Formalin-fixed Paraffin-embedded Colorectal Cancer Tissue Research Articles

High Wnt2 Expression Confers Poor Prognosis in Colorectal Cancer, and Represents a Novel Therapeutic Target in BRAF-Mutated Colorectal Cancer.

Background and Objectives: We aimed to investigate the role of Wnt2 expression in colorectal cancer (CRC) prognosis and evaluate its potential as a therapeutic target in BRAF-mutated CRC. Materials and Methods: Exactly 136 samples of formalin-fixed paraffin-embedded CRC tissue specimens were obtained from patients who underwent surgical resection for CRC. The gene mutation status of the samples was detected using fluorescence PCR. Wnt2 expression was detected using immunohistochemistry. Survival curves with high Wnt2 expression and BRAF mutations were compared using the Kaplan-Meier method. A nomogram was constructed to determine the estimated overall survival probability. We also predicted the 3-year and 5-year survival rates for patients with high Wnt2 expression and BRAF mutations. In total, 50 samples of BRAF-mutated CRC were collected and detected Wnt2 expression by immunohistochemistry. The Chi-squared test was used to analyze the association between Wnt2 expression and BRAF-mutated CRC. Results: High Wnt2 expression and BRAF mutations are associated with poor prognosis of CRC. Multivariate survival analyses indicated that high Wnt2 expression and BRAF mutations are significant independent predictors of CRC prognosis. Furthermore, high Wnt2 expression was significantly associated with BRAF-mutated CRC, and Wnt2 may be a potential therapeutic target for BRAF-mutated CRC. Conclusions: High Wnt2 expression confers poor prognosis in colorectal cancer and represents a novel therapeutic target in BRAF-mutated CRC.

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

Abstract 1960: Invasive potential of colorectal cancer through tumor budding in 3D

Abstract Background and Objectives: Tumor budding (TB) is defined as isolated single cancer cells or clusters of up to four cells located at the invasive tumor front of colorectal cancer (CRC) and other solid cancers. Despite the growing body of evidence establishing TB as a strong independent prognostic marker in CRC, further investigation is needed to elucidate the biological mechanisms behind TB and its invasive potential. Pseudobudding (PsB), in contrast to TB, is defined as single cancer cells or clusters of up to four cells that occur due to gland disruption and external influences such as inflammation. We hypothesize that the invasive potential of a tumor is therefore better reflected by the active process of TB rather than through the reactive process of PsB. By improved visualization of the invasive tumor front, we can characterize TB and PsB, elucidate their underlying biological processes, and improve future TB scoring accuracy in routine diagnostic practice. Methods: Cases containing TB and PsB were selected from the pathology archives. Thick sections (80-90µm) of formalin-fixed paraffin-embedded CRC tissue were taken using a microtome and stained using a free-floating immunostaining protocol for Pan-cytokeratin (epithelium) and DAPI (nuclei). Thick sections were imaged via confocal microscopy. Three-dimensional z-stacks were processed and analyzed using FIJI software. Tissue microarrays (TMAs) of regions with TB and PsB were created and RNA spatial transcriptomics was conducted on thin TMA sections (5µm) using the Nanostring GeoMx Digital Spatial Profiling Platform. Results: For sections where the invasive margin contained TB, both single cells and cell clusters appear isolated from the core tumor in 3D. These single cells and cell clusters exhibit migratory and invasive features associated with epithelial-mesenchymal transition (EMT), as evident by complete loss of cell-to-cell adhesions and shape shifting through elongation of the entire cell. In contrast, PsB is characterized by cellular debris surrounding a discontinuous epithelial lining. Undivided cells and cell clusters within the damaged area are observed as involuntarily disassociated with lagging remnants of cell-to-cell adhesions. This may be due to inflammatory cells disrupting the extracellular matrix and pulling the cells apart from one another. The identification of differing morphologies allows for further study of the RNA spatial transcriptome (of which the data will be presented during the meeting). Conclusion: Our findings show that TB and PsB are independent features within the invasive tumor margin which represent two separate biological processes. Considering the strong predictive capacity of TB as a marker of poor prognosis in CRC, TB scoring within routine diagnostic practice benefits from avoiding areas with a strong inflammatory reaction and PsB. Citation Format: Tariq Haddad, Luuk van den Dobbelsteen, Alessandro Lugli, Shannon van Lent-van Vliet, Femke Simmer, Inti Zlobec, Iris Nagtegaal. Invasive potential of colorectal cancer through tumor budding in 3D [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 1960.

Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues.

Simple SummaryThe analysis of colorectal cancer (CRC) gut microbiota can reveal crucial aspects of carcinogenesis and variation of treatment responses. Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable resource for studies in cancer genomics; however, their use in high-throughput metagenomic studies has been questioned due to several limitations in the DNA quality. In this study, we evaluated the impact of sample preservation on CRC-associated microbiota characterization. Using 16S rRNA sequencing and RNA in situ hybridization (RNA-ISH), we found differences in the comparison between paired FFPE and fresh frozen (FF) tissues, mostly derived from contamination issues. A quality index was also outlined to potentially assess the reliability of microbiome profiling obtained from FFPE DNA samples. These results suggest that tissular CRC microbiome studies should preserve internal coherence by using either FFPE or FF samples but not necessarily both.Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most widely available clinical material to study colorectal cancer (CRC). However, the accuracy and clinical validity of FFPE microbiome profiling in CRC is uncertain. Here, we compared the microbial composition of 10 paired fresh-frozen (FF) and FFPE CRC tissues using 16S rRNA sequencing and RNA-ISH. Both sample types showed different microbial diversity and composition. FF samples were enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Conversely, FFPE samples were mainly enriched in typical contaminants, such as Sphingomonadales and Rhodobacterales. RNA-ISH in FFPE tissues confirmed the presence of CRC-associated bacteria, such as Fusobacterium and Bacteroides, as well as Propionibacterium allowing discrimination between tumor-associated and contaminant taxa. An internal quality index showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. Given the importance of FFPE specimens for larger studies in human cancer genomics, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible metagenomics analyses.

Retrieving high-quality genomic DNA from formalin-fixed paraffin-embedded tissues for multiple molecular analyses

Formalin-fixed paraffin-embedded (FFPE) tissues represent the biggest source of archival materials for molecular biology research and pathology investigations. Nevertheless, fixation by formalin may cause denaturation and modification of macromolecules constraining DNA quality and its downstream applications. In this study, we developed a fast, simple, and cost-effective phenol/chloroform-based protocol for the extraction of high-quality DNA from 101 FFPE colorectal cancer tissue blocks that can be used in multiple molecular studies. DNA samples extracted using this phenol/chloroform protocol and the QIAamp DNA FFPE Tissue kit were evaluated for the quantity, quality, and amplificability. Spectrophotometer analyses revealed significantly higher quality and quantity of DNA samples obtained with the phenol/chloroform protocol as compared to those of the QIAamp DNA FFPE Tissue kit. In addition, the amplificability of these samples as assessed by conventional and multiplex polymerase chain reaction (PCR), followed by sequencing and fragment analyses presented an absolute success rate. Additionally, it is able to amplify a DNA fragment of 725 base-pairs at an adequate amount for downstream applications. This fast, simple, and cost-effective method may facilitate the molecular analyses of a large number of FFPE specimens that best suits the needs of the overall study design in terms of the quality and quantity of the extracted DNA.

Clinical and Prognostic Relevance of B7-H3 and Indicators of Glucose Metabolism in Colorectal Cancer.

ObjectiveThis study aimed to investigate the clinical and prognostic relevance of B7-H3 expression and indicators of glucose metabolism in patients with colorectal cancer (CRC).MethodsUsing immunohistochemistry, the expression of B7-H3 was detected in a total of 213 formalin-fixed paraffin-embedded CRC tissue specimens. Furthermore, levels of fasting blood glucose (FBG), lactic dehydrogenase (LDH), and fructosamine (FMN) as indicators of glucose metabolism were analyzed in CRC patients and stratified into high or low expression sub-groups based on Youden Index. The relationship between B7-H3, FBG, LDH, FMN expression, and clinicopathological characteristics were also evaluated to establish their prognostic significance in patients with CRC.ResultsB7-H3 was highly expressed in CRC tissue. The positive rates of B7-H3 expression was 63.8% (136/213). We found a linear correlation between B7-H3 and FBG in depth of tumor invasion (T3/4) (p = 0.037, r = 0.259), lymph node metastasis (N0) (p = 0.004, r = 0.259), and TNM stage (I/II) (p = 0.009, r = 0.242). High expression of FBG, LDH, FMN [hazard ratio (HR) = 1.916, 95% CI: 1.223–3.00, p = 0.005; HR = 1.801, 95% CI: 1.153–2.813, p = 0.010; HR = 2.154, 95% CI: 1.336–3.472, p = 0.002], respectively, was identified as a significant independent predictor of poor overall survival (OS). Although B7-H3 expression did not affect OS, CRC patients expressing both high B7-H3 and high FMN contributed to a significant decrease in OS (HR = 1.881, 95%CI: 1.059–3.339, p = 0.031). Moreover, with low expression of B7-H3, high expression of FBG, LDH and FMN were also recognized as predictors of inferior OS (HR = 3.393, 95% CI: 1.493-7.709, p = 0.004; HR = 7.107, 95% CI: 2.785–18.138, p = 0.000; HR = 2.800, 95% CI: 1.184–6.625, p = 0.019).ConclusionB7-H3 combined with FBG, LDH, or FMN, could reflect the clinical outcomes of patients with CRC.

Loss of Yes-associated Protein Represents an Aggressive Subtype of Colorectal Cancer.

Background: Yes-associated protein (YAP) is a downstream effecter of Hippo signaling pathway, and has been linked to the initiation and development of colorectal cancer (CRC). However, the clinical significance of YAP in CRC remains controversial. This study was designed to investigate the clinical significance of YAP in CRC.Methods: We selected 206 eligible patients diagnosed with CRC from 2003 to 2007. Tissue microarray (TMA) blocks were made from 206 formalin-fixed paraffin-embedded CRC tissues and 158 corresponding normal colonic tissues. Using the TMA blocks, we performed immunohistochemical staining of YAP and assessed its expression status in different subcellular locations. The patients were divided into four groups according to the expression status of YAP in the cytoplasm and nucleus. Statistical analysis was performed to explore the correlation between YAP expression and clinicopathological features and overall survival (OS) in CRC patients.Results: Our results showed that both cytoplasmic YAP and nuclear YAP were overexpressed in CRC tissues compared to normal colonic tissues. Complete loss of YAP expression in CRC was significantly correlated with larger tumor size (p=0.023), proximal tumor location (p=0.038), higher tumor grade (p=0.022) and worse OS (p<0.001). Univariate and multivariate Cox regression analyses revealed that complete loss of YAP expression was an independent indicator of poor prognosis in CRC (p<0.001).Conclusions: Loss of YAP expression correlates with poor prognosis and may represent a subgroup with more aggressive biological features in CRC.

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues.

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

Clinical application of targeted next-generation sequencing for colorectal cancer patients: a multicentric Belgian experience.

International guidelines made RAS (KRAS and NRAS) status a prerequisite for the use of anti-EGFR agents for metastatic colorectal cancer (CRC) patients. Daily, new data emerges on the theranostic and prognostic role of molecular biomarkers; this is a strong incentive for a validated, sensitive, and broadly available molecular screening test. Next-generation sequencing (NGS) has begun to supplant other technologies for genomic profiling. We report here our 2 years of clinical practice using NGS results to guide therapeutic decisions.The Ion Torrent AmpliSeq colon/lung cancer panel, which allows mutation detection in 22 cancer-related genes, was prospectively used in clinical practice (BELAC ISO 15189 accredited method). The DNA of 741 formalin-fixed paraffin-embedded CRC tissues, including primary tumors and metastasis, was obtained from 14 different Belgian institutions and subjected to targeted NGS.Of the tumors tested, 98% (727) were successfully sequenced and 89% (650) harbored at least one mutation. KRAS, BRAF and NRAS mutations were found in 335 (46%), 78 (11%) and 32 (4%) samples, respectively. These mutation frequencies were consistent with those reported in public databases. Moreover, mutations and amplifications in potentially actionable genes were identified in 464 samples (64%), including mutations in PIK3CA (14%), ERBB2 (0.4%), AKT1 (0.6%), and MAP2K1 (0.1%), as well as amplifications of ERBB2 (0.3%) and EGFR (0.3%). The median turnaround time between reception of the sample in the laboratory and report release was 8 calendar days.Overall, the AmpliSeq colon/lung cancer panel was successfully applied in daily practice and provided reliable clinically relevant information for CRC patients.

Clinical performance evaluation of the Idylla NRAS-BRAF mutation test on retrospectively collected formalin-fixed paraffin-embedded colorectal cancer tissue

AimsUnderstanding the molecular mechanisms of underlying disease has led to a movement away from the one-drug-fits-all paradigm towards treatment tailored to the genetic profile of the patient. The Biocartis Idylla...

Validation of targeted next-generation sequencing for RAS mutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR

ObjectiveTo validate the targeted next-generation sequencing (NGS) platform-Ion Torrent PGM for KRAS exon 2 and expanded RAS mutations detection in formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) specimens, with comparison of...

Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue using tandem mass tag protein labeling

In clinical research, repositories of biological samples form a rich source of clinical material for biomarker studies. Banked material, however, is often not stored in optimal conditions regarding the technology used for biomarker research. A case in point is formalin-fixed paraffin-embedded (FFPE) tissue that could be used to obtain large cohorts of samples over a short period of time, as these tissues are routinely prepared for pathological analysis. However, in the context of mass spectrometry based peptide-centric proteomics, protein extraction and identification can be hampered by formalin-induced crosslinking. Furthermore, the molecular formalin crosslinks might be entangled differently across various samples, making it more difficult to reproducibly extract the same proteins from different samples. In this study, we establish the crosslink variability using Tandem Mass Tag (TMT) protein labeling followed by digestion, separation, identification and quantification of proteins extracted from FFPE colorectal cancer and paired healthy tissues. Moreover, by applying de novo interpretation of tandem mass spectra and subsequent analysis by Peaks PTM, unspecified modifications could be elucidated, leading to increased protein and proteome coverage. This approach might be useful for future FFPE proteomics studies.

Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab

Background Anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) treatment are only effective in patients with KRAS wild type tumours. Here we assess the predictive value of other potential relevant markers involved in the epidermal growth factor receptor (EGFR) signalling pathways for response to cetuximab-based treatment. Materials and methods Formalin-fixed paraffin-embedded colorectal cancer tissue of the primary tumour was obtained from 559 mCRC patients treated with chemotherapy and bevacizumab with or without cetuximab (phase III CAIRO2 study). DNA was isolated for mutation analysis of BRAF (V600E) , KRAS (codon 12 and 13) and PIK3CA (exon 9 and 20). Tissue microarray’s (TMA’s) were constructed for the assessment of EGFR and HER2 gene copy number (GCN), and EGFR and PTEN protein expression. The results of these markers, individually or in combination, were correlated with progression-free survival (PFS) and overall survival (OS) in the subgroup of patients with a KRAS wild type tumour treated in the cetuximab-arm. KRAS wild type patients treated without cetuximab were used as a control group. Results A total of 208 tumours (39.4%) contained a KRAS mutation, 8.7% a BRAF mutation and 9.9% a PIK3CA mutation. Loss of PTEN expression and the presence EGFR protein expression were observed in 42.0% and 61.7% of the samples, respectively. An increased EGFR GCN was observed in 15.3% of the samples, and 11.5% of the evaluable samples contained an increased HER2 GCN. In KRAS wild type patients treated with cetuximab a BRAF mutation was significantly and independently associated with PFS and OS. In patients treated without cetuximab the PFS and OS were also associated with the BRAF genotype. No prognostic or predictive value was observed for any of the other markers when tested individually or in combination. Conclusions BRAF genotype is correlated with PFS and OS in KRAS wild type mCRC patients, which is independent of cetuximab treatment. PIK3CA mutation, loss of PTEN expression, EGFR GCN and HER2 GCN have no predictive value for response to treatment with cetuximab, neither individually nor in combination with other markers.

Abstract 4655: Gene expression signatures of microsatellite-stable stage III colorectal adenocarcinomas based on the p53 status

Abstract Background: Microsatellite (MS) instability and p53 mutations have been implicated in development, progression and metastasis of colorectal cancer (CRCs). Thus, to identify aggressive tumor subsets, the gene expression profiles of Stage- III MS-stable (MSS), sporadic CRCs were analyzed based on their p53 gene status. Methods: DNA samples extracted from 136 Stage-III formalin-fixed paraffin embedded CRC tissues were analyzed for MSS and for p53 mutations. Further, mRNA samples extracted from 20 (10 each of p53-mutated and wild-type) corresponding snap-frozen Stage-III-MSS-CRCs were profiled for differential gene expression. Affymetrix Human Genome U133 Plus 2.0 arrays were used for gene expression profiling; and Affymetrix Microarray Suite (MAS 5.0), gene ontology annotation, and KEGG pathways were used for data analysis. Differentially expressed (DE) genes were confirmed by measuring mRNA levels by RT-PCR and by the quantitative nuclease protection assay. Results: A higher incidence of p53 mutations was found in MSS (65 of 113, 58%) than in MSI (6 of 23, 30%) phenotypes. Kaplan-Meier analyses demonstrated that patients with MSS-CRCs exhibiting p53 mutations had poor overall survival when compared to p53-wt-phenotypes (log rank, p=0.031). Gene expression analyses identified 116 DE genes (73 down-regulated and 43 up-regulated). All of these passed the stringent background filter (intensity p-value ≤0.001 and intensity ≥3 SD above mean background) and DE ratio (χ2 p-value ≤0.01 &amp; fold change ≥2.0). GBP1, PSMB9, TRIM29, BST2 and TFF1, that have prognostic value in CRCs, were up-regulated in p53-mutated-MSS-CRCs but down-regulated in p53-wt-MSS-CRCs. Further, genes that regulate cell-cycle arrest (CDKN1B) and tumor growth suppression (ACVR1B, SESN1, WFDC1 and IRF7) were down-regulated in p53-mutated-MSS-CRCs, but up-regulated in p53-wt-MSS-CRCs. The genes presented here are the top-ranked (top-5) genes in their respective groups. The mRNA expression levels of these DE genes in all 136 CRCs were confirmed on two different platforms. Conclusion: Our findings suggest that mutant- and wt-p53 Stage-III-MSS-CRCs have distinct gene expression profiles and appear to be useful in identifying aggressive Stage III subsets and for the assessment of clinical outcomes. These studies are supported by grants from the National Institute of Health/National Cancer Institute (U54-CA118948, RO1-CA98932, and R03CA139629). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4655.

Matrix-comparative genomic hybridization from multicenter formalin-fixed paraffin-embedded colorectal cancer tissue blocks

BackgroundThe identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses.MethodsTo verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III) from systemically advanced (UICC stage IV) colorectal tumours.ResultsThe majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes.ConclusionArchival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.