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

Discrimination of grade 2 and 3 cervical intraepithelial neoplasia by means of analysis of water soluble proteins recovered from cervical biopsies

<p>Abstract</p> <p>Background</p> <p>Cervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value.</p> <p>Aim</p> <p>To investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3.</p> <p>Methods</p> <p>Fresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications.</p> <p>Results</p> <p>The age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification.</p> <p>Conclusions</p> <p>114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.</p

Validation of Expression Patterns for Nine miRNAs in 204 Lymph-Node Negative Breast Cancers

INTRODUCTION: Although lymph node negative (LN-) breast cancer patients have a good 10-years survival (∼85%), most of them still receive adjuvant therapy, while only some benefit from this. More accurate prognostication of LN- breast cancer patient may reduce over- and under-treatment. Until now proliferation is the strongest prognostic factor for LN- breast cancer patients. The small molecule microRNA (miRNA) has opened a new window for prognostic markers, therapeutic targets and/or therapeutic components. Previously it has been shown that miR-18a/b, miR-25, miR-29c and miR-106b correlate to high proliferation. METHODS: The current study validates nine miRNAs (miR-18a/b miR-25, miR-29c, miR-106b, miR375, miR-424, miR-505 and let-7b) significantly correlated with established prognostic breast cancer biomarkers. Total RNA was isolated from 204 formaldehyde-fixed paraffin embedded (FFPE) LN- breast cancers and analyzed with quantitative real-time Polymerase Chain Reaction (qPCR). Independent T-test was used to detect significant correlation between miRNA expression level and the different clinicopathological features for breast cancer. RESULTS: Strong and significant associations were observed for high expression of miR-18a/b, miR-106b, miR-25 and miR-505 to high proliferation, oestrogen receptor negativity and cytokeratin 5/6 positivity. High expression of let-7b, miR-29c and miR-375 was detected in more differentiated tumours. Kaplan-Meier survival analysis showed that patients with high miR-106b expression had an 81% survival rate vs. 95% (P = 0.004) for patients with low expression. CONCLUSION: High expression of miR-18a/b are strongly associated with basal-like breast cancer features, while miR-106b can identify a group with higher risk for developing distant metastases in the subgroup of Her2 negatives. Furthermore miR-106b can identify a group of patients with 100% survival within the otherwise considered high risk group of patients with high proliferation. Using miR-106b as a biomarker in conjunction to mitotic activity index could thereby possibly save 18% of the patients with high proliferation from overtreatment

Mitotic activity index and CD25+ lymphocytes predict risk of stage progression in non-muscle invasive bladder cancer

In urothelial cell type non-muscle invasive urinary bladder carcinoma, TNM stage and WHO grade are widely used to classify patients into low and high-risk groups for prognostic and therapeutic decision-making. However, stage and grade reproducibility and prediction accuracy are wanting. This may lead to suboptimal treatment. We evaluated whether proliferation features, nuclear area of the epithelial cancer cells and the composition of stromal and tumor infiltrating lymphocytes have independent prognostic value. In 183 primary non-muscle invasive bladder cancer patients with long follow-up (median for stage progression cohort: 119 months, range 5-173; median for tumor recurrence cohort: 82, range 3-165) proliferation features Ki67, PPH3 and Mitotic Activity Index (MAI), Mean Nuclear Area (MNA), lymphocyte subsets (CD8+, CD4+, CD25+) and plasma cells (CD138+) were assessed on consecutive sections. Post-resection instillation treatments (none, mitomycin, BCG) were strictly standardized during the intake period. Risk of recurrence was associated with expression of Ki67 (�39 vs.>39) and Multifocality (p = 0.01). Patients with low Ki67 had a higher recurrence rate than those with high Ki67. Lymphocyte composition did not predict recurrence. Stage progression was strongly associated with high values for MAI (>15) and CD25+ (>0.2%). In a multivariate analysis the combination of MAI and CD25+ was the single most prognostic feature (p<0.001). Validation of these results in additional, independent studies 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

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