Histopathological Growth Pattern in Colorectal Liver Metastasis and The Tumor Immune Microenvironment

Almost half of all patients with colorectal cancer present with or eventually develop metastasis, most frequently in the liver. Understanding the histopathological growth patterns and tumor immune microenvironment of colorectal liver metastases may help determine treatment strategies and assess prognosis. A literature search was conducted to gather information on cancer biology, histopathological growth patterns, and the tumor immune microenvironment in colorectal liver metastases, including their mechanisms and their impact on clinical outcomes. A first consensus on histopathological growth patterns emerged in 2017, identifying five growth patterns. Later studies found benefits from a two-tier system, desmoplastic and non-desmoplastic, incorporated into the updated 2022 consensus. Furthermore, the tumor immune microenvironment shows additional characteristic features with relevance to cancer biology. This includes density of T-cells (CD8+), expression of claudin-2, presence of vessel co-option versus angiogenesis, as well as several other factors. The relation between histopathological growth patterns and the tumor immune microenvironment delineates distinct subtypes of cancer biology. The distinct subtypes are found to correlate with risk of metastasis or relapse, and hence to clinical outcome and long-term survival in each patient. In order to optimize personalized and precision therapy for patients with colorectal liver metastases, further investigation into the mechanisms of cancer biology and their translational aspects to novel treatment targets is warranted.publishedVersio

Digital Image Analysis of the Proliferation Markers Ki67 and Phosphohistone H3 in Gastroenteropancreatic Neuroendocrine Neoplasms: Accuracy of Grading Compared with Routine Manual Hot Spot Evaluation of the Ki67 Index

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are rare epithelial neoplasms. Grading is based on mitotic activity or the percentage of Ki67-positive cells in a hot spot. Routine methods have poor intraobserver and interobserver consistency, and objective measurements are lacking. This study aimed to evaluate digital image analysis (DIA) as an objective assessment of proliferation markers in GEP-NENs. A consecutive cohort of patients with automated DIA measurement of Ki67 (DIA Ki67) and phosphohistone H3 (DIA PHH3) on immunohistochemical slides was analyzed using Visiopharm image analysis software (Hoersholm, Denmark). The results were compared with the Ki67 index from routine pathology reports (pathology Ki67). The study included 159 patients (57% males). The median pathology Ki67 was 2.0% and DIA Ki67 was 4.1%. The interclass correlation coefficient of the DIA Ki67 compared with the pathology Ki67 showed an excellent agreement of 0.96 [95% confidence interval (CI): 0.94-0.96]. The observed kappa value was 0.86 (95% CI: 0.81-0.91) when comparing grades based on the same methods. PHH3 was measured in 145 (91.2%) cases. The observed kappa value was 0.74. (95% CI: 0.65-0.83) when comparing grade based on the DIA PHH3 and the pathology Ki67. The DIA Ki67 shows excellent agreement with the pathology Ki67. The DIA PHH3 measurements were more varied and cannot replace other methods for grading GEP-NENs.publishedVersio

Prevalence of PD‑L1 expression is associated with EMAST, density of peritumoral T‑cells and recurrence‑free survival in operable non‑metastatic colorectal cancer

Introduction Microsatellite instability (MSI) predict response to anti-PD1 immunotherapy in colorectal cancer (CRC). CRCs with MSI have higher infiltration of immune cells related to a better survival. Elevated Microsatellite Alterations at Tetranucleotides (EMAST) is a form of MSI but its association with PD-L1 expression and immune-cell infiltration is not known. Methods A consecutive, observational cohort of patients undergoing surgery for CRC. EMAST and clinicopathological characteristics were investigated against PD-L1, as well as CD3 and CD8 expression in the invasive margin or tumour centre (Immunoscore). Difference in survival between groups was assessed by log rank test. Results A total of 149 stage I–III CRCs patients, with a median follow up of 60.1 months. Patients with PD-L1+ tumours (7%) were older (median 79 vs 71 years, p = 0.045) and had EMAST+ cancers (OR 10.7, 95% CI 2.2–51.4, p = 0.001). Recurrence-free survival was longer in cancers with PD-L1+ immune cells (HR 0.35, 95% CI 0.16–0.76, p = 0.008, independent of EMAST) and high Immunoscore (HR 0.10, 95% CI 0.01–0.72, p = 0.022). Patients expressing PD-L1 in immune cells had longer disease-specific survival (HR 0.28, 95% CI 0.10–0.77, p = 0.014). Conclusions Higher Immunoscore (CD3/CD8 cells) and expression of tumour PD-L1 is found in CRCs with EMAST. Lymphocytic infiltrate and peritumoral PD-L1 expression have prognostic value in CRC.publishedVersio

A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

Background: In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available ‘Immunoscore®’ exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. Methods: A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. Results: Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I–II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. Conclusion: A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.publishedVersio

A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

Background In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available ‘Immunoscore®’ exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. Methods A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. Results Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I–II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. Conclusion A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.publishedVersio

Programmed death ligand-1 (PD-L1) clone 22C3 expression in resected colorectal cancer as companion diagnostics for immune checkpoint inhibitor therapy: A comparison study and inter-rater agreement evaluation across proposed cut-offs and predictive (TPS, CPS and IC) scores

Background: Expression of programmed death ligand-1 (PD-L1) guides the use of immune checkpoint inhibitors (ICI) in several cancers. In colorectal cancer (CRC), ICI are only approved for metastatic CRC, while several studies suggest high efficacy even in operable CRC. The aim of this study was to investigate the inter-rater agreement of PD-L1 as a companion diagnostic marker. Methods: Specimens from resected stage I-III CRC (n = 166 tumors) were stained with PD-L1 22C3 clone. PD-L1 expression was scored by two pathologists as tumor proportion score (TPS), combined positive score (CPS) and immune cell score (IC). Inter-rater agreement was tested using three different agreement coefficients. Results: Raw scores of the two pathologists had ‘good’ to ‘excellent’ correlation. Spearman's rho for TPS=0.917 (95 %CI 0.839–0.995), for CPS=0.776 (95 %CI 0.726–0.826) and IC=0.818 (95 %CI 0.761–0.875). For TPS, kappa (κ)-agreements for both the ≥1 % and ≥10 % cutoffs had excellent correlation. For CPS the ≥1 % and ≥10 % cutoffs demonstrated κ=0.32 (95 %CI 0.12–0.51) and κ=0.36 (95 %CI 0.25–0.48) respectively. Cutoffs for IC showed κ=0.53 (95 %CI 0.18–0.79) for the ≥1 % cutoff, and κ=0.61 (95 %CI 0.48–0.73) for the ≥10 % cutoff. Gwet's agreement coefficient (AC1) showed higher agreement coefficients than κ-values for most, but not all cut-offs. Conclusion: Agreement for PD-L1 was good to excellent for raw scores. Agreement variation across several criteria and cut-offs suggests the need for more robust criteria for PD-L1 as a companion diagnostic marker

Elevated microsatellite alterations at selected tetranucleotides in early-stage colorectal cancers with and without high-frequency microsatellite instability: same, same but different?

Microsatellite instability (MSI) is associated with better prognosis in colorectal cancer (CRC). Elevated microsatellite alterations at selected tetranucleotides (EMAST) is a less-understood form of MSI. Here, we aim to investigate the role of EMAST in CRC±MSI related to clinical and tumor-specific characteristics. A consecutive, population-based series of stage I–III colorectal cancers were investigated for MSI and EMAST using PCR primers for 10 microsatellite markers. Of 151 patients included, 33 (21.8%) had MSI and 35 (23.2%) were EMAST+, with an overlap of 77% for positivity, (odds ratio [OR] 61; P < 0.001), and 95% for both markers being negative. EMAST was more prevalent in colon versus rectum (86% vs. 14%, P = 0.004). EMAST+ cancers were significantly more frequent in proximal colon (77 vs. 23%, P = 0.004), had advanced t-stage (T3–4 vs. T1–2 in 94% vs. 6%, respectively; P = 0.008), were larger (≥5 cm vs. <5 cm in 63% and 37%, respectively; P = 0.022) and had poorly differentiated tumor grade (71 vs. 29%, P < 0.01). Furthermore, EMAST+ tumors had a higher median number of harvested lymph nodes than EMAST− (11 vs. 9 nodes; P = 0.03). No significant association was found between EMAST status and age, gender, presence of distant metastases or metastatic lymph nodes, and overall survival. A nonsignificant difference toward worse survival in node-negative colon cancers needs confirmation in larger cohorts. EMAST+ cancers overlap and share features with MSI+ in CRC. Overall, survival was not influenced by the presence of EMAST, but may be of importance in subgroups such as node-negative disease of the colon.publishedVersio

Digital Image Analysis of the Proliferation Markers Ki67 and Phosphohistone H3 in Gastroenteropancreatic Neuroendocrine Neoplasms: Accuracy of Grading Compared with Routine Manual Hot Spot Evaluation of the Ki67 Index

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are rare epithelial neoplasms. Grading is based on mitotic activity or the percentage of Ki67-positive cells in a hot spot. Routine methods have poor intraobserver and interobserver consistency, and objective measurements are lacking. This study aimed to evaluate digital image analysis (DIA) as an objective assessment of proliferation markers in GEP-NENs. A consecutive cohort of patients with automated DIA measurement of Ki67 (DIA Ki67) and phosphohistone H3 (DIA PHH3) on immunohistochemical slides was analyzed using Visiopharm image analysis software (Hoersholm, Denmark). The results were compared with the Ki67 index from routine pathology reports (pathology Ki67). The study included 159 patients (57% males). The median pathology Ki67 was 2.0% and DIA Ki67 was 4.1%. The interclass correlation coefficient of the DIA Ki67 compared with the pathology Ki67 showed an excellent agreement of 0.96 [95% confidence interval (CI): 0.94-0.96]. The observed kappa value was 0.86 (95% CI: 0.81-0.91) when comparing grades based on the same methods. PHH3 was measured in 145 (91.2%) cases. The observed kappa value was 0.74. (95% CI: 0.65-0.83) when comparing grade based on the DIA PHH3 and the pathology Ki67. The DIA Ki67 shows excellent agreement with the pathology Ki67. The DIA PHH3 measurements were more varied and cannot replace other methods for grading GEP-NENs