Total Number Of Tumor Cells Research Articles

A Cancer Subpopulation Competition Model Reveals Optimal Levels of Immune Response that Minimize Tumor Size.

Breast cancer is a complex disease with significant phenotypic heterogeneity of cells, even within a single breast tumor. Emerging evidence underscores the significance of intratumoral competition, which can serve as a key contributor to cancer drug resistance, imparting substantial clinical implications. Understanding the competitive dynamics is paramount as it can significantly influence disease progression and treatment outcomes. In the present work, a mathematical model was developed using a system of differential equations to describe the dynamic interactions between two cancer subtypes (each further classified into cancer stem cells and tumor cells) and innate immune cells. The purpose of the model is to comprehensively understand the competitive interactions between the heterogeneous subpopulations. The equilibrium points and stability analysis for each equilibrium point were established. Model simulations showed that the competition between two cancer subtypes directly affects the number of both species. When competition between two cancer subtypes is strong, increasing the immune response rate specific to the more competitive species effectively reduces the tumor size. However, if the competition is relatively weak, an optimal immune response rate is required to minimize the total number of tumor cells. Rates below the optimal level fail to reduce the population of the stronger species, whereas rates above the optimal level can lead to the recurrence of the weaker species. Overall, this model provides insights into breast cancer dynamics and guides the development of effective treatment strategies.

Research on the application of mathematical modeling in tumor immunology in the context of chemotherapy

Cancer is not only a highly detrimental disease but also a particularly grave health concern. Moreover, the current incidence and mortality rates in our country are far from encouraging, making the prevention and control situation very challenging. Therefore, identifying the most scientific and effective treatment methods has become one of our primary research focuses. This paper, building upon previous models and incorporating resistance factors, categorizes tumor cells into those that are sensitive to chemotherapy drugs and those that become resistant. Using MATLAB, we have adjusted various sensitivity parameters in the model to simulate the number of tumor cells over 40 days. This simulation aims to analyze the sensitivity levels of tumor cells to different parameters upon the inclusion of resistance factors. The initial data used for the simulation were derived from the original paper. Ultimately, our findings indicate that tumor cells are most sensitive to the chemotherapy drugs killing rate for normal tumor cells and the decay rate of the chemotherapy drug. Due to the drug resistance factor, the sensitivity of different parameters is influenced. For parameters related to chemotherapy drugs, the final results, when incorporating this factor, may deviate significantly from those of previous models without this factor. For instance, the decay rate of chemotherapy drugs might result in a larger total number of tumor cells or a steeper trend compared to previous findings.

High PD-L1 Expression May be Associated with Worse Overall Prognosis in Acute Myeloid Leukemia

Background: PD-L1 expression has been explored in solid tumours and in classical Hodgkin's lymphoma, however its expression in acute leukemias has been minimally studied. PD-L1 over-expression in AML blasts has previously been associated with worse outcomes in patients with mutated FTL3 genes, and positively correlated with TP53 expression. Over-expression of PD-L1 may contribute to failed response to hypo-methylating agents, leading to the rationale behind combination therapy with immune-checkpoint inhibitors. Despite evidence that the PD-1/PDL-1 axis may promote myeloid neoplasms by modulating immune escape, clinical trials studying the role of immunotherapy in MDS/AML have produced disappointing results. The lack of evidence to support the role of PD-1-targeted therapy in AML may be due to lack of identification of the subset of patients who may benefit from immunotherapy. We previously demonstrated that PD-L1 expression is significantly enhanced in patients with JAK2/STAT mutations compared to patients with wild-type (WT) JAK2/STAT. Here, we investigate the clinical correlate of our findings and compare clinical outcomes of AML patients with respect to PD-L1 expression. Methods: Immunohistochemical staining for PD-L1 was previously performed on bone marrow biopsies of 31 AML patients, including 11 patients with JAK2/STAT mutations (nine with JAK2 V617F exon 14 mutations, one with JAK2 I540-E543delinsRG exon 12 mutation and one with STAT5B N642H mutation) and 20 patients with WT JAK2/STAT. PD-L1 CPS was calculated by summation of all PD-L1-stained cells divided by total number of tumour cells, and multiplied by 100. Retrospective chart review was performed to extract data regarding patient demographics, cytogenetics, mutations, treatment course, as well as clinical outcomes. Low and high PD-L1 expression was defined as CPS score <10 and > 20 respectively. Kaplan-Meier analysis was used for analysing overall survival (OS) and event-free survival (EFS), with statistical significance determined using a 2-tailed log-rank test. EFS was defined as duration of time between diagnosis and the first occurrence of either going off protocol induction without achieving complete remission (CR), relapse from CR, or death due to any cause. Patients who were alive at the time of study analysis were censored at date of last follow-up. Results: A total 15 patients demonstrated high PD-L1 expression (lowest PD-L1 CPS 20), including four patients with WT JAK2/STAT and all 11 patients with mutant JAK2/STAT. 16 patients demonstrated low PD-L1 expression (highest PD-L1 CPS 6). Only three patients in the low PD-L1 expression group never achieved complete remission compared to eight patients in the high PD-L1 expression group. Median OS in the low PD-L1 expression group was significantly longer than median OS in the high PD-L1 expression group (29.1 months [95% CI 1.4-7.2] vs 9.3 months [95% CI 0.1-0.7]; HR 2.4 [95% CI 1.1-5.6], p = 0.0253, Figure 1). Similarity, EFS was significantly longer in the low PD-L1 expression group (13.1 months [95% CI 1.3-6.7] vs. 4.4 months [95% CI 0.1-0.8]; HR 2.4 [95% CI 1.1-5.6], p = 0.0253). When analysis was separated by European LeukemiaNet (ELN) risk stratification, significant differences in OS and EFS were re-demonstrated in patients with favourable ELN risk, but not in patients with intermediate or adverse risks. When wild-type JAK2/STAT patients were analyzed separately, there was no significant differences between OS and EFS with respect to PD-L1 expression. Conclusions: We previously demonstrated that PD-L1 is significantly up-regulated in AML patients who carry mutant JAK2/STAT when compared to WT JAK2. Using a proposed PD-L1 CPS cut-off score of 20, we demonstrated that high PD-L1 expression was associated with significantly worse outcomes with respect to OS and EFS. These results may indicate a therapeutic role of immunotherapy in the treatment of AML, particularly in patients with JAK2/STAT mutations.

Polycystin-2 Associates With Malignancy in Meningiomas.

The involvement of polycystin-2 (PC2) in cell survival pathways raises questions about its role in carcinogenesis. Aberrant expression of PC2 has been associated with malignancy in various tumors. No evidence exists referring to PC2 expression in meningiomas. The aim of this study was to investigate the expression levels of PC2 in meningiomas and compare them with normal brain samples including leptomeninges. PC2 immunohistochemical expression was quantitatively analyzed in archival tissue from 60 patients with benign (WHO grade 1) and 22 patients with high-grade (21: WHO grade 2 and 1: grade 3) meningiomas. Specifically, the labeling index [the percentage of positive (labeled) cells out of the total number of tumor cells counted] was determined. PC2 mRNA levels were evaluated by quantitative real-time polymerase chain reaction. PC2 immunostaining was not detected in the leptomeninges. Gene expression analysis revealed increased levels of PC2 in WHO grade 1 ( P = 0.008) and WHO grade 2 ( P = 0.0007) meningiomas compared with that of normal brains. PC2 expression was significantly associated with an ascending grade of malignancy by both immunohistochemistry and quantitative real-time polymerase chain reaction ( P < 0.05). Recurrent meningiomas displayed higher levels of PC2 compared with primary meningiomas ( P = 0.008). Although no significant association of PC2 with the overall survival of the patients was found ( P > 0.05), it was noticed that the patients with WHO grade 2 meningiomas with low expression of PC2 survived longer compared with the patients with WHO grade 1 meningioma with high expression of PC2 (mean survival 49.5 and 28 months, respectively). The above results indicate a possible association of PC2 with malignancy in meningiomas. However, the mechanisms underlying PC2 implication in meningioma pathogenesis should be further elucidated.

Correlation between C-MYC and BAX expression with various Ann Arbor stages in B-cell large cell type of Non-Hodgkin lymphoma

Diffuse large B-cell lymphoma (DLBCL) is one of the B-cell large cell types of non-Hodgkin lymphoma (NHL) that has poor prognosis with highly variable clinical course. Various prognostic factors have been proposed to predict this, but the results were variable. C-MYC is a proto-oncogen that can cause overexpression leading to the increased of tumor cells proliferation. BAX is a main proapoptotic member of the BCL-2 family proteins that regulates apoptotic function. The study aimed to analyze correlation of c-MYC and BAX protein with various Ann Arbor stages in B-cell large cell type of NHL. This cross-sectional study was performed on 39 formalin fixed paraffin-embedded tissue of patients diagnosed as B-cell large cell type of NHL during January 2017 - December 2019 in Anatomical Pathology Laboratory at Dr. Soetomo General Hospital, Surabaya. To assess the expression of c-MYC and BAX, the immunohistochemistry examination was performed. Immunoexpression of C-MYC and BAX were evaluated according to the number of positive tumor cells divided by the total number of tumor cells and calculated in percentage. There was no difference in C-MYC (p = 0.877) and BAX (p = 0.093) expression with various Ann Arbor stages in B-cell large cell type of NHL. There was no correlation between c-MYC with BAX expression in various Ann Arbor stages in B-cell large cell type of NHL (rs = 0.206, p = 0.209). This indicated that C-MYC and BAX expression alone could not to be used as parameters to predict the outcome of the B-cell large cell type of NHL via Ann Arbor stages.

Expression of substance P, neurokinin 1 receptor, Ki-67 and pyruvate kinase M2 in hormone receptor negative breast cancer and evaluation of impact on overall survival

BackgroundChronic inflammation is a hallmark of cancer, and it can be stimulated by many factors. Substance P (SP), through binding to neurokinin 1 receptor (NK1R), and pyruvate kinase M2 (PKM2) play critical roles in cancer development and progression via modulating the tumor microenvironment. This study aimed to investigate the prognostic significance of SP and PKM2 in combination with NK1R and Ki-67 in hormone receptor negative (HR-ve) breast cancer.MethodsImmunohistochemical expression levels of SP, NK1R, PKM2, and Ki-67 were measured in 144 paraffin-embedded breast cancer tissues (77 h -ve and 67 h + ve). SP, NK1R, and PKM2 were scored semiquantitatively, while Ki-67 was obtained by the percentage of total number of tumor cells with nuclear staining. The optimal cutoff value for SP, NK1R, PKM2, and Ki-67 were assessed by Cutoff Finder.ResultsHigh SP expression in HR -ve breast cancer was associated with TNM stage (p = 0.020), pT stage (p = 0.035), pN stage (p = 0.002), axillary lymph node metastasis (p = 0.003), and NK1R expression level (p = 0.010). In HR + ve breast cancer, SP expression was associated with HER2 status (p = 0.001) and PKM2 expression level (p = 0.012). Regarding PKM2 expression level, it significantly associated with HER2 status (p = 0.001) and history of DCIS (p = 0.046) in HR-ve tumors, and with HER2 status (p < 0.001) and SP expression level (p = 0.012) in HR + ve tumors. Survival analysis revealed that high SP level negatively impacted overall survival in HR-ve tumors that had low NK1R level (p = 0.021). Moreover, high SP negatively impacted overall survival in HR-ve tumors that had low Ki-67 level (p = 0.005). High PKM2 negatively impacted overall survival in HR-ve cases with low SP (p = 0.047).ConclusionCombined expression levels of SP with NK1R or Ki-67, and PKM2 with SP could be used to predict survival in breast cancer patients with HR-ve tumors. Our findings suggest a role of SP/NK1R pathway and PKM2 in HR-ve breast cancer pathogenesis which should be further investigated to unveil the underlying molecular mechanisms.

Digital Quantification of Tumor Cellularity as a Novel Prognostic Feature of Non–Small Cell Lung Carcinoma

Non-small cell lung carcinoma is currently staged based on the size and involvement of other structures. Tumor size may be a surrogate measure of the total number of tumor cells. A recently revised reporting system for adenocarcinoma incorporates high-risk histologic patterns, which may have increased cellular density. Modern digital image analysis tools can be utilized to automate the quantification of cells. In this study, we tested the hypothesis that tumor cellularity can be used as a novel prognostic tool for lung cancer. Digital slides from The Cancer Genome Atlas lung adenocarcinoma (ADC) data set (n= 213) and lung squamous cell carcinoma (SCC) data set (n= 90) were obtained and analyzed using QuPath. The number of tumor cells was normalized with the surface area of the tumor to provide a measure of tumor cell density. Tumor cellularity was calculated by multiplying the size of the tumor with the cell density. Major histologic patterns and grade were compared with the tumor density of the lung ADC and lung SCC cases. The overall and progression-free survival were compared between groups of high and low tumor cellularity. High-grade histologic patterns in the ADC and SCC cases were associated with greater tumor densities compared with low-grade patterns. Cases with lower tumor cellularity had improved overall and progression-free survival compared with cases with higher cellularity. These results support tumor cellularity as a novel prognostic tool for non-small cell lung carcinoma that considers tumor stage and grade elements.

MPC-10 RECURRENT ASTROCYTOMA WITH SOMATIC IDH MOSAICISM: A CASE REPORT AND WARRANT FOR MOLECULAR DIAGNOSTICS

Abstract Introduction Acquisition of somatic mutations is essential for the development of malignant tumors such as diffuse gliomas. Recent single-cell analyses have pointed out the importance of somatic mosaicism in the pathogenesis of cancer. Here we report a case of IDH (isocitrate dehydrogenase)-mutant astrocytoma that has relapsed after intensive chemoradiation with somatic mosaicism for the IDH1 R132H mutation. Clinical and Pathological Findings A 36-year-old male presented with epileptic symptoms and a mass lesion in the right frontal lobe. A needle biopsy demonstrated IDH-mutant astrocytoma (grade 2). At the age of 41, relapsed tumor was completely resected, and the tumor cells were diffusely positive for IDH1 R132H with grade 4 morphology. Chemoradiotherapy (PAV + IMRT 60Gy/30fr) was added, but contrast-enhanced lesions reappeared 5 months after the initial surgery. The re-excised specimen was grade 4 astrocytoma, and the number of IDH1 R132H-positive tumor cells was very small compared to the total number of tumor cells, suggesting somatic mosaicism for IDH mutations. Conclusion It is imperative to clarify the induction mechanism and significance of somatic cell mosaicism in cancer, and molecular genetic assessment with sequencing and digital PCR would be necessary in addition to immunostaining for accurate molecular diagnoses of the brain tumors.

Oncogenic JAK2/STAT Signaling Molecules Activation Is Associated with PD-L1 Upregulation in Acute Myeloid Leukemia

Background: Increased evidence suggests the PD-1/PD-L1 axis is involved in the oncogenesis of myeloid neoplasms through promoting cancer immune escape, which suggests potential benefits by blockade of this axis in these neoplasms by immune checkpoint inhibitors. However, the ongoing clinical trials on PD-1/PD-L1 blocker in MDS/AML have reported controversial results, unlike the promising therapeutic efficacy shown in solid tumors. Identifying reliable biomarkers and scoring of PD-L1 expression and targeting the subgroup of patients who might benefit the most from PD-1/PD-L1 blockade are promising directions. Janus kinase 2 (JAK2) pathway activation has been shown to enhance PD-L1 expression in malignancies by in vitro studies. Here, we investigated PD-L1 expression associated with JAK2/STAT oncogenic pathway in AML patients using the combined positive score (CPS) algorithm. Methods: Immunohistochemical staining (IHC) for PD-L1 and pSTAT3 was performed on the marrow biopsies of 31 AML patients (39 cases), including 11 patients (17 cases) with JAK2/STAT mutation and 20 patients (22 cases) with wild-type JAK2/STAT. For PD-L1 IHC, the cases were evaluated for variable staining intensity (weak, moderate, strong) and membrane staining pattern (partial, complete) in tumor and mononuclear immune cells. The PD-L1 CPS was calculated by summing the number of all PD-L1-stained cells and dividing by the total number of tumor cells, multiplied by 100. The pSTAT3 was evaluated by percentage of positive tumor cells among the total tumor cells. The status of JAK2 / STAT mutations and other molecular abnormalities was evaluated by next generation sequencing (NGS) study. Results: Among patients of JAK2/STAT mutations, nine with JAK2 V617F exon 14 mutations, one with JAK2 I540-E543delinsRG exon 12 mutation and one with STAT5B N642H mutation. Evaluation of PD-L1 expression by IHC revealed four major cell staining patterns in our study cohort: 1. Negative (Figure 1A); 2. Partial weak membrane staining (Figure 1B, CPS<10); 3. Partial-complete, weak-moderate membrane staining (Figure 1C, CPS≥10); 4. Complete, moderate-strong membrane staining (Figure 1D, CPS≥10). Patterns 3-4 were classified as positive PD-L1 staining with a CPS cut-off of 10 (CPS≥10). Comparison of the PD-L1 CPS score in the JAK2/STAT wild-type versus the JAK2/STAT mutant groups revealed significantly higher PD-L1 CPS score in the JAK2/STAT mutant group (Figure 1K, P<0.0001, CPS≥10). One patient showed wild-type JAK2 at initial AML diagnosis. At two subsequent relapses, gain of JAK2 exon 12 (I540-E543delinsRG) mutation with increased VAF from 17% to 41% was detected. Interestingly, PD-L1 expression was correspondingly increased with CPS from 25 to 75 in these subsequent biopsies (Figure 1E, F). In another case with wild-type JAK2, STAT5B N642H mutation was detected and strong PD-L1 expression with CPS of 80 was obtained (Figure 1G). To address if JAK2/STAT3 axis is involved in PD-L1 upregulation, we assessed STAT3 phosphorylation by IHC in JAK2 wild-type and mutated groups. The results revealed significantly increased p-STAT3 expression in the JAK2 mutant compared with the wild-type cases, supporting the idea that oncogenic JAK2 mutation leads to STAT3 phosphorylation (Figure 1H-J). Furthermore, p-STAT3 expression showed a significant positive correlation with the PD-L1 CPS score (Figure 1H, r=0.8663, P<0.0001). Conclusion: In the AML patients with oncogenic JAK2 or its downstream signaling molecule STAT5B mutations, there is significantly enhanced PD-L1 expression. In addition, we proposed that the PD-L1 CPS positivity cut-off score of 10 being used as a quantitative measure of PD-L1 expression in this heterogeneous myeloid neoplasm. In the patient with relapsing AML, PD-L1 expression showed a positive correlation with increased JAK2 mutant VAF. Independent of the oncogenic JAK2 mutation, PD-L1 expression upregulation was also noted with STAT5B N642H mutation. Lastly, we demonstrated enhanced p-STAT3 expression in JAK2 mutant cases and a positive correlation between p-STAT3 and PD-L1 expression. Taken together, these data support the model that JAK2/STAT pathway activation is associated with PD-L1 upregulation in AML patients (Figure 1I), together with the proposed PD-L1 CPS cut-off of 10, can serve as scanning biomarkers to select relevant subgroups of AML patients for PD-1/PD-L1 immunotherapeutic study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

STAT3 Expression and Correlation Between Ki-67 and PHH3 in Meningiomas: Is it Possible to Predict Recurrence?

Aim: The aim of this study was to investigate the correlation between PHH3 and ki-67 labeling index, and the association of STAT3 expression with mitotic index, grade by World Health Organization 2016 classification, and clinicopathological features of meningioma cases. Material and Methods: A total of 25 meningioma cases from the archives of the Department of Pathology, Düzce University School of Medicine, diagnosed between 2012 and 2021 were included in the study. The mitotic count from the ten fields with the highest number of mitotic figures was determined. Immunohistochemistry was performed on the formalin-fixed, paraffin-embedded tissue blocks to determine STAT3, ki-67, and PHH3 expression. STAT3 was scored between 0 and 3 points according to staining intensity. Staining percentages for STAT3 were determined using a manual count of stained cells and the total number of tumor cells. The ki-67 labeling index was determined as a percentage by a manual count. For PHH3, the total number of immunostained mitotic figures per 10 high-power fields were evaluated in each case. Results: A statistically significant difference was found in terms of the percentage of STAT3 staining between the tumor grades (p=0.047). STAT3 expression was significantly higher in cases with high tumor grades. A moderate positive correlation was found between ki-67 and PHH3 when calculated as a percentage in the area with the highest mitotic index by manual counting (r=0.621, p=0.001). Conclusion: A combination of ki-67, PHH3, and STAT3 will be useful in the grading of meningiomas and predict the recurrence.

A Quantitative Digital Analysis of Tissue Immune Components Reveals an Immunosuppressive and Anergic Immune Response with Relevant Prognostic Significance in Glioblastoma.

Objectives: Immunostimulatory therapies using immune checkpoint blockers show clinical activity in a subset of glioblastoma (GBM) patients. Several inhibitory mechanisms play a relevant role in the immune response to GBM. With the objective of analyzing the tumor immune microenvironment and its clinical significance, we quantified several relevant immune biomarkers. Design: We studied 76 primary (non-recurrent) GBMs with sufficient clinical follow-up, including a subgroup of patients treated with a dendritic cell vaccine. The IDH-mutation, EGFR-amplification, and MGMT methylation statuses were determined. Several relevant immune biomarkers, including CD163, CD8, PD1, and PDL1, were quantified in representative selected areas by digital image analysis and semiquantitative evaluation. The percentage of each immune expression was calculated with respect to the total number of tumor cells. Results: All GBMs were wild-type IDH, with a subgroup of classical GBMs according to the EGFR amplification (44%). Morphologically, CD163 immunostained microglia and intratumor clusters of macrophages were observed. A significant direct correlation was found between the expression of CD8 and the mechanisms of lymphocyte immunosuppression, in such a way that higher values of CD8 were directly associated with higher values of CD163 (p < 0.001), PDL1 (0.026), and PD1 (0.007). In a multivariate analysis, high expressions of CD8+ (HR = 2.05, 95%CI (1.02–4.13), p = 0.034) and CD163+ cells (HR 2.50, 95%CI (1.29–4.85), p = 0.007), were associated with shorter survival durations. The expression of immune biomarkers was higher in the non-classical (non-EGFR amplified tumors) GBMs. Other relevant prognostic factors were age, receipt of the dendritic cell vaccine, and MGMT methylation status. Conclusions: In accordance with the inverse correlation between CD8 and survival and the direct correlation between effector cells and CD163 macrophages and immune-checkpoint expression, we postulate that CD8 infiltration could be placed in a state of anergy or lymphocytic inefficient activity. Furthermore, the significant inverse correlation between CD163 tissue concentration and survival explains the relevance of this type of immune cell when creating a strong immunosuppressive environment. This information may potentially be used to support the selection of patients for immunotherapy.

Abstract 6399: SP in relation to NK1R and Ki67 predicts survival in hormone receptor negative breast cancer

Abstract Background: Substance P (SP) is a tachykinin that has been implicated in carcinogenesis in several types of cancer through binding to neurokinin 1 receptor (NK1R). This study aimed to investigate the expression levels of SP and NK1R in hormone receptor negative (HR -ve) versus positive (HR +ve) breast cancer tissues and their relationship with clinicopathological characteristics of patients, Ki-67 index, and overall survival rate. Methods: Immunohistochemical expression levels of SP, NK1R and Ki-67 were measured in 144 paraffin-embedded breast cancer tissues (77 HR -ve and 67 HR +ve). SP and NK1R were scored semiquantitatively, while Ki-67 was obtained by the percentage of total number of tumor cells with nuclear staining. The optimal cutoff value for SP, NK1R and Ki-67 were assessed by Cutoff Finder. Pearson Chi-square (χ2) and Fisher’s exact tests were used to compare the staining scores between groups. ANOVA and student’s t-test were used to compare group means. Kaplan-Meier method with log-rank test was used for survival analysis. Results: High expression of SP, NK1R and Ki-67 was observed in 41%, 36.8% and 38.9% of cases, respectively. Association analyses revealed that SP expression in HR -ve breast cancer was higher in pN3 tumors (p=0.002), positive axillary lymph node metastasis (p=0.003), and high NK1R expression (p=0.010). In HR+ve breast cancer, SP was lower in HER2 positive tumors (p=0.001). Regarding NK1R, the association analysis showed that low NK1R expression level in HR +ve tumors was related to stage IV (p=0.023), pN1 (p=0.006), and low Ki-67 index (p=0.038). Survival analysis revealed that high SP level negatively impacted overall survival in HR-ve tumors that had low NK1R level (p=0.021). Moreover, high SP negatively impacted overall survival in HR-ve tumors that had low Ki-67 level (p=0.005). Nevertheless, NK1R expression did not impact survival in HR-ve or HR+ve cases despite stratification by SP and Ki-67 expression (p&amp;gt;0.05). Conclusion: Up to our knowledge, this is the first study to investigate the prognostic potential of SP and NK1R in breast cancer. Combined expression levels of SP and NK1R or Ki-67 could be used to predict survival in breast cancer patients with HR-ve tumors. Our findings suggest the potential role of SP and NK1R as therapeutic targets in breast cancer. Citation Format: Maha S. Al-Keilani, Rana Elstaty, Mohammad A. Alqudah. SP in relation to NK1R and Ki67 predicts survival in hormone receptor negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6399.

Immunohistochemical assessment of PD-L1 expression using three different monoclonal antibodies in triple negative breast cancer patients

BackgroundPD-L1 receptor expression in breast cancer tissue can be assessed with different anti-human PD-L1 monoclonal antibodies. The performance of three specific monoclonal antibodies in a head-to-head comparison is unknown. In addition, a potential correlation of PD-L1 expression and clinico-pathological parameters has not been investigated.MethodsThis was a retrospective study on tissue samples of patients with histologically confirmed triple negative breast cancer (TNBC). PD-L1 receptors were immune histochemically stained with three anti-human PD-L1 monoclonal antibodies: 22C3 and 28-8 for staining of tumor cell membranes (TC) and cytoplasm (Cyt), SP142 for immune cell staining (IC). Three different tissue samples of each patient were evaluated separately by two observers in a blinded fashion. The percentage of PD-L1 positive tumor cells in relation to the total number of tumor cells was determined. For antibodies 22C3 and 28-8 PD-L1 staining of 0 to < 1% of tumor cells was rated "negative", 1–50% was rated "positive" and > 50% was rated "strong positive". Cyt staining was defined as “negative” when no signal was observed and as “positive”, when any positive signal was observed. For IC staining with SP142 all samples with PD-L1 expression ≥ 1% were rated as “positive”. Finally, the relationship between PD-L1 expression and clinico-pathological parameters was analyzed.ResultsTissue samples from 59 of 60 enrolled patients could be analyzed. Mean age was 55 years. Both the monoclonal antibodies 22C3 and 28-8 had similar properties, and were positive for both TC in 13 patients (22%) and for Cyt staining in 24 patients (40.7%). IC staining with antibody SP142 was positive in 24 patients (40.7%), who were also positive for Cyt staining. The differences between TC and Cyt staining and TC and IC staining were significant (p = 0.001). Cases with positive TC staining showed higher Ki67 expression compared to those with negative staining, 40 vs 30%, respectively (p = 0.05). None of the other clinico-pathological parameters showed any correlation with PDL1 expression.ConclusionsAntibodies 22C3 and 28-8 can be used interchangeably for PD-L1 determination in tumor cells of TNBC patients. Results for Cyt staining with 22C3 or 28-8 and IC staining with SP142 were identical. In our study PD-L1 expression correlates with Ki67 expression but not with OS or DFS.

The Prognostic Potential of Neurokinin 1 Receptor in Breast Cancer and Its Relationship with Ki-67 Index.

Background Neurokinin 1 receptor (NK1R) is a promising biomarker and therapeutic target in breast cancer. This study was aimed at investigating the expression level of NK1R in breast cancer tissues and its relationship with proliferation index as measured by Ki-67, clinicopathological characteristics of patients, and overall survival rate. Methods Immunohistochemical expression of NK1R and Ki-67 was measured in 164 paraffin-embedded breast cancer tissues of four molecular subtypes (42 HER2-enriched, 40 luminal A, 42 luminal B, and 40 triple negative). NK1R was scored semiquantitatively, while Ki-67 was obtained by the percentage of total number of tumor cells with nuclear staining. The optimal cutoff values for NK1R and Ki-67 were assessed by Cutoff Finder. Pearson's Chi-square (χ2) and Fisher's exact tests were used to compare the staining scores between groups. The Kaplan-Meier method with log-rank test was used for survival analysis. ANOVA and Student's t-test were used to compare group means. Results A total of 164 patients were included in the study which represented females with invasive ductal carcinoma. NK1R was expressed at high levels in about 34% of investigated cases. The mean Ki-67 level was about 27% and 41.5% of sample had high Ki-67 (expression level > 22%). NK1R expression levels were associated with higher tumor grade (p = 0.021) and high Ki-67 (p = 0.012). NK1R expression negatively impacted overall survival in grade II tumors (p = 0.027). Conclusion NK1R contributes to cellular proliferation and is associated with negative prognosis in breast cancer. These findings suggest the potential role of NK1R as a therapeutic target in breast cancer.

Assessing the Utility of Actual Proliferation Index Measured by Expression of ‘Agnor and Ki-67’ in Histopathologically Negative Surgical Margins of Oral Squamous Cell Carcinoma as a Modality for Prediction of Recurrence

Background: Oral squamous cell carcinoma is most common malignancy of head and neck which is the major cause of death. Recurrence is the major important prognostic factor in the survival of patients and tumor stage, degree of differentiation of neoplastic cells, pN stage resected margins, and lymphovascular invasion are the factors for recurrence. The rate of recurrence varies from 18 to76% for patients who underwent standard treatment. Histological assessment of surgical margins has a major role to predict the recurrence. But in spite of negative histological surgical margins recurrence rate is high, as there are genetic alterations in these margins which are not detected by routine histopathlogy. Even when surgical margins are free histopathologically the local recurrence rate is 10 to 30 percent.&#x0D; That’s why molecular evaluation of these margins should be done. Many immunohistochemical studies are done to assess surgical margins of OSCC but no study is carried out to assess the proliferative index in surgical margins. Therefore this study will help to improve management and predict the recurrence of OSCC.&#x0D; Objectives: In this study we aim to assess the utility of “actual proliferation index” measured by expression of “Agnor” and “Ki-67” in histopathologically negative “surgical margins” of “oral sqaumous cell carcinoma” as a modality for prediction of recurrence. &#x0D; Methodology: The resected surgical margins of the oral squamous cell carcinoma cases which are operated in Sharad Pawar dental college will be selected as samples. The anterior, posterior, inferior and superior margin of resected specimens will be stained by H&amp;E staining. Immunohistochemical staining by ki-67 antibody will be done in the negative histopathological surgical margins. The same sections will be stained with AgNOR staining. Both sections will be evaluated. Actual proliferative index will be calculated with the given formula&#x0D; Ki67 Labeling Index: Ki67 L1= “Number of Ki67 positive cells” x 100&#x0D; Total number of tumor cells observed&#x0D; “Actual Proliferative Index (PI)” = “ Ki-67” or MIB-1 LI × AgNOR count ’&#x0D; Recurrence of tumor will be observed during first two years period after surgical procedure.&#x0D; Expected Results: The “actual proliferative index” calculated by “Ki-67 labelling index” and “AgNOR count expression in histopathologically” negative surgical margins of OSCC will be more in recurrence patients as compared to patients who do not have recurrence.&#x0D; Conclusion: The API values measured by assessing the Agnor and Ki67 expression, in histopathological negative surgical margins of OSCC will be compared between with values of patient with recurrence.

Tumor cell death visualization of renal cell carcinoma under the combined effect of theGratiola officinalisextract and cyclophosphamide using fluorescent staining methods

Objective of the study: We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide). Materials and methods: The dyes were propidium iodide and acridine orange, which were used in the “alive and dead” test. This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started, it was characterized by the appearance of apoptotic bodies or nucleus pyknosis. Results: We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1:1. The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2:1. When the ratio of concentration of the extract relative to the cytostatic increased to 3:1, the cytostatic activity of the extract began to appear, the total number of tumor cells decreased. The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3:1 ratio of extract and cytostatic concentrations, which confirms the presence of pro-apoptotic activity of the studied combination. This trend indicates the dependence of a certain form of cell death (apoptosis, necrosis) on the ratio of extract and cytostatic doses, and it also demonstrates the cytostatic and cytotoxic effects of this combination. Conclusion: Fluorescence methods of investigation in the “alive and dead” test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide) 24 h after exposure. We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.

Contribution of tumour and immune cells to PD-L1 as a predictive biomarker in metastatic triple-negative breast cancer (mTNBC): analysis from keynote-119

Background: We evaluated whether PD-L1 expression on tumour cells contributes to PD-L1 as a predictive biomarker for pembrolizumab in mTNBC. Methods: Patients with mTNBC received pembrolizumab or chemotherapy. PD-L1 expression (PD-L1 IHC 22C3 pharmDx) was quantified per tumour proportion score (TPS; percentage of PD-L1-expressing tumour cells [partial or complete membrane staining] relative to total number of tumour cells) and combined positive score (CPS; number of PD-L1-staining cells [tumour cells, lymphocytes, macrophages] divided by total number of viable tumour cells, multiplied by 100). Quantitative immune cell density (QID) was CPS minus TPS. TPS, CPS, and QID ability to predict ORR (ROC analysis) and overall survival (OS) hazard ratio (HR) (pembrolizumab vs chemotherapy) were evaluated. Results: With pembrolizumab, area under ROC curve (95% CI) was 0.69 (0.58–0.80) for CPS, 0.67 (0.56–0.77) for QID, and 0.55 (0.46–0.64) for TPS. At each cutoff, QID had lower sensitivity and Youden Index and larger OS HR than CPS. QID appeared to be orthogonal to TPS. Conclusions: Trends suggest tumour cell expression is an important component of PD-L1 as a predictive biomarker of pembrolizumab in mTNBC. When immune cells alone were used, a meaningful number of responders was missed, and OS benefit trended toward higher HR estimates.

Pan-tumour analysis of the association between PD-L1 combined positive score (CPS) and response to pembrolizumab monotherapy

PD-L1 is expressed on tumour and immune cells; PD-L1 CPS captures tumour and immune cell expression in one aggregate score. We performed a retrospective, exploratory analysis of CPS as an enrichment biomarker of pembrolizumab monotherapy across multiple tumour types. PD-L1 expression was assessed using PD-L1 IHC 22C3 pharmDx and measured using CPS (number of PD-L1-staining cells [tumour cells, lymphocytes, macrophages] divided by total number of tumour cells, multiplied by 100). Data were pooled from 11 studies across tumour types for pembrolizumab and for standard-of-care (in controlled studies). Estimates of ORR, prevalence, and receiver operating characteristics (ROC) analysis was performed over various CPS cut-points. CPS distribution by response, tumour type, and line of therapy were also assessed. 3769 patients had available PD-L1 CPS (pembrolizumab, n=2678; standard-of-care, n=1091). Area under the ROC curve for ORR was 0.63 (95% CI, 0.61–0.66; pembrolizumab) versus 0.48 (95% CI, 0.43–0.53; standard-of-care); cut-points of 1, 10, 20, and 50 were also examined. This pan-tumour analysis demonstrates that CPS is an effective method for scoring PD-L1 expression and can be used as a predictive biomarker to identify patients likely to respond to pembrolizumab monotherapy. CPS demonstrated enrichment of response to pembrolizumab monotherapy across most tumour types.

Protein expression of programmed cell death ligand 1 and ligand 2 and their prognostic values in extensive-stage small cell lung cancer.

e20593 Background: The receptor-ligand interaction between programmed cell death protein-1 (PD-1) and its ligands (PD-L1 and PD-L2) plays a critical role in tumor immune evasion and has become a therapeutic target in a number of cancer types, including extensive stage small cell lung cancer (ES-SCLC). While there are some published data on PD-L1 expression in ES-SCLC, the results vary with different laboratory assays or thresholds of positivity employed. Data on PD-L2 expression is very limited in ES-SCLC. The prognostic values of these biomarkers are not well understood. The current study aims to address this data gap. Methods: A retrospective cohort study of patients with ES-SCLC receiving usual care in the clinical setting in Denmark was conducted. Formalin-fixed paraffin embedded (FFPE) tumor tissue samples and data on patient characteristics and clinical outcomes were obtained for the patient population. Protein expression of PD-L1 and PD-L2 was determined by immunohistochemistry (IHC), and a combined positive score (CPS, the percentage of tumor cells and mononuclear inflammatory cells expressing the biomarker at any intensity out of the total number of tumor cells) of ≥1 was used to define biomarker positivity. Kaplan-Meier plots and Cox proportional hazard models were employed to assess the relationship between PD-L1 and PD-L2 protein expression and overall survival (OS). Results: Among the 80 patients with ES-SCLC included in this study, 25 and 29 were positive for PD-L1 and PD-L2, respectively (Table). A total of 68 patients had concordant expression status of PD-L1 and PD-L2 (21 as double positive and 47 as double negative). There was a significant association between PD-L1 positivity and longer OS, which persisted after adjustment for age and stage at SCLC diagnosis, performance score, prior exposure to chemotherapy, and lactate dehydrogenase levels (Table). Similar results were observed for PD-L2 (Table), and the data did not suggest any additional prognostic value of PD-L2 expression independent of PD-L1 expression. Conclusions: PD-L1 and PD-L2 positivity was both observed, and highly correlated with each other, in approximately one third of the ES-SCLC tumor samples tested. Patients with PD-L1 or PD-L2 positive tumors have significantly longer OS than those with negative tumors in this study, independent of other known prognostic factors.[Table: see text]

Abstract PD14-04: Contribution of tumor and immune cells to PD-L1 as a predictive biomarker in triple-negative breast cancer (TNBC): Analysis from KEYNOTE-119

Abstract Background: Pembrolizumab monotherapy did not significantly improve overall survival (OS) as second- or third-line treatment for metastatic TNBC vs chemotherapy in the randomized, open-label, phase 3 KEYNOTE-119 study (NCT02555657; N = 622). However, the benefit of pembrolizumab compared with chemotherapy appeared to be greater with increasing PD-L1 expression as quantified by combined positive score (CPS; defined as the number of PD-L1–staining cells [tumor cells, lymphocytes, macrophages] divided by the total number of viable tumor cells, multiplied by 100). In the current exploratory analysis, we aimed to determine whether expression of PD-L1 on tumor cells contributes to the value of PD-L1 as a predictive biomarker in metastatic TNBC.Methods: Patients with centrally confirmed TNBC and 1 or 2 prior systemic treatments for metastatic disease were enrolled in KEYNOTE-119. Patients were randomly assigned 1:1 to pembrolizumab 200 mg Q3W or investigator’s choice of single-agent chemotherapy (capecitabine, eribulin, gemcitabine, or vinorelbine). PD-L1 expression in tumor samples was assessed using PD-L1 IHC 22C3 pharmDx and quantified per tumor proportion score (TPS; defined as the percentage of PD-L1–expressing tumor cells [partial or complete membrane staining] relative to total number of tumor cells) and CPS. Quantitative immune cell density (QID) was defined as CPS minus TPS. QID isolates immune cells but may be truncated when TPS is high. The ability of each scoring method (TPS, CPS, and QID) to predict objective response rate (ORR) with pembrolizumab, including receiver operating characteristics (ROC) analysis, and OS hazard ratios (HRs; pembrolizumab vs chemotherapy) was evaluated.Results: Tumor samples were available for 601 patients (pembrolizumab, 309; chemotherapy, 292) in KEYNOTE-119. ORR was 9.7% (30/309) with pembrolizumab and 11.3% (33/292) with chemotherapy when PD-L1 expression status was not considered. In the pembrolizumab arm, the area under the ROC curve (AUROC; 95% CI) was 0.69 (0.58-0.80) for tumor samples scored for CPS, 0.66 (0.55-0.77) for QID, and 0.55 (0.46-0.64) for TPS. ROC analysis is shown in the Table. At each cutoff, QID had lower estimated sensitivity (ie, missed responders) and a lower Youden Index compared with CPS. The number of missed responders (of 30 total in the pembrolizumab arm) for QID relative to CPS were 2, 5, 5, 6, 2, and 2 at cutoffs of 1, 10, 20, 30, 40, and 50, respectively. Across all practical cutoffs, the OS HR tended to be slightly smaller for CPS than QID. At cutoffs corresponding to the upper percentiles of 10, 20, 40, and 60, OS HRs were 0.497, 0.658, 0.758, and 0.850, respectively, for CPS vs 0.572, 0.712, 0.814, and 0.863, respectively, for QID. QID appeared to be orthogonal to TPS (r = -0.03 for all 601 observations; r = -0.04 after eliminating 7 potentially truncated values).Conclusions: Trends estimated using KN119 suggest that tumor cell expression is an important component of PD-L1 as a predictive biomarker of pembrolizumab efficacy in metastatic TNBC. In this exploratory analysis, when immune cells alone were used to measure PD-L1 expression, a meaningful number of responders was missed and OS benefit trended toward higher HR estimates. Tumor and immune cell PD-L1 expression may represent distinct (presumably negative modulatory) mechanisms. Table. ROC AnalysisCutoffCPS SensCPS SpecCPS YICPS PrevQID SensQID SpecQID YIQID PrevTPS SensTPS SpecTPS YITPS Prev010011001100110.8330.3660.1990.6540.7670.4160.1820.6020.3000.7890.0890.220100.5670.7170.2840.3110.4000.8140.2140.2070.2000.8920.0920.117200.5000.8490.3490.1840.3330.9250.2580.1000.1670.9320.0990.078300.3670.8920.2590.1330.1670.9570.1240.0550.1000.9430.0430.061400.2000.9350.1350.0780.1330.9860.1190.0260.0670.9530.0200.049500.2000.9570.1570.0580.1330.9930.1260.0190.0670.9680.0340.036 Citation Format: Eric P. Winer, Oleg Lipatov, Seock-Ah Im, Anthony Goncalves, Eva Muñoz-Couselo, Keun Seok Lee, Zbigniew Nowecki, Peter Schmid, Kenji Tamura, Laura Testa, Isabell Witzel, Shoichiro Ohtani, Stephanie Hund, Karina Kulangara, Vassiliki Karantza, Jaime A. Mejia, Junshui Ma, Petar Jelinic, Lingkang Huang, Kenneth Emancipator, Javier Cortes. Contribution of tumor and immune cells to PD-L1 as a predictive biomarker in triple-negative breast cancer (TNBC): Analysis from KEYNOTE-119 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD14-04.