Colorectal Cancer Resection Specimens Research Articles

Stromal CD4 (+) T Cell Subsets Mediate Antitumor Cytotoxic Immune Responses in Human Colorectal Carcinoma.

The local immune response in colorectal cancer is closely related to prognosis and therapeutic efficacy. In this study, histological analyses were performed to determine the phenotype of tumor-infiltrating lymphocytes (TILs) and their infiltration in the stromal and intratumoral regions, aiming to elucidate their interactions and prognostic effects. Multiplex fluorescent labeling was performed using surgically resected colorectal cancer specimens to investigate the infiltration of CD45RO (+) TILs, which exhibit cytotoxicity, and subsets of CD4 (+) TILs, identified by their characteristic transcription factor expression. The degree of CD45RO (+) TIL infiltration in the entire observation field or stromal area was not associated with prognosis. However, a high degree of infiltration in the tumor nest (intratumoral area) was significantly associated with a favorable prognosis. CD4 (+) TILs and their subsets were not associated with prognosis. However, stratified analyses revealed that a high degree of infiltration of stromal CD4 (+) TILs and the subsets T helper (Th)1, Th2, Th17, and regulatory T cells is necessary for the association between high intratumoral CD45RO (+) TIL infiltration and favorable prognosis. A sufficient degree of infiltration of stromal CD4 (+) TIL subsets is required for intratumoral CD45RO (+) TILs to exert toxicity against cancer cells. This highlights the significance of stromal immune reactions in achieving effective cytotoxic immune responses in the intratumoral area and demonstrates the critical role of the spatial distribution pattern of TILs in exerting their functions.

Dual excitation spectral autofluorescence lifetime and reflectance imaging for fast macroscopic characterization of tissues.

Advancements in optical imaging techniques have revolutionized the field of biomedical research, allowing for the comprehensive characterization of tissues and their underlying biological processes. Yet, there is still a lack of tools to provide quantitative and objective characterization of tissues that can aid clinical assessment in vivo to enhance diagnostic and therapeutic interventions. Here, we present a clinically viable fiber-based imaging system combining time-resolved spectrofluorimetry and reflectance spectroscopy to achieve fast multiparametric macroscopic characterization of tissues. An essential feature of the setup is its ability to perform dual wavelength excitation in combination with recording time-resolved fluorescence data in several spectral intervals. Initial validation of this bimodal system was carried out in freshly resected human colorectal cancer specimens, where we demonstrated the ability of the system to differentiate normal from malignant tissues based on their autofluorescence and reflectance properties. To further highlight the complementarity of autofluorescence and reflectance measurements and demonstrate viability in a clinically relevant scenario, we also collected in vivo data from the skin of a volunteer. Altogether, integration of these modalities in a single platform can offer multidimensional characterization of tissues, thus facilitating a deeper understanding of biological processes and potentially advancing diagnostic and therapeutic approaches in various medical applications.

National screening for colorectal cancer is associated with stage shift to earlier diagnosis.

Australia's National Bowel Cancer Screening Program consists of an immunohistochemical faecal occult blood test, targeting adults aged 50-74. Existing literature supports the principle of early detection of colorectal cancer (CRC) via national screening, but little is known about the association between colonoscopy or polypectomy rates and CRC stage over time. The aim of this study is to identify the longitudinal change to colonoscopy and polypectomy rates, and any stage shift associated with this screening program. A retrospective data-linkage study was performed using the Australian national health database (Medicare) to obtain colonoscopy and polypectomy rates between 1998 and 2017. A second prospective database of CRC resection specimens was analysed for this period. The cohort was divided based on time intervals related to the National Bowel Cancer Screening Program: pre-commencement 1998-2006 (Period A), immediately post-commencement 2007-2011 (Period B), and subsequent years 2012-2017 (Period C). Linear regression was used to test relation between annualized predictor and response variables. Annual colonoscopy rates doubled, and polypectomy rates tripled during the study (P < 0.001). Annual colonoscopy rate correlated to a lower T-stage (P = 0.038) and lower N-stage (P = 0.026), and there was a 7% increase in early CRC (stage I-II) in Period C (P < 0.001). Across the study period there was also a significant increase in right-sided tumours, and concurrent MMR deficiency and BRAF mutation. Polypectomy and colonoscopy rates increased after the introduction of the National Bowel Cancer screening program. There was a clinically significant shift to earlier CRC stage which manifested 5 years after its implementation.

221 Lynch Syndrome - Do We Really Screen for It?

Abstract Aim Firstly, assess the compliance of our Trust with the DG27 NICE Guidelines (2017) recommending universal screening of patients with Colorectal Cancer (CRC) for Lynch Syndrome (LS), using Immunohistochemistry or Microsatellite Instability testing. Secondly, provide a cost estimate for the Trust to implement universal screening for LS. Method Retrospective data collection for all CRC resection specimens reported by the Trust's Pathology Lab in 2019, and check of pathology reports, Clinic letters, and lab results through the Trust's electronic system for screening for LS. Cost estimates were calculated using information from the Histopathology Department and similar Audits by other NHS Trusts. Results From the 361 colorectal resection specimens examined at the Trust's Pathology lab, 340 were not screened for LS (NICE Guidelines compliance = 5.8%). Of the 21 screened, 12 patients did not have LS, and for 9 screening was not completed. By categorising the patients into groups by age, location of cancer, and presence of synchronous or metachronous cancers, we found that there was no consistent rationale behind screening (i.e., patients with phenotypical characteristics traditionally related to LS were not screened). We estimated that universal LS screening would cost the Trust approximately £25,000 annually. Conclusions There is extremely poor compliance with the NICE Guidelines in a Trust with one of the highest numbers of CRC resections annually. Even the Amsterdam Criteria are not used to screen patients for LS. Screening could change the surgical and oncological management of the patients and also suggest different surveillance and prophylaxis for the affected family members.

Molecular Basis of Extramural Vascular Invasion (EMVI) in Colorectal Carcinoma

Extramural vascular invasion (EMVI) is a known poor prognostic factor in colorectal carcinoma; however, its molecular basis has not been defined. This study aimed to assess the expression of molecular markers in EMVI positive colorectal carcinoma to understand their tumor microenvironment. Immunohistochemistry was performed on tissue microarrays of surgically resected colorectal cancer specimens for immunological markers, and BRAFV600E mutation (and on the tissue blocks for mismatch repair proteins). Automated quantification was used for CD8, LAG3, FOXP3, PU1, and CD163, and manual quantification was used for PDL1, HLA I markers (beta-2 microglobulin, HC10), and HLA II. The Wilcoxon rank-sum test was used to compare EMVI positive and negative tumors. A logistic regression model was fitted to assess the predictive effect of biomarkers on EMVI. There were 340 EMVI positive and 678 EMVI negative chemo naïve tumors. PDL1 was barely expressed on tumor cells (median 0) in the entire cohort. We found a significantly lower expression of CD8, LAG3, FOXP3, PU1 cells, PDL1 positive macrophages, and beta-2 microglobulin on tumor cells in the EMVI positive subset (p ≤ 0.001). There was no association of BRAFV600E or deficient mismatch repair proteins (dMMR) with EMVI. PU1 (OR 0.8, 0.7-0.9) and low PDL1 (OR 1.6, 1.1-2.3) independently predicted EMVI on multivariate logistic regression among all biomarkers examined. There is a generalized blunting of immune response in EMVI positive colorectal carcinoma, which may contribute to a worse prognosis. Tumor-associated macrophages seem to play the most significant role in determining EMVI.

P-43 Correlation of mesothelin expression with recurrence in colorectal cancer (CRC) patients

Mesothelin (MSLN) is a membrane bound protein that can promote survival, proliferation and invasion of cancer cells. Our previous work had shown a significant association between MSLN expression on resected localized CRC (Stage I-III) and future development of peritoneal carcinomatosis (PC) (p < 0.001). MSLN expression in early stage CRC was also associated with an inferior overall survival. We expanded this work by examining an additional cohort of patients at West Virginia University (WVU), WV, USA. This is a retrospective study of patients with localized CRC who underwent surgical resection from 2005-2019 at WVU. The patients were categorized into two groups based on whether they had a recurrence. Metastatic group comprised of patients who recurred and control group constituted patients who did not have a recurrence after a minimum of 5 years of surveillance. Metastatic group is further subdivided into 2 subgroups; SOM: patients who developed metastases in solid organs only; PC group: patients who developed PC at any time. MSLN staining with immunohistochemistry was performed using Rockland (MN1) mouse monoclonal antibody and scored based on intensity (0, 1+, 2+, and 3+) and percent positivity (0: 50%). MSLN total score was defined as the sum of intensity and percent positivity, with a total score of 3 or greater considered “positive” for MSLN expression. Fisher’s exact test was performed to determine the association of MSLN score and future recurrence of CRC. Out of a total of 484 patients diagnosed with localized CRC, 88 patients had a recurrence. Out of 88 patients, SOM and PC groups comprised 48 (54%) and 19 (22%) patients respectively while 21 patients (24%) had local recurrence. Control group comprised 126 patients. MSLN staining was completed on 70 patients in the entire cohort and the results are presented here. Among 70 patients, 14 patients had a recurrence in either solid organs (n-9) or peritoneum (n-5) and 56 patients did not recur. Positive MSLN score was demonstrated on resected CRC tissues in 8 out of 14 patients who had a recurrence; Five of them from the SOM group (5 out of 9 patients (55.6%)) and 3 from the PC group (3 out of 5 patients (60%)). Contrastingly, only 16 out of 56 patients (28.5%) in the control group had a positive MSLN score. Based on Fisher’s exact test, a near significant association was observed between positive MSLN score and future development of cancer recurrence (p: 0.06). No statistical significance was observed between positive MSLN score and location of metastases (SOM vs PM) likely due to limited sample size. A higher proportion of patients who had a recurrence (SOM+PC groups) had a positive MSLN score on their resected CRC specimens compared to the patients who did not recur (control group) (p: 0.06). MSLN staining results on the remaining patients is ongoing. Precision medicine informatics to identify patients at highest risk of development of future recurrence is key to personalize treatment for patients with colorectal cancer.

5-fluorouracil treatment of patient-derived scaffolds from colorectal cancer reveal clinically critical information

BackgroundColorectal cancer is a commonly diagnosed cancer worldwide. Unfortunately, many patients do not respond to standard chemotherapy treatments and develop disease relapse and metastases. Besides cancer cell specific genetic changes, heterogeneity in the tumor microenvironment contribute to the clinical presentation of the disease and can potentially also influence drug resistance. By using a recently developed patient-derived scaffold method monitoring how a standardized reporter cancer cell line adapts to various microenvironments treated with chemotherapy, we wanted to clarify how individual patient specific microenvironments influence the chemotherapy response in colorectal cancer.MethodsSurgically resected colorectal cancer specimens from 89 patients were decellularized to produce patient-derived scaffold, which were seeded with HT29 cells, cultured for 3 weeks, and treated with 5-fluorouracil. Gene expression changes of adapted and treated HT29 cells were monitored by qPCR and compared with clinical parameters including disease-free survival.ResultsThe effects of 5-fluorouracil treatment varied between different patient-derived scaffold, but generally induced a reduced expression of proliferation genes and increased expression of pluripotency and epithelial-to-mesenchymal transition genes. Interestingly, patient-derived scaffold cultures obtained from patients with disease recurrences showed a significantly less pronounced anti-proliferative effect of 5-fluorouracil and more pronounced increase of pluripotency, with MKI67 and POU5F1 being among the most significant genes linked to disease relapse in colorectal cancer.ConclusionsColorectal patient-derived scaffold can decode clinically relevant tumor microenvironmental influence of 5-fluorouracil treatment effects opening up for optimized precision medicine in colorectal cancer treatment.

Dataset for Pathology Reporting of Colorectal Cancer: Recommendations From the International Collaboration on Cancer Reporting (ICCR).

Objective:The aim of this study to describe a new international dataset for pathology reporting of colorectal cancer surgical specimens, produced under the auspices of the International Collaboration on Cancer Reporting (ICCR).Background:Quality of pathology reporting and mutual understanding between colorectal surgeon, pathologist and oncologist are vital to patient management. Some pathology parameters are prone to variable interpretation, resulting in differing positions adopted by existing national datasets.Methods:The ICCR, a global alliance of major pathology institutions with links to international cancer organizations, has developed and ratified a rigorous and efficient process for the development of evidence-based, structured datasets for pathology reporting of common cancers. Here we describe the production of a dataset for colorectal cancer resection specimens by a multidisciplinary panel of internationally recognized experts.Results:The agreed dataset comprises eighteen core (essential) and seven non-core (recommended) elements identified from a review of current evidence. Areas of contention are addressed, some highly relevant to surgical practice, with the aim of standardizing multidisciplinary discussion. The summation of all core elements is considered to be the minimum reporting standard for individual cases. Commentary is provided, explaining each element's clinical relevance, definitions to be applied where appropriate for the agreed list of value options and the rationale for considering the element as core or non-core.Conclusions:This first internationally agreed dataset for colorectal cancer pathology reporting promotes standardization of pathology reporting and enhanced clinicopathological communication. Widespread adoption will facilitate international comparisons, multinational clinical trials and help to improve the management of colorectal cancer globally.

Improved universal tumor screening program with paired testing: Experience at a large community-based teaching hospital.

e22505 Background: Lynch syndrome (LS) is the most common cause of hereditary predisposition to colon, endometrial, and other cancers. The universal tumor screening program for LS at Beaumont Health (BH) utilizes immunohistochemistry (IHC) of the mismatch repair (MMR) proteins to identify patients for genetics evaluation. We present the 8-year experience of the program at a large community-based teaching hospital. Methods: The MMR IHC results for all colorectal cancer (CRC) resection specimens screened from August 2012 to September 2020 were reviewed. Specimens with absent MLH1 and PMS2 were evaluated for MLH1 promoter hypermethylation with reflex to BRAF V600E mutation analysis. All abnormal results were referred for cancer genetics evaluation. The distribution of abnormal MMR and germline genetic testing results was analyzed. Results: Specimens from 2361 CRC resections were screened, and 511 specimens had abnormal MMR IHC (22%). Most cases of absent MLH1 and PMS2 were explained by hypermethylation or BRAF analysis (n = 338, 66% of all abnormal, 89% of MLH1 and PMS2). Of the remaining cases showing MMR deficiency (n = 173), the most common result was absence of MSH2 and MSH6 (n = 67), followed by absence of MLH1 and PMS2 (n=41, see table). Germline genetic testing of 83 individuals with abnormal MMR IHC revealed 49 cases of Lynch syndrome [MLH1 (n=9), MSH2 (n= 25), MSH6 (n=7), PMS2 (n=7), EPCAM (n=1)]. A significant proportion of cases (n=34, 40%) had negative germline testing, and had unexplained MMR deficiency. Paired germline/tumor testing was implemented in 2017, and 14 patients had this analysis. Using this approach, 5 individuals were identified to have somatic mutations explaining their result, and the proportion of unexplained cases was reduced to 29% (n = 4). Conclusions: Recent advances in cancer screening and therapeutics have underscored the importance of analyzing tumors for mismatch repair deficiency (dMMR), with known challenges to implementation and interpretation of results. The BH cancer genetics program has demonstrated successful growth of a universal tumor screening program over 8 years. This has led to the identification of a large number of dMMR tumors (22% of all CRC resections) and LS cases (2% of all CRC resections), which impacts management for patients and their families. Recent implementation of paired germline/tumor testing has improved the testing algorithm, and resulted in more accurate interpretation of a greater proportion of abnormal IHC results. This combined approach allows for focused high-risk screening for LS patients, access to novel therapeutic interventions including immune therapies for patients with dMMR tumors, and future precision oncology approaches.[Table: see text]

Factors Affecting Lymph Node Yield in Surgically Resected Colorectal Cancer Specimens

Background: Currently, the CAP protocol mandates evaluation of ≥12 Lymph nodes as a quality indicator for the adequacy of pathologic examination of colorectal cancer resection specimens. Aim: To identify factors that may influence the lymph node yield in colorectal cancer specimens and to compare with the relevant publications. &#x0D; &#x0D; Methods: The retrospective study of seventy patients with loco-regional colorectal adenocarcinomas treated by standard surgical resection from April 2015- April 2017 was included. All cases with inadequate lymph nodes had been re-grossed by another pathologist. Variables like age, gender, primary site, type of surgery, specimen length, tumour size, grade and stage, neoadjuvant therapy and tumour site perforation were evaluated for their impact on the average total number of nodes examined.&#x0D; Results: Out of seventy, eleven [15.71%] patients had inadequate mean nodal yield [MNY]. Of these eleven patients, MNY was greater in males [6.6] than in females [6.4]. MNY was lesser in patients with age &gt;50years [5.71] than patients ≤ 50 years [8]. The yield increased exponentially with increasing tumour stage and tumour size. Yield was higher in tumours with perforation. Specimens longer than 20cm had a higher yield [7.29] than in shorter specimens [5.25]. The yield was lesser when tumour is located more distally [APR:4.5 and AR &amp;sigmoid colectomy:7.7]. Seven patients had taken neoadjuvant therapy [63.6%] of whom, six had moderately differentiated adenocarcinoma &amp; one had no tumour.&#x0D; Conclusion: Factors like neoadjuvant therapy, age &amp; gender of the patient, type of surgery, length of the specimen, tumour size, grade, stage, site &amp; perforation, affect the MNY in colorectal cancers.

Feasibility and Performance of Elastin Trichrome as a Primary Stain in Colorectal Cancer Resection Specimens: Results of an Interobserver Variability Study.

Venous invasion (VI) is a powerful prognostic factor in colorectal cancer (CRC) that is widely underreported. The ability of elastin stains to improve VI detection is now recognized in several international CRC pathology protocols. However, concerns related to the cost and time required to perform and evaluate these stains in addition to routine hematoxylin and eosin (H&E) stains remains a barrier to their wider use. We therefore sought to determine whether an elastin trichrome (ET) stain could be used as a "stand-alone" stain in CRC resections, by comparing the sensitivity, accuracy, and reproducibility of detection of CAP-mandated prognostic factors using ET and H&E stains. Representative H&E- and ET-stained slides from 50 CRC resections, including a representative mix of stages and prognostic factors, were used to generate 2 study sets. Each case was represented by H&E slides in 1 study set and by corresponding ET slides from the same blocks in the other study set. Ten observers (3 academic gastrointestinal [GI] pathologists, 4 community pathologists, 3 fellows) evaluated each study set for CAP-mandated prognostic factors. ET outperformed H&E in the assessment of VI with respect to detection rates (50% vs. 28.6%; P<0.0001), accuracy (82% vs. 59%, P<0.0001), and reproducibility (k=0.554 vs. 0.394). No significant differences between ET and H&E were observed for other features evaluated. In a poststudy survey, most observers considered the ease and speed of assessment at least equivalent for ET and H&E for most prognostic factors, and felt that ET would be feasible as a stand-alone stain in practice. If validated by others, our findings support the use of ET, rather than H&E, as the primary stain for the evaluation of CRC resections.

Low level of stromal lectin-like oxidized LDL receptor 1 and CD8+ cytotoxic T-lymphocytes indicate poor prognosis of colorectal cancer.

BackgroundLectin‐like oxidized LDL receptor‐1 (LOX‐1) has been identified as a new marker for functional myeloid‐derived suppressor cells (MDSCs) that exhibit an immunosuppressive phenotype in the tumor microenvironment (TME). However, the role of LOX‐1+ cells in the TME of colorectal cancer (CRC) remains unknown.AimThis study aimed to determine the expression and significance of LOX‐1 in the TME of clinical CRC specimens.Methods and resultsWe performed immunohistochemical and genetic analyses of LOX‐1, CD8, KRAS, and BRAF in 128 resected CRC specimens and determined the expression of IFN‐γ and IL‐10 using real‐time reverse transcription‐polymerase chain reaction. We analyzed the correlation between LOX‐1, TME factors, gene alteration, clinicopathological factors, and disease prognosis. The co‐expression pattern of LOX‐1, hematopoietic markers, and a fibroblast marker was evaluated using multiplex immunofluorescence staining. Low stromal LOX‐1 expression and low intratumoral CD8+ cytotoxic T‐lymphocyte (CTL) status correlated with poor prognosis. Moreover, stromal LOX‐1‐low/CD8+ CTL‐low status was the most important independent prognostic factor of poor overall survival. Most of the LOX‐1+ stromal cells were positive for CD163+, indicating they were CD163+ M2 macrophages.ConclusionsThe MDSC marker, LOX‐1, was mainly expressed by M2 macrophages in CRC tissues. LOX‐1+ macrophages and CD8+ CTLs may serve as useful biomarkers for predicting the prognosis of CRC.

MicroRNA-552 expression in colorectal cancer and its clinicopathological significance

BackgroundMicroRNA-552 (miR-552) has been reported to correlate with the development and progression of various cancers, including colorectal cancer (CRC). This study aimed to investigate miR-552 expression in cancer tissue samples compared to normal mucosal tissue and its role as a diagnostic or prognostic marker in CRC patients.MethodsNormal mucosal tissues and primary cancer tissues from 80 surgically resected CRC specimens were used. Quantitative real-time polymerase chain reaction was performed for miR-552 and U6 small nuclear RNA to analyze miR-552 expression and its clinicopathological significance. Immunohistochemistry for p53 and phosphatase and tension homolog (PTEN) was performed to evaluate their association with miR-552 expression.ResultsmiR-552 expression was significantly higher in primary cancer tissues compared to normal mucosal tissues (p<.001). The expression level of miR552 was inversely correlated with that of PTEN (p=.068) and p53 (p=.004). Survival analysis showed that high miR-552 expression was associated with worse prognosis but this was not statistically significant (p=.255). However, patients with CRC having high miR-552 expression and loss of PTEN expression had significantly worse prognosis than others (p=.029).ConclusionsOur results suggest that high miR-552 expression might be a potential diagnostic biomarker for CRC, and its combined analysis with PTEN expression can possibly be used as a prognostic marker.

Time dependent effect of cold ischemia on the phosphoproteome and protein kinase activity in fresh-frozen colorectal cancer tissue obtained from patients

BackgroundBased on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics.MethodsBiopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT.ResultsUnsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p < 0.01 and fold change > 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p < 0.01). Only two phosphopeptides were significantly changed in all time points, including one peptide (PKP3) with a fold change > 2.ConclusionsThe vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

SIRT3-Mediated SOD2 and PGC-1α Contribute to Chemoresistance in Colorectal Cancer Cells.

Anticancer drugs generate excessive reactive oxygen species (ROS), which can cause cell death. Cancer cells can resist this oxidative stress, but the mechanism of resistance and associations with chemoresistance are unclear. Here, we focused on Sirtuin 3 (SIRT3), a deacetylating mitochondrial enzyme, in oxidative stress resistance in colorectal cancer (CRC). To evaluate SIRT3-related changes in mitochondrial function, ROS (mtROS) induction, and apoptosis, we used the human CRC cell lines HT29 and HCT116 transfected with short-hairpin RNA targeting SIRT3 and small interfering RNAs targeting superoxide dismutase 2 mitochondrial (SOD2) and peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α). In 142 clinical specimens from patients with CRC, we also assessed the association of SIRT3 protein levels (high/low) and prognosis. SIRT3 expression correlated with mtROS generation and apoptosis induction in cells treated with anticancer agents. Suppressing SIRT3 increased mtROS levels and cell sensitivity to anticancer agents. SIRT3 knockdown decreased SOD2 expression and activity, and suppressing SOD2 also improved sensitivity to anticancer drugs. In addition, SIRT3 was recruited with PGC-1α under oxidative stress, and suppressing SIRT3 decreased PGC-1α expression and mitochondrial function. PGC-1α knockdown decreased mitochondrial activity and increased apoptosis in cells treated with anticancer drugs. In resected CRC specimens, high vs low SIRT3 protein levels were associated with significantly reduced cancer-specific survival. SIRT3 expression affected CRC cell chemoresistance through SOD2 and PGC-1α regulation and was an independent prognostic factor in CRC. SIRT3 may be a novel target for CRC therapies and a predictive marker of sensitivity to chemotherapy.

Flow cytometry detection of cell type-specific expression of programmed death receptor ligand-1 (PD-L1) in colorectal cancer specimens

AimPD-1/PD-L1 blockade therapy is now widely used for the treatment of advanced malignancies. Although PD-L1 is known to be expressed by various host cells as well as tumor cells, the role of PD-L1 on non-malignant cells and its clinical significance is unknown. We evaluated cell type-specific expression of PD-L1 in colorectal cancer (CRC) specimens using multicolor flow cytometry. MethodsSingle cell suspensions were made from 21 surgically resected CRC specimens, and immunostained with various mAbs conjugated with different fluorescent dyes. Tumor cells, stromal cells, and immune cells were identified as CD326(+), CD90(+) and CD45(+) phenotype, respectively. CD11b(+) myeloid cells, CD19(+) B cells and CD4(+) or CD8(+) T cells were also stained in different samples, and their frequencies in the total cell population and the ratio of PD-L1(+) cells to each phenotype were determined. ResultsPD-L1 was expressed by all the cell types. The ratio of PD-L1(+) cells to CD326(+) tumor cells was 19.1% ± 14.0%, lower than those for CD90(+) stromal cells (39.6% ± 16.0%) and CD11b(+) myeloid cells (31.9% ± 14.3%). The ratio of PD-L1(+) cells in tumor cells correlated strongly with the ratio in stromal cells, while only weakly with that in myeloid cells. Tumor cells were divided into two populations by CD326 expression levels, and the PD-L1 positive ratios were inversely correlated with the rate of CD326 highly expressing cells as well as mean fluorescein intensity of CD326 in tumor cells, while positively correlated with the frequencies of stromal cells or myeloid cells in CRC. ConclusionPD-L1 is differentially expressed on various cell types in CRC. PD-L1 on tumor cells may be upregulated together with CD326 downregulation in the process of epithelial mesenchymal transition. Quantification of cell type-specific expression of PD-L1 using multicolor flow cytometry may provide useful information for the immunotherapy of solid tumors.

Using the Fat-Clearing Technique to Improve Lymph Node Retrieval in Colorectal Cancer.

College of American Pathologists recommended that at least 12 lymph nodes should be harvested for adequate staging of colorectal carcinoma. Lymph node harvesting is routinely performed by a manual technique of inspection and palpation, which is laborious and time-consuming. The study assessed the influence of the improved fat-clearing technique on the number of lymph nodes retrieved from colorectal cancer specimens and the clinical efficacy. Seventy colorectal cancer resection specimens were examined and assessed by 4 pathology residents. Thirty-five specimens were handled with the conventional manual technique by inspection and palpation, and the other 35 specimens with the improved fat-clearing technique to retrieve lymph nodes. As a result, compared with the conventional manual technique, the numbers of lymph nodes retrieved with the improved fat-clearing technique were significantly increased from 14.7 ± 6.2 lymph nodes to 20.8 ± 9.0 lymph nodes per specimen (P < .05). Besides, the percentage of cases with at least 12 lymph nodes retrieved increased from 80% to 91%. The result of this study pointed out that using the improved fat-clearing technique to process colorectal specimens could increase the lymph node yield effectively, and was effective, practical, and suitable for routine gross examination.

Impact on colorectal cancer pathology reporting practice of migration from TNM 5 to TNM 8.

The introduction of TNM 8 into UK pathology practice in January 2018 considers tumour deposits in colorectal cancer staging for the first time. The impact of this new classification on pathology reporting practices has yet to be evaluated. A clinical audit was conducted, comparing consecutive colorectal cancer resection specimens reported under TNM 5 classification guidelines in 2017 (n=177) and TNM 8 guidelines in 2018 (n=234). Tumour features (venous invasion, perineural invasion, lymph node metastatic disease, tumour deposits) and changes in reporting practices were evaluated among four specialist gastrointestinal pathologists working within a large pathology department. Adoption of TNM 8 practice led to an approximate doubling in the use of ancillary stains (41.0% of TNM 8 versus 22.0% of TNM 5 cases, P<0.001) to help evaluate tumours. A narrowing of the range between pathologists was observed in reporting cases as having one or more form of regional, extramural, discontinuous tumour (TNM 5 range=50.0-79.0%, TNM 8 range=57.8-65.7%), with no change in the overall proportion of cases reported as such (62.7% versus 62.4%, P=0.95). However, significant interobserver variation in reporting rates for individual parameters remained. TNM 8 colorectal cancer staging offers potentially greater reproducibility in pathology reporting of regional, extramural, discontinuous disease with similar proportions of patients reported as having one or more of these forms of tumour spread compared with TNM 5. Further guidance in defining individual features is required to reduce interobserver variation in pathology assessments and to help elucidate the clinical significance of each parameter.

Detection of Mismatch Repair and Microsatellite Instability in Colorectal Cancer Patients

Introduction: Colorectal cancer is the third most common cancer worldwide. Microsatellite instability (MSI) contributes to be one of the main mechanisms in colorectal cancer. Individuals with MSI tumors have loss of expression of one or more Mismatch Repair proteins. MSI tumors have better survival rate than microsatellite stable (MSS) tumors, poor response to 5FU-based adjuvant chemotherapy and relatively successful immunotherapy in metastatic MSI tumors. Immunohistochemistry recognizes altered gene by recognizing loss of its protein product. Based on the presence or absence of Mismatch repair proteins, groups are classified into Mismatch repair proficient (MMR-p) and Mismatch repair deficient (MMR-d). Aim: To investigate the immunohistochemical profile of Mismatch repair proteins namely: hMLH1, hMSH2, hMSH6, and hPMS2 in surgically resected colorectal cancer specimens. Materials and Method: A total of 76 cases were selected from the Histopathology Department of HTAA to determine MMR protein expression status. Cases were either MMR-p or MMR-d. Results: Of the specimens which were properly immunostained, seventeen out of seventy-six cases (22.37%) showed loss of one or more MMR proteins expression and thus were MMR-d. MLH1, MSH2, MSH6 and PMS2 protein expression was detected as 85.53% (65/76), 81.6% (62/76), 88.16% (67/76), and 76.32% (58/76), respectively. Conclusion: Mismatch repair proteins profile should be done using immunohistochemistry in local laboratories on these selected cases before referring for the expensive molecular test.

Association of p53, VEGF and Microvascular Density in Colorectal Carcinoma

Introduction: Colorectal Cancer (CRC) is third most common malignancy worldwide. Various genomic alterations play fundamental role in initiation and progression of CRC. Among these, p53 mutation has a crucial role in survival and metastasis and its point mutation induces Vascular Endothelial Growth Factor (VEGF) promoting vascular permeability, migration and differentiation. The degree of angiogenesis can be measured by Microvascular Density (MVD) using CD34, which is helpful in identifying high risk patients for recurrence and metastasis. Aim: The aim of the study was to analyse the expression of p53, VEGF and MVD in CRC and their association with clinicopathological parameters. Materials and Methods: The ambispective study of 2 year duration was conducted from September 2015 to July 2017 in the Department of Pathology, Kalinga Institute of Medical Sciences and PBMH, Bhubaneswar. It included CRC resection specimens and archival tissue blocks. Tissue microarray blocks were prepared manually for IHC application in total 70 cases (58 (82.9%) adenocarcinomas and 12 (17.1%) adenomas) which were histologically staged and graded as per American Joint Committee on Cancer (AJCC) and World Health Organisation (WHO) guidelines. Pearson chi-square test and fisher’s-exact method were used to find significance of p53, VEGF and CD34 expression in adenomas and adenocarcinomas with respect to clinicopathological parameters. Results: No significant statistical association was found between p53, VEGF and MVD with tumour grade and nodal status. Majority, 41 (70.69%) cases were hypervascular (MVD-High). Adenomas 9 (75%) cases, were mostly hypovascular (MVD-Low) with p-value of 0.003. There was significant statistical association between VEGF and MVD with a p-value of 0.01. VEGF and MVD were more expressed on left-sided colon cancers. There was significant statistical association (p=0.01) between p53 graded expression and diagnosis in the present study. MVD and tumour nodal status had an inversely significant relationship (p=0.03). Conclusion: p53 and VEGF expressed more on carcinomas than adenomas. Both p53 and VEGF induce angiogenesis which can be effectively measured by CD34 expression (MVD). There is a directly proportional relationship of angiogenesis and malignant transformation. So these three IHC markers together can be considered a significant prognostic factor involved in CRC.