Use of quantitative pathology to improve grading and predict prognosis in tumours of the gastrointestinal tract

Cancer represents a formidable health burden and was the second leading cause of death globally in 2018. In Norway, almost 35000 new cancer cases were reported in 2019. For colon cancer, the incidence and mortality rates in Norway are among the highest in the world. Furthermore, the tumour-node-metastasis (TNM) system used today is not optimal for selecting which patients should receive adjuvant therapy or not. With the implementation of digital pathology in different pathology departments, there will be better opportunities for digital image analysis, a tool aimed at giving a more reproducible and objective diagnosis than subjective evaluation in a microscope. In digital image analysis, a computer programme is used for the quantification of different biomarkers. This can improve cancer diagnostics because several biases in manual evaluation can be reduced or avoided. One of the challenges in pathology is intra-and inter-observer variability of prognostic and predictive biomarkers. This especially applies for gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs), in which the proliferation marker Ki67 is important for grading (1–3), prognosis and treatment of patients. Several studies have shown interand intra-observer variations in the manual evaluation of Ki67 positivity, which can be improved with digital image analysis. This is important because the interpretation of the immunohistochemical staining of different biomarkers, such as Ki67, often influences patient prognosis and treatment. The immune system, especially the number of T-cells in and around the tumour, has been investigated as a promising biomarker for predicting prognosis and survival in colorectal cancer (CRC). The immune system is closely linked to microsatellite instability (MSI) in CRC, and MSI-high CRC has been shown to respond well to immune therapy. A TNM-immune is suggested based on scoring of the number of T-cells in the tumour centre and the invasive margin using digital image analysis. In this study, we explored the correlation between T-cells in presurgical blood samples and T-cells in the invasive margins and the tumour centres in CRC with digital image analysis in a feasibility study and found a correlation. Furthermore, we used digital image analysis to calculate the immune score in colon cancer patients based on immunohistochemical (IHC) staining of cluster of differentiation (CD)3+ and CD8+ T-cells in invasive margins and tumour centres in a prospective cohort. This immune score corresponded strongly with known clinicopathological features, such as stage and MSI status. Also, we evaluated digital image analysis as an objective assessment tool for two different proliferation markers in GEP-NENs: Ki67 and Phosphohistone 3 (PHH3). We compared manual (visual) evaluation of Ki67 from pathology reports with digital image analysis of Ki67 and found excellent agreement, but there is a tendency to upgrade cases from grade 1 to grade 2 with digital image analysis. For the digital image analysis of PHH3, the measurements were more diverging. The data presented show the use of digital image analysis in two settings: developing an immune score as a prognostic marker in colon cancer and providing an objective and reproducible evaluation of proliferation in neuroendocrine neoplasms. With the transition to digital pathology, digital image analysis can be implemented in daily diagnostics. This implementation requires more research for the validation of the different methods. With time, digital image analysis is expected to be utilized for tasks performed by pathologists today.Doktorgradsavhandlin

Clinical trials of neoadjuvant immune checkpoint inhibitors for early-stage operable colon and rectal cancer

Background: Immune checkpoint inhibitors (ICI) have become first-line treatment for metastatic colorectal cancer (CRC) with deficient mismatch repair (dMMR). Despite the remarkable response reported in preliminary trials, the role of ICI in patients with early-stage, operable CRC remains unclear. The aim of this study was to investigate trials on neoadjuvant ICI in operable CRC. Materials and methods: Scoping review of clinical trial registries (Clinicaltrials.gov and EU clinical trial registers) and PubMed/Medline database of trials on neoadjuvant ICI for operable CRC was done up to December 2022. Results: Some 40 trials investigating neoadjuvant ICI for early-stage, operable CRC were identified, including five published trials and three conference abstracts. Preclinical phase I/II trial predominated with only three clinical phase III trials. Few trials investigated neoadjuvant ICI as the only intervention (monotherapy). Trials in rectal cancer were designed for combined ICI with chemo(radio)therapy, only 8 trials stating an MSI/dMMR status for inclusion, one designed for MSS/pMMR only and, the rest agnostic for MMR status. Thirty-eight (95%) trials investigated programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitors. PD-1/PD-L1 inhibitors were combined with vascular endothelial growth factor (VEGF) inhibitor or with cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) inhibitor, in two trials each, respectively. Pathological complete response as primary outcome after surgery was the most frequently used study endpoint. In rectal cancer, six trials included a “watch and wait” strategy for patients with complete clinical response. No “watch and wait” study design for colon cancer after neoadjuvant ICI were identified. Conclusion: High response rates from neoadjuvant ICI in early-stage colon and rectal cancer are reported in phase I/II studies. Contemporary trial designs are heterogeneous, with few comparable inclusion criteria, use of several drug combinations and durations and, wide variation of endpoints reported. Harmonizing clinical and translational aspects including survival data is needed for improved future trial designs with clinical impact.publishedVersio

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

Use of quantitative pathology to improve grading and predict prognosis in tumours of the gastrointestinal tract

Cancer represents a formidable health burden and was the second leading cause of death globally in 2018. In Norway, almost 35000 new cancer cases were reported in 2019. For colon cancer, the incidence and mortality rates in Norway are among the highest in the world. Furthermore, the tumour-node-metastasis (TNM) system used today is not optimal for selecting which patients should receive adjuvant therapy or not. With the implementation of digital pathology in different pathology departments, there will be better opportunities for digital image analysis, a tool aimed at giving a more reproducible and objective diagnosis than subjective evaluation in a microscope. In digital image analysis, a computer programme is used for the quantification of different biomarkers. This can improve cancer diagnostics because several biases in manual evaluation can be reduced or avoided. One of the challenges in pathology is intra-and inter-observer variability of prognostic and predictive biomarkers. This especially applies for gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs), in which the proliferation marker Ki67 is important for grading (1–3), prognosis and treatment of patients. Several studies have shown interand intra-observer variations in the manual evaluation of Ki67 positivity, which can be improved with digital image analysis. This is important because the interpretation of the immunohistochemical staining of different biomarkers, such as Ki67, often influences patient prognosis and treatment. The immune system, especially the number of T-cells in and around the tumour, has been investigated as a promising biomarker for predicting prognosis and survival in colorectal cancer (CRC). The immune system is closely linked to microsatellite instability (MSI) in CRC, and MSI-high CRC has been shown to respond well to immune therapy. A TNM-immune is suggested based on scoring of the number of T-cells in the tumour centre and the invasive margin using digital image analysis. In this study, we explored the correlation between T-cells in presurgical blood samples and T-cells in the invasive margins and the tumour centres in CRC with digital image analysis in a feasibility study and found a correlation. Furthermore, we used digital image analysis to calculate the immune score in colon cancer patients based on immunohistochemical (IHC) staining of cluster of differentiation (CD)3+ and CD8+ T-cells in invasive margins and tumour centres in a prospective cohort. This immune score corresponded strongly with known clinicopathological features, such as stage and MSI status. Also, we evaluated digital image analysis as an objective assessment tool for two different proliferation markers in GEP-NENs: Ki67 and Phosphohistone 3 (PHH3). We compared manual (visual) evaluation of Ki67 from pathology reports with digital image analysis of Ki67 and found excellent agreement, but there is a tendency to upgrade cases from grade 1 to grade 2 with digital image analysis. For the digital image analysis of PHH3, the measurements were more diverging. The data presented show the use of digital image analysis in two settings: developing an immune score as a prognostic marker in colon cancer and providing an objective and reproducible evaluation of proliferation in neuroendocrine neoplasms. With the transition to digital pathology, digital image analysis can be implemented in daily diagnostics. This implementation requires more research for the validation of the different methods. With time, digital image analysis is expected to be utilized for tasks performed by pathologists today

Elevated microsatellite alterations at selected tetranucleotides (EMAST) is not attributed to MSH3 loss in stage I-III colon cancer: An automated, digitalized assessment by immunohistochemistry of whole slides and hot spots

INTRODUCTION: EMAST is a poorly understood form of microsatellite instability (MSI) in colorectal cancer (CRC) for which loss of MSH3 has been proposed as the underlying mechanism, based on experimental studies. We aimed to evaluate whether MSH3 loss is associated with EMAST in CRC. METHODS: A consecutive cohort of patients with stage I-III CRC. Digital image analysis using heatmap-derived hot spots investigated MSH3 expression by immunohistochemistry. Fragment analysis of multiplex PCR was used to assess MSI and EMAST, and results cross-examined with MSH3 protein expression. RESULTS: Of 152 patients, EMAST was found in 50 (33%) and exclusively in the colon. Most EMAST-positive cancers had instability at all 5 markers, and EMAST overlapped with MSI-H in 42/50 cases (84%). The most frequently altered tetranucleotide markers were D8S321 (38.2% of tumors) and D20S82 (34.4%). Subjective evaluation of MSH3 expression by IHC in tumor found ≤10% negative tumor cells in all samples, most being ≤5% negative. Digital analysis improved the detection but showed a similar spread of MSH3 loss (range 0.1–15.7%, mean 2.2%). Hotspot MSH3 negativity ranged between 0.1 to 95.0%, (mean 8.6%) with significant correlation with the whole slide analysis (Spearman's rho = 0.677 P < .001). Loss of MSH3 expression did not correlate with EMAST. CONCLUSIONS: In a well-defined cohort of patients with CRC, loss of MSH3 was not associated with EMAST. Further investigation into the mechanisms leading to EMAST in CRC is needed.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