (43) Citation Format: Heidi Savage, Wendy Lin, Mafalda Oliveira, Carlos Barrios, Joyce O’Shaughnessy, Nicholas Turner, Rebecca Dent, Steven J Isakoff, Shigehira Saji, Qinshu Lian, Denise Bradley, Sarah-Jayne Reilly, Heather Hinton, Matthew J Wongchenko, Aruna Mani, Sung-Bae Kim. Prospective testing for PIK3CA/AKT1/PTEN alterations in tumor tissue from 1440 patients with advanced hormone receptor-positive HER2-negative breast cancer (HR+/HER2- BC) or triple-negative breast cancer (TNBC) screened for the IPATunity130 randomized phase 3 trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-06.

Abstract PS18-27: Integrated immuno-genomic analyses in early breast cancer: Results from the Scandinavian breast group 2004-1 (SBG-2004-1) randomized phase II trial

Abstract Background: Given the emerging role of immune-related biomarkers in breast cancer (BC), little is known about specific immune cell composition patterns and genomic alterations and how these could interact in early BC setting. The aim of this proof-of-principle study is to describe immuno-genomic correlates within the tumor microenvironment using newly developed techniques for multiplexed fluorescent immunohistochemistry (mfIHC, Mezheyeuski et al., J Pathol. 2018) and high-throughput low-input genome sequencing (CUTseq, Zhang et al., Nat Commun 2019). Methods: The randomized SBG-2004-1 phase II trial evaluated the feasibility of tailored and dose-dense epirubicin and cyclophosphamide followed by docetaxel (EC→T) as adjuvant chemotherapy for early BC, enrolling a total of 124 patients. CUTseq was applied to gDNA extracted from archival low-input formalin-fixed paraffin-embedded (FFPE) tissue as well as peripheral blood samples of BC patients. mfIHC was performed on FFPE tissue microarrays, allowing simultaneous detection of six immune markers (CD4, CD8, PD-L1, PD-1, FoxP3, CD68) and quantification in an automated and tissue-compartment manner (Vectra® Polaris™ automated quantitative pathology imaging system and inForm® software, Akoya Biosciences). Patient characteristics and 10-year follow-up data were also available. Associations of different immune cell patterns and DNA copy number alterations (CNAs) with clinicopathological parameters and survival outcomes were evaluated using standard statistical methods.Results: 69/124 (55.6%) and 82/124 (66.1%) FFPE samples were available for CNA profiling (blood gDNA for normalization, n=33) and mfIHC, respectively. MYC gene was the most commonly amplified cancer-associated gene (n=44, 63.8%), and other frequent alterations included ERBB2 (n=31, 44.9%) and PIK3CA (n=14, 20.3%) amplification as well as TP53 (n=29, 42%) and PTEN (n=16, 23.2%) deletion. CNA burden (i.e., the percentage of the genome either amplified or deleted) was significantly correlated with higher tumor grade (p=0.013) but was not prognostic for relapse-free survival. CD4+ T-cells were the most abundant immune cell subpopulation, followed by CD68+ macrophages. The immune checkpoint markers PD-L1 and PD-1 were mostly expressed in CD4+ T-cells in both tumor and stroma compartments, while stromal PD-1+CD8+ and PD-1+CD68+ cell subsets were also among the ones with the higher cell density. CD4+ T-cell density was significantly correlated with tumor size, whereas the different immune cell infiltration patterns were not found to be prognostic. Combined immuno-genomic analyses revealed that high CNA burden was inversely associated with intra-tumoral CD4+ (Spearman’s r=-0.33, p=0.01) and CD8+ infiltration (Spearman’s r=-0.37, p=0.004). Conclusions: The present study indicates the feasibility of CNA profiling by CUTseq and immuno-profiling by mfIHC in FFPE samples obtained from BC patients. Moreover, our data provide a link between genomic alterations and the immune landscape in early BC and set the basis for further application of CUTseq and mfIHC in larger patient cohorts. Citation Format: Ioannis Zerdes, Michele Simonetti, Alexios Matikas, Luuk Harbers, Ning Zhang, Dimitrios Salgkamis, Susanne Agartz, Markella Zacharouli, Pablo Moreno-Ruiz, Johan Hartman, Artur Mezheyeuski, Jonas Bergh, Nicola Crosetto, Theodoros Foukakis. Integrated immuno-genomic analyses in early breast cancer: Results from the Scandinavian breast group 2004-1 (SBG-2004-1) randomized phase II trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-27.

Immunohistochemical Expression of Oestrogen and Epidermal Growth Factor Receptors in Endometrial Cancerous in Sudanese Patients

Endometrial carcinoma is the most prevalent gynecologic cancer in industrialized countries and the second most common in developing countries after cervical cancer; it is the fourth most commonly diagnosed cancer. The purpose of this study is to evaluate the immunohistochemistry expression of oestrogen receptor (ER) and Her-2/neu in Sudanese patients with endometrial carcinoma (EMC). A retrospective&nbsp;descriptive study of archival formalin-fixed paraffin-embedded tissue from hysterectomy spec

3'RNA Sequencing Accurately Classifies Formalin-Fixed Paraffin-Embedded Uterine Leiomyomas.

Simple SummaryUterine leiomyomas are benign smooth muscle tumors affecting millions of women globally. On a molecular level, leiomyomas can be classified into three main subtypes, each characterized by mutations affecting either MED12, HMGA2, or FH. Leiomyomas are still widely regarded as a single entity, although early observations suggest that different subtypes behave differently, in terms of both clinical outcomes and therapeutic requirements. The majority of classification studies on leiomyomas have been performed using fresh frozen tissue. Archival formalin-fixed paraffin-embedded (FFPE) tissue represents an invaluable source of biological material that can be studied retrospectively. Methods capable of generating high-quality data from FFPE material are in high demand. Here, we show that 3′RNA sequencing can accurately classify leiomyomas that have been stored as FFPE tissue in hospital archives for years. A targeted 3′RNA sequencing panel could provide researchers and clinicians with a cost-effective and scalable diagnostic tool for classifying smooth muscle tumors.Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3′RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3′RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3′RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.

Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives.

Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed for studying endogenous lysine acetylation, which allows identification and localization of the lysine acetylation together with quantitative comparison between samples. We demonstrated that FFPE tissues are equivalent to frozen tissues to study the degree of acetylation between patients. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with isobaric labeling and for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives. The MS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020157, PXD021986, and PXD021964.