High Percentage Of Tumor Cells Research Articles

Spatial characterization and quantification of CD40 expression across cancer types

BackgroundCD40, a TNF receptor family member, is expressed by a variety of immune cells and is involved in the activation of both adaptive and innate immune responses. Here, we used quantitative immunofluorescence (QIF) to evaluate CD40 expression on the tumor epithelium of solid tumors in large patient cohorts of lung, ovarian, and pancreatic cancers.MethodsTissue samples from nine different solid tumors (bladder, breast, colon, gastric, head and neck, non-small cell lung cancer (NSCLC), ovarian, pancreatic and renal cell carcinoma), constructed in tissue microarray format, were initially assessed for CD40 expression by QIF. CD40 expression was then evaluated on the large available patient cohorts for three of the tumor types demonstrating high CD40 positivity rate; NSCLC, ovarian and pancreatic cancer. The prognostic impact of CD40 expression on tumor cells was also investigated.ResultsCD40 expression on tumor cells was found to be common, with 80% of the NSCLC population, 40% of the ovarian cancer population, and 68% of the pancreatic adenocarcinoma population displaying some degree of CD40 expression on cancer cells. All of three of these cancer types displayed considerable intra-tumoral heterogeneity of CD40 expression, as well as partial correlation between expression of CD40 on tumor cells and on surrounding stromal cells. CD40 was not found to be prognostic for overall survival in NSCLC, ovarian cancer, or pancreatic adenocarcinoma.ConclusionsThe high percentage of tumor cells expressing CD40 in each of these solid tumors should be considered in the development of therapeutic agents designed to target CD40.

Analysis of Immune Intratumor Heterogeneity Highlights Immunoregulatory and Coinhibitory Lymphocytes as Hallmarks of Recurrence in Stage I Non–Small Cell Lung Cancer

Our understanding of the molecular mechanisms underlying postsurgical recurrence of non–small cell lung cancer (NSCLC) is rudimentary. Molecular and T cell repertoire intratumor heterogeneity (ITH) have been reported to be associated with postsurgical relapse; however, how ITH at the cellular level impacts survival is largely unknown. Here we report the analysis of 2880 multispectral images representing 14.2% to 27% of tumor areas from 33 patients with stage I NSCLC, including 17 cases (relapsed within 3 years after surgery) and 16 controls (without recurrence ≥5 years after surgery) using multiplex immunofluorescence. Spatial analysis was conducted to quantify the minimum distance between different cell types and immune cell infiltration around malignant cells. Immune ITH was defined as the variance of immune cells from 3 intratumor regions. We found that tumors from patients having relapsed display different immune biology compared with nonrecurrent tumors, with a higher percentage of tumor cells and macrophages expressing PD-L1 (P =.031 and P =.024, respectively), along with an increase in regulatory T cells (Treg) (P =.018), antigen-experienced T cells (P =.025), and effector-memory T cells (P =.041). Spatial analysis revealed that a higher level of infiltration of PD-L1+ macrophages (CD68+PD-L1+) or antigen-experienced cytotoxic T cells (CD3+CD8+PD-1+) in the tumor was associated with poor overall survival (P =.021 and P =.006, respectively). A higher degree of Treg ITH was associated with inferior recurrence-free survival regardless of tumor mutational burden (P =.022), neoantigen burden (P =.021), genomic ITH (P =.012) and T cell repertoire ITH (P =.001). Using multiregion multiplex immunofluorescence, we characterized ITH at the immune cell level along with whole exome and T cell repertoire sequencing from the same tumor regions. This approach highlights the role of immunoregulatory and coinhibitory signals as well as their spatial distribution and ITH that define the hallmarks of tumor relapse of stage I NSCLC.

Synergistic Therapeutic Effects of Low Dose Decitabine and NY-ESO-1 Specific TCR-T Cells for the Colorectal Cancer With Microsatellite Stability.

Patients of colorectal cancer (CRC) with microsatellite stability (MSS) show poor clinical response and little beneficial result from the immune-checkpoint inhibitors, due to the ‘cold’ tumor microenvironment. Meanwhile, decitabine can drive the ‘cold’ microenvironment towards ‘hot’ in multiple ways, such as upregulating the tumor associated antigen (TAA) and human leukocyte antigen (HLA) molecular. NY-ESO-1, one of the most important TAAs, can be observably induced in tumors by low dose decitabine, and present itself as ideal targets for antigen specific T cell receptor engineered T (TCR-T) cells. We innovatively used a synergistic tactic, combining decitabine and NY-ESO-1 specific TCR-T cells, for fighting the MSS CRC. Firstly, we confirmed the lysing effect of the NY-ESO-1 TCR-T cells on the NY-ESO-1+ and HLA-A2+ cells in vitro and in vivo. In A375 tumor-bearing mice, the results showed that NY-ESO-1 TCR-T cell therapy could inhibit A375 tumor growth and prolonged the survival time. Furthermore, the synergistic effect of decitabine and NY-ESO-1 TCR-T cells was shown to induce an even higher percentage of tumor cells being lysed in vitro than other control groups, and more potent tumor inhibition and longer survival time were observed in vivo. The innovative synergistic therapeutic strategy of decitabine and TCR-T cells for the CRC with MSS may be also effective in the treatment of other epithelial malignancies. Decitabine may likewise be adopted in combination with other cellular immunotherapies.

Expression of connexin-43 in surgical resections of primary tumor and lymph node metastases of squamous cell carcinoma and adenocarcinoma of the lung: a retrospective study.

BackgroundConnexins are transmembrane proteins forming gap junctions between the cells, which allow intercellular communication. Significance of gap junctions and connexins in lung carcinoma is not yet understood. The objective of the study was to investigate immunohistochemical expression and the localization of connexin-43 (Cx43) in primary lung carcinoma and its lymphatic metastases.MethodsSurgical specimens of excised tumors from 88 patients (45 men and 43 women, 61.9 ± 7.4 years) with lung carcinoma (52 adenocarcinoma (AC), 36 squamous cell carcinoma (SqCC)) who were operated on at the University Hospital “Bezanijska Kosa” in a five-year period (2012–2016) were used. We conducted immunohistochemical staining for Cx43 and measured the degree of expression (percentage of positive cells and staining intensity) as well as localization of Cx43 in primary tumor and in lymphatic metastases.ResultsImmunohistochemical analysis of the primary tumors revealed that SqCC showed significantly higher percentage of tumor cells expressing Cx43 as well as higher staining intensity than AC (p < 0.001). Almost 70% of samples with SqCC showed high Cx43 expression, whereas AC showed no expression in more than 50% of cases. Localization of Cx43 expression was most often cytoplasmic (AC and SqCC) and combined membranous and cytoplasmic (SqCC) with very rare instances of nuclear localization (AC). Almost the same pattern in distribution, intensity, and localization of Cx43 expression was observed in the lymph node metastases; however, almost a third of AC cases changed the pattern of Cx43 expression in the metastasis compared to primary tumor.ConclusionThe results of this study showed that lung carcinomas express Cx43 in more than 65% of cases and that it was aberrantly localized (not membranous localization). We highlighted that SqCC expressed Cx43 more than did AC, both in primary tumor and lymphatic metastases. Further research is needed to establish whether Cx43 could be used as a prognostic biomarker in lung carcinoma.

Epithelial Mesenchymal Transition and Immune Response in Metaplastic Breast Carcinoma.

Metaplastic breast carcinoma (MBC) is a heterogeneous group of infrequent triple negative (TN) invasive carcinomas with poor prognosis. MBCs have a different clinical behavior from other types of triple negative breast cancer (TNBC), being more resistant to standard chemotherapy. MBCs are an example of tumors with activation of epithelial–mesenchymal transition (EMT). The mechanisms involved in EMT could be responsible for the increase in the infiltrative and metastatic capacity of MBCs and resistance to treatments. In addition, a relationship between EMT and the immune response has been seen in these tumors. In this sense, MBC differ from other TN tumors showing a lower number of tumor-infiltrating lymphocytes (TILS) and a higher percentage of tumor cells expressing programmed death-ligand 1 (PD-L1). A better understanding of the relationship between the immune system and EMT could provide new therapeutic approaches in MBC.

Impact of copy number variant and single nucleotide polymorphism of the programmed death-ligand 1 gene, programmed death-ligand 1 protein expression and therapy regimens on overall survival in a large group of Caucasian patients with non-small cell lung carcinoma.

Anti-programmed death-1 or anti-programmed death-ligand 1 (PD-L1) blockade may be ineffective in some patients with non-small cell lung cancer (NSCLC) with high percentage of tumor cells with PD-L1 expression. In addition, immunotherapy may provide great benefits in patients without PD-L1 expression. The present study assessed PD-L1 protein expression by immunohistochemistry, copy number variation (CNV) of PD-L1 and two single nucleotide polymorphisms (SNPs), rs822335 and rs822336, in the promoter of PD-L1 by quantitative PCR in 673 patients with NSCLC. Overall survival time of patients with NSCLC depending on the assessed predictive factors (PD-L1 CNV or SNP) and the treatment methods (immunotherapy in first/second line of treatment or chemotherapy) was analyzed. The present study revealed significantly higher PD-L1 copies number in patients with ≥10% and ≥50% of tumor cells with PD-L1 expression compared to patients with lower percentage of PD-L1-positive tumor cells (P=0.02 and P=0.0002, respectively). There was a significant positive correlation (R=0.2; P=0.01) between number of PD-L1 copies and percentage of tumor cells with PD-L1 protein expression. Percentage of tumor cells with PD-L1 expression was lower in patients with TT genotype of the rs822335 polymorphism compared to those with CC genotype (P=0.03). The present study observed significantly higher risk of death in patients treated with chemotherapy compared to those treated with immunotherapy (P<0.0001; hazard ratio=2.4768; 95% confidence interval, 2.0120–3.0490). The present study demonstrated a close relationship between PD-L1 copies number, genotype of rs822335 PD-L1 polymorphism and PD-L1 protein expression on tumor cells. However, the impact of CNV and SNPs of PD-L1 on overall survival of patients with NSCLC requires further investigation.

ESR1 genetic alterations and their association with clinicopathologic characteristics in advanced breast cancer: a single academic institution experience

Estrogen receptor (ER) alpha, a ligand-dependent nuclear transcription factor encoded by the ESR1 gene, is expressed in 70% of breast carcinomas (BCs) and is used as a target for endocrine-based therapies. However, some patients develop resistance to endocrine-based therapies due to ESR1 mutation, which leads to constitutive activation in the absence of ligand. We retrospectively analyzed 223 clinically advanced BCs using the FoundationOne CDX assay and found 13.9% (31/223) of cases had ESR1 genetic alterations (26 mutations and 5 amplifications). All ESR1 mutations occurred within the ligand binding domain, with the most prevalent being Y537S (42.3%) and D538G (38.5%), and all ESR1-mutated cases had a history of aromatase inhibitor use. No significant difference in clinicopathologic features was identified between ESR1-mutated and ESR1-amplified cases except higher frequency of HER2 positivity and TP53 mutations in ESR1-amplified cases. The prevalence of ESR1 mutations in ER-positive BCs was 19.1% (26/136). In comparison to ESR1-nonmutated ER-positive cases, ESR1-mutated cases demonstrated significantly higher percentage of tumor cells with ER and progesterone receptor expression, an increased tendency for overall distant metastasis and liver metastasis, higher frequency of FGF3/4/19 mutations, lower frequency of TP53 mutation, but no difference in overall survival and metastatic/recurrent intervals. In conclusion, our findings suggest that development of ESR1 mutations are selected for under the influence of estrogen deprivation, and a positive correlation between ESR1 mutations and ER protein expression may exist.

Single cell dissociation from mouse tumor samples and analysis on BD FACSLyric Flow Cytometer.

e15245 Background: Single cell multiomics analysis of tumor cells and tumor infiltrating immune cells using high parameter flow cytometry and single cell genomics technologies is a powerful approach in the investigation of tumor biology and in the discovery of new drug targets and biomarkers. The preparation of single cells from solid tumor samples often involves a physical dissociation step in combination with an enzymatic digestion step. Methods: The effect of the tumor cell dissociation process on the phenotypic properties of single cells isolated from solid tumor samples were analyzed using a 12-color reagent panel on the BD FACSLyric flow cytometer. Specifically, spontaneous pancreatic tumor samples from genetically modified mice were processed using a manual dissociation process with a scalpel blade as well as an integrated tumor sample dissociation process with the prototype Singulator instrument with various protocol settings. The BD Horizon Dri Tumor &amp; Tissue Dissociation Reagent (TTDR) reagent was used for the enzymatic step in all sample preparations. The yield, viability, and frequency of tumor cells and different subsets of leukocytes were measured for each tumor-derived single cell sample preparation. On the Singulator system, cell yield reached a plateau with highest viability at the 30-min process time and a higher percentage of tumor cells was obtained compared to the manual sample processing method. Results: The relative frequencies of T cells, B cells, myeloid cells and macrophages also changed in different sample preparation methods. However, the spatial heterogeneity in the tumor section and cell composition may contribute to these differences and would require more in-depth analysis. In summary, these results suggest that there are many variables in the tumor sample single cell dissociation process that could impact the downstream study results, particularly for multiomics studies. Conclusions: This study highlights the need for optimization and standardization of the sample processing methodologies for downstream analysis. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD, the BD Logo, FACSLyric, and Horizon are trademarks of Becton, Dickinson and Company. © 2020 BD and its subsidiaries. All rights reserved.

PD-L1 gene copy number and promoter polymorphisms regulate PD-L1 expression in tumor cells of non-small cell lung cancer patients

Most drugs targeting PD-1 or PD-L1 are more effective when cancer cells of non-small cell lung cancer (NSCLC) patients express PD-L1 protein. The polymorphisms of PD-L1 gene and PD-L1 gene copy number could be responsible for PD-L1 mRNA and protein expression.We analyzed PD-L1 protein expression using two IHC assays, mRNA (PD-L1) expression by qRT-PCR, PD-L1 gene promoter region polymorphisms (rs822335 and rs822336) by qPCR and PD-L1 gene copy number by fluorescence in situ hybridization method.Patients with CC genotype in rs822335 had significantly (p = 0.043) higher percentage of tumor cells with PD-L1 expression (test with 22C3 antibody) than patients with CT or TT genotypes. PD-L1 gene copy number significantly positively correlated with percentage of tumor cells with PD-L1 expression detected in tests with 22C3 antibody (p = 0.005, R = +0.442) and with SP142 antibody (p = 0.021, R = +0.369). PD-L1 gene copy number did not correlate with PD-L1 mRNA expression. Patients with PD-L1 expression tested with 22C3 antibody had significantly higher expression of PD-L1 mRNA (p = 0.023), number of chromsosme 9 centromeres (p = 0.023) and PD-L1 gene copy number (p = 0.003) than patients without PD-L1 expression on tumor cellsPD-L1 gene polymorphisms and PD-L1 gene copy number may be a predictor for PD-L1 protein expression on tumor cells.

Influence of Retronectin-Mediated T-Cell Activation on Expansion and Phenotype of CD19-Specific Chimeric Antigen Receptor T Cells.

Enhanced in vivo expansion, long-term persistence of chimeric antigen receptor T (CART) cells, and efficient tumor eradication through these cells are linked to the proportion of less-differentiated cells in the CART cell product. Retronectin is well established as an adjuvant for improved retroviral transduction, while its property to enrich less-differentiated T cells is less known. In order to increase these subsets, this study investigated the effects of retronectin-mediated T-cell activation for CD19-specific CART cell production. Peripheral blood mononuclear cells of healthy donors and untreated chronic lymphocytic leukemia (CLL) patients without or with positive selection for CD3+ T cells were transduced with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Activation of peripheral blood mononuclear cells was performed by CD3/CD28, CD3/CD28/retronectin, or CD3/retronectin. Interleukin-7 and -15 were supplemented to all cultures. Retronectin was used in all three activation protocols for retroviral transduction. Expansion was assessed by trypan blue staining. Viability, transduction efficiency, immune phenotype, and cytokine production were longitudinally analyzed by flow cytometry. Cytotoxic capacity of generated CART cells was evaluated using a classical chromium-51 release assay. Retronectin-mediated activation resulted in an enrichment of CD8+ cytotoxic CART cells and less-differentiated naïve-like T cells (CD45RA+CCR7+). Retronectin-activated CART cells showed increased cytotoxic activity. However, activation with retronectin decreased viability, expansion, transduction efficiency, and cytokine production, particularly of CLL patient-derived CART cells. Both retronectin-mediated activation protocols promoted a less-differentiated CART cell phenotype without comprising cytotoxic properties of healthy donor-derived CART cells. However, up-front retronectin resulted in reduced viability and expansion in CLL patients. This effect is probably attributed to the retronectin-mediated activation of B cells with prolonged CLL persistence. Consequently, CART cell expansion and generation failed. In summary, activation with retronectin should be performed with caution and may be limited to patients without a higher percentage of tumor cells in the peripheral blood.

Fine Needle Aspiration Biopsy of Primary Pulmonary Adenocarcinoma Yields Higher Percentage of Tumor Cells for Molecular Studies: A 5-year Comparison Study to Needle Core Biopsy Sampling

Fine Needle Aspiration Biopsy of Primary Pulmonary Adenocarcinoma Yields Higher Percentage of Tumor Cells for Molecular Studies: A 5-year Comparison Study to Needle Core Biopsy Sampling

Abstract 2615: Heterogeneity of HER2 expression in gastric cancer (GC) leads to high deviation rates between local and central testing and hampers efficacy of anti-HER2 therapy: Survival results from the VARIANZ study

Abstract Background: HER2 is a membrane-bound receptor tyrosine kinase that belongs to the epidermal growth factor receptor family. The anti-HER2 directed monoclonal antibody trastuzumab is approved for 1st-line treatment of stage IV HER2+ GC, while other drugs addressing HER2 (pertuzumab, TDM1, lapatinib) failed to improved survival outcomes. Until today, resistance against HER2-directed treatment of GC is poorly understood. The VARIANZ study aims to assess the biologic background of resistance to anti-HER2 therapy in GC. Methods: This academic network study funded by the German Federal Ministry of Education and Research (BMBF 01ZX1610E) recruited patients (pts) who received medical treatment for stage IV GC in 34 sites. HER2 expression was verified centrally by two dedicated GI pathologists using immunohistochemistry (DCS, HI608C0I) and chromogenic in situ hybridization (Zytomed Systems, C-3022-40). Treatment and survival outcomes were reported by investigators. Results: 502 pts were enrolled from May 2014 to Oct 2017. At present, 442 samples were fully characterized for HER2. According to criteria from the ToGA study, 73 of 442 samples were found HER2+ in central testing. In 57 samples with a HER2+ status diagnosed by local pathologists, HER2 positivity could not be confirmed centrally. Deviation rate between local and central testing was 23%. Centrally confirmed HER2-positive GC displayed a higher percentage of tumor cells staining positive for HER2 (49.72 ± 28.58% [SD] vs. 11.35 ± 17.74% [SD]; p&amp;lt;0.001) and a higher HER2/CEP17 ratio (5.78 ± 2.55 [SD] vs. 1.43 ± 0.41 [SD]; p&amp;lt;0.001). Survival outcomes indicate that only pts with centrally confirmed HER2+ status benefit from trastuzumab with an overall survival of 30.7 months (95%-CI 10.7 - 50.8; n=28) versus 7.9 months (95%-CI 2.8 - 12.9, n=33); HR for death was 3.9 in pts with unconfirmed vs. confirmed HER2+ GC receiving trastuzumab plus chemotherapy; p&amp;lt;0.0001. Conclusions: Variability between local and central HER2 assessment in GC is significant. Pts with centrally unconfirmed HER2+ status have no benefit from anti-HER2 therapy. HER2 status should be assessed in highly qualified laboratories and cut-offs for defining HER2+ should be reconsidered. Alternative treatment options–other than trastuzumab–should be investigated in pts with unconfirmed or borderline HER2 expression. Citation Format: Florian Lordick, Ivonne Haffner, Birgit Luber, Dieter Maier, Elba Raimundez, Jan Hasenauer, Albrecht Kretzschmar, Ludwig Fischer von Weikersthal, Miriam Ahlborn, Jorge Riera Knorrenschild, Gabriele Siegler, Beate Rau, Stefan Fuxius, Thomas Decker, Katrin Schierle, Christian Wittekind. Heterogeneity of HER2 expression in gastric cancer (GC) leads to high deviation rates between local and central testing and hampers efficacy of anti-HER2 therapy: Survival results from the VARIANZ study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2615.

Dermoscopy in a Lymphangiosarcoma of Stewart–Treves Syndrome

Stewart–Treves syndrome is a rare invasive lymphangiosarcoma linked with chronic lymphedema after mastectomy for breast cancer. Given the aggressive nature of the tumor, preventative measures and early diagnosis are important. This case is the first dermoscopic description of the lymphangiosarcoma of Stewart–Treves syndrome; it shares similar dermoscopic findings with angiosarcoma of the scalp after irradiation, with findings of a pink-white background, white lines and a combination of red, blue and black structureless areas containing red dots and globules. The lack of conspicuous lacunae make dermoscopy useful for ruling out other differentials such as benign vascular proliferations. Color heterogeneity and vascularization in dermoscopy might inform the clinician of a high percentage of tumor cells on histology.

Abstract 3570: Differential expression of the melanoma inhibitor of apoptosis protein (ML-IAP)/Livin in patients with ulcerated and non-ulcerated melanomas

Abstract Background: Cutaneous malignant melanoma is one of the most aggressive forms of skin cancer with an extremely poor prognosis for the patients diagnosed with metastatic disease. Ulceration has been correlated with an increased risk of death within a given thickness range, however, the biologic basis for the development of ulceration is poorly understood. Livin, an inhibitor of apoptosis protein (IAP), is overexpressed in various cancers and possesses both the ability to protect from cell death and to promote it once it is cleaved. It is unknown whether there is a differential cellular expression of livin in ulcerated versus non-ulcerated melanomas. Methods: To explore the role of livin in relation to ulceration, other clinicopathological factors and survival of melanoma patients, we have performed semiquantitative immunohistochemical analysis of livin expression as measured by percentage and intensity (levels) of cells immunoreactive for livin in tumor specimens having both ulcerated and non-ulcerated areas from 50 melanoma lesions. Results: Superficial as well as deep melanoma cells located in non-ulcerated areas of tumor had higher percentage of cytoplasmic immunopositivity for livin when compared with melanocytes in the ulcerated areas of the melanoma (p&amp;lt;0.0001). We also observed nuclear expression of livin protein in this subset of melanoma patients. Compared to the ulcerated areas, the superficially located melanoma cells from the non-ulcerated areas showed significantly higher intensity of livin immunoreactivity in their nuclei (p = 0.048) but not those from the deep non-ulcerated areas (p = 0.32). Ulcerated melanomas with low tumor stage (I/II) had higher percentage of tumor cells with nuclear livin expression than those from higher tumor stage (III/IV) (p = 0.002). Interestingly, we observed that patients with higher intensity of cytosolic expression of livin protein in the ulcerated area of tumor were more likely to have a brisk tumor-infiltrating lymphocytes (TILs) response (p = 0.001). Patients with higher intensity of cytoplasmic livin protein of tumor cells in ulcerated areas had higher risk of death compared to those with lower levels (p = 0.005). On the other hand, patients with higher intensity of cytoplasmic expression of livin in melanoma cells located superficially in the non-ulcerated areas of tumor had better prognosis (p = 0.045). Conclusion: Our data indicate that there is a decrease of nuclear as well as cytoplasmic expression of livin protein in melanoma cells from ulcerated when compared to non-ulcerated areas. Also, cytoplasmic livin expression is an indicator of poor prognosis, but apparently only in patients with ulcerated lesions. Although IAPs regulate caspases, they also regulate signaling pathways that activate NF-κB modulating immunity. The prominent TILs response in livin-positive tumors suggests a possible involvement in tumor immunity in melanoma. Citation Format: Nitin Chakravarti, Dawen Sui, Denái R. Milton, Wen-Jen Hwu, Elizabeth A. Grimm, Victor G. Prieto. Differential expression of the melanoma inhibitor of apoptosis protein (ML-IAP)/Livin in patients with ulcerated and non-ulcerated melanomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3570.

Abstract P6-04-12: Immunoprofile of TIL, genetics of Fc receptor gama and apoptosis activation in patients with Her2-positive breast cancer – Pilot study

Abstract Possible markers helping us to detect molecular-immunologically patients with Her2 positivity are Fc-gamma receptors (FcR). FcR binds to CH3 section of Fc trastuzumab fragment and is the key factor for activation of cell lysis via immune system cells (NK cells, macrophages). Tumors, where the H131R mutation decreased the effect of NK cells and macrophages, show a higher percentage of tumor cells entering apoptosis and activation of tumor infiltrating lymphocytes (TIL) (CD8+), which can result in tumor volume reduction and inhibition and thus extend overall survival. In the pilot study included 50 patients with histologically verified breast cancer treated in the Department of Oncology (First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague) participated in our pilot study. 40 patients were with a median age of 49.4 years (14 postmenopausal [46.7%] and 16 premenopausal [53.3%]) with detected HER-2 FISH amplification (histologically 18 patients with HER-2 subtype which means without hormone receptor positivity and 12 patients with luminal B subtype and thus hormone receptor positive). All patients were without evidence of distal metastases (6 in stage I, 13 in stage II and 11 in stage III). A control group was formed of 10 patients with a median age of 49.2 years (4 postmenopausal [40.0%] and 6 premenopausal [60.0%]) and without HER-2 amplification (histologically 7 patients with luminal A subtype, 2 patients with luminal B subtype and 1 patient with basal-like subtype). All patients of the control group were without evidence of distal metastases (2 in stage I, 6 in stage II and 2 in stage III). All patients signed the written informed consent. Our aim was to carry out the H131R mutation analysis using the StripAssay (ViennaLab, Austrian) method in correlation with clinical data, to quantify cells entering apoptosis using molecular method TUNEL (DeadEnd, Fluorometric TUNEL System, USA) and to immunohistochemically determine the expression and ratio of CD8 (Clone C8/144B) to CD4 (Clone 4B12) (Dako, Denmark) in a tumor. Results showed that in Her2-positive patients responding badly to biological treatment according to the clinical data the H131R mutation in the FcR gene was detected in 34/40 (85%) cases (26 heterozygotes / 8 homozygotes). Kaplan-Meier analysis of patients with the H131R mutation (FcR mutation / activation of apoptosis / CD8 positivity) showed significant differences in patients' survival from the generalization of a tumor – 28 vs 83 months. In patients with longer survival molecular analysis TUNEL confirmed a large number of cells entering apoptosis (20-30% in the field of vision) and activated antitumor immunity (CD8+ positivity) in more than 60% of a tumor and concurrently no expression (CD4-). Our study showed that in Her2-positive patients with detected H131R mutation in the FcR gene the function of NK cells and macrophages in tumor inhibition was decreased (blocked). However, in some patients the apoptotic pathway was activated and the process of programmed cell death was induced. Increased expression of CD8 was a positive prognostic factor, with a longer survival of these patients. Dedication: PRVOUK-P-27/LF1/1., MZ 00064203. Citation Format: Stanek L, Tesarova P, Vocka M, Musil Z, Kasparova K, Petruzelka L. Immunoprofile of TIL, genetics of Fc receptor gama and apoptosis activation in patients with Her2-positive breast cancer – Pilot study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-12.

Immunoexpression patterns for Hypoxia-inducible Factor-1α and von Hippel-Lindau protein, in relation to Hsp90, of human brain tumors.

The pathogenesis of many tumors, including brain tumors, has been associated with hypoxia, which induces the transcriptional activity of hypoxia-inducible Factor-1α (HIF-1α). HIF-1α is normally degradated by the von Hippel-Lindau protein (pVHL) but, in hypoxia, pVHL/HIF-1α interaction is inhibited resulting in the nuclear accumulation of HIF-1α. Hsp90 (Heat shock protein 90), as a chaperone protein, plays a critical role for both stabilization of HIF-1α and degradation of pVHL. The aim of this study was to estimate immunohistochemically the expression levels of HIF-1α and pVHL, in relation to Hsp90, in different types of human brain tumors (42 gliomas, 9 medulloblastomas, and 38 meningiomas) using specific antibodies. The tumors were further divided into two groups according to the age of patients (≥19 years old or ⟨19 years old). Nuclear, for HIF-1α, and cytoplasmic, for pVHL and Hsp90, localization was detected in a high percentage of tumor cells in the majority of tumors. In astrocytomas, a significant, grade-dependent relationship for HIF-1α immunoexpression was observed (p⟨0.05). Furthermore, there was a significant correlation between pVHL and Hsp90 immunoexpression (p⟨0.01). The group of ≥19 years old patients with glioblastomas (WHO grade IV) demonstrated significantly increased immunoexpression for HIF-1α compared to pVHL (p⟨0.0001) and Hsp90 expression (p⟨0.01). In medulloblastomas, a significant correlation of HIF-1α with Hsp90 immunoexpression (p⟨0.05) was found. In meningiomas, no significant correlation for the expression of the three proteins was detected (p≥0.05). These results indicate that HIF-1α/pVHL/Hsp90 interactions may be implicated in biology of different types of brain tumors through different signaling mechanisms.

A novel bicompartmental mathematical model of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common primary CNS neoplasm, and continues to have a dismal prognosis. A widely-used approach to the mathematical modeling of GBM involves utilizing a reaction-diffusion model of cell density as a function of space and time, which accounts for both the infiltrative nature of the tumor using a diffusion term, and the proliferation of tumor cells using a proliferation term. The current paper extends the standard models by incorporating an advection term to account for the so-called 'cell streaming' which is often seen with GBM, where some of the tumor cells seem to stream widely along the white matter pathways. The current paper introduces a bicompartmental GBM model in the form of coupled partial differential equations with a component of dispersive cells. The parameters needed for this model are explored. It is shown that this model can account for the rapid distant dispersal of GBM cells in the CNS, as well as such phenomena as multifocal gliomas with tumor foci distant from the core tumor site. The model suggests a higher percentage of tumor cells below the threshold of MRI images in comparison to the standard model. By incorporating an advection component, the proposed model is able to account for phenomena such as multicentric gliomas and rapid distant dispersion of a small fraction of tumor cells throughout the CNS, features important to the prognosis of GBM, but not easily accounted for by current models.

Aggressive B-cell lymphomas of gastrointestinal tract: a clinicopathologic analysis of 54 cases

To study the histological features, diagnosis, differential diagnoses of aggressive B-cell lymphomas of the gastrointestinal tract and to correlate clinical prognosis with pathologic parameters and immunophenotypes with an emphasis on c-myc, Tcl-1 and CD38 expression and their values in predicting the status of c-myc gene translocation. Fifty-four cases of aggressive B-cell lymphomas of the gastrointestinal tract with complete clinical and pathologic data were retrospectively collected. The clinical data, histologic and immunohistochemical findings and follow-up results were analyzed. Predictive immunohistochemical stains including c-myc, Tcl-1 and CD38 were performed and ROC curve analysis was used to confirm the accuracy of these markers in predicting c-myc translocation. Of 54 cases, there were 33 males and 21 females with median age of 56 years. Histological types of lymphomas included 49 cases of DLBCL (11 cases of germinal central B cell like and 38 cases of activated B cell like by Hans classification), 4 cases of DLBCL/BL and 1 case of BL. Eleven of 54 patients died within 97 months, with median survival of 42 months. Histologically, full-thickness infiltration of the gastrointestinal tract by large atypical cells with evident phagocytosis of karyorrhexis by macrophages ("starry sky") were seen in 18/54 cases. The lymphoma cells were positive for CD20 (54/54), CD79a (54/54), CD43 (4/54), CD5 (7/54), bcl-2 (26/54), Tcl-1 (17/54) and CD38 (15/54), but all negative for CD3 and CD30. The proliferative index by Ki-67 ranged from 40% to 100%. The univariate survival analysis indicated that B symptoms, general performance, high LDH, high IPI, distant metastasis, high clinical stage and tumors with over 90% of cells positive for c-myc were negative predictors for the patient's survival. In addition, cases of DLBCL positive for CD5 had an unfavorable prognosis. Cox regression analysis showed c-myc translocation, distant metastasis and high LDH were independent predictors for unfavorable prognosis. ROC curve revealed the percentage of c-myc positivity predicted the presence of c-myc gene translocation, with 75% as the optimal threshold. Aggressive B-cell lymphomas of the gastrointestinal tract with a prognosis influenced by variable clinicopathologic factors. DLBCL and DLBCL/BL may possess c-myc translocation and tend to be Burkitt-like or atypical Burkitt lymphoma. As independent prognostic indicator, c-myc expression may be used for selection of therapeutic regimens and prognostication. High percentage of tumor cells with c-myc positivity may be used to predict the presence of c-myc gene translocation.

Abstract 856: Biomarkers of sensitivity and resistance to the BTK inhibitor PCI-32765 in mantle cell lymphoma

Abstract Bruton's tyrosine kinase (Btk) is a central regulator of BCR signaling in B-cell lymphomas. PCI-32765 is a potent and selective inhibitor of Btk which has shown good clinical activity, particularly in mantle cell lymphoma. But its mechanism of action in this disease is poorly understood. In this study, we explored the role of Btk and the activity of PCI-32765 on BCR signaling in PBMC samples from patients, which contain a high percentage of tumor cells, as well as in several MCL lines, including Granta-519, Jeko-1, JVM-2, JVM-13, Maver-1, Mino, NCEB-1, Rec-1 and Z-138. In this work, we present a detailed characterization of signalling pathways inhibited by PCI-32765 downstream of the B-cell receptor in cell lines and tumor cells, studied by gene expression profiling, flow cytometry, and proteomic analyses of signaling pathways and effectors. Correlation with PCI-32765 activity in tumor cells and patients leads to the identification of biomarkers of biomarkers of sensitivity and resistance. Results: Btk and surface IgM protein expression was detected in all MCL lines at variable levels. In a 3-day proliferation assay, JVM-2 &amp; MINO emerged as the most sensitive lines to PCI-32765 (GI50: 1.75-4.4uM). Rec-1 was resistant to PCI-32765 alone (11.3uM) but became much more sensitive (1.45uM) upon BCR stimulation using anti-IgM (10ug/mL). Other lines such as Maver-1, Granta-519 &amp; Jeko-1 all required &amp;gt;10uM of PCI-32765 for inhibition and BCR stimulation did not make much difference. When signaling pathways downstream of BCR activation were studied, intracellular calcium flux following stimulation with IgM was observed in all lines (except JVM-2) and was inhibited at &amp;lt;100nM PCI-32765 in most of them, but no correlation between this and growth inhibition was observed. Constitutive BTK autophosphorylation was observed in all lines and was completely abolished by PCI-32765. BCR stimulation increased p-BTK which was also blocked by PCI-32765 in all lines. Mino and JVM-2 showed constitutive p-ERK activity, which was slightly increased upon BCR stimulation and could be blocked with PCI-32765, whereas the more resistant lines such as Maver-1 and Rec-1 had low endogeneous levels of p-ERK, but which was increased by BCR stimulation and only partially or not reversed by PCI-32765 at 5uM. Little change was observed in levels of p-PLCg1 or p-NF-kB p65. Additionally, in all cell lines stimulation with anti-IgM led to an increased secretion of the chemokines CCL3 and CCL4, which was inhibited by PCI-32765. Blockade of Btk function using PCI-32765 inhibits MCL cell proliferation, BCR signaling and chemokine secretion. These findings highlight the importance of BCR signaling and Btk in MCL, help explain the activity of the Btk inhibitor PCI-32765 in MCL patients, and provide biomarkers that may be of value in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 856. doi:1538-7445.AM2012-856

Peptide Based Vaccine Approaches for Cancer-A Novel Approach Using a WT-1 Synthetic Long Peptide and the IRX-2 Immunomodulatory Regimen.

Therapeutic cancer vaccines have the potential to generate a long lasting immune response that will destroy tumor cells with specificity and safety, in contrast to many other current cancer therapies. Clinical success to date has been limited by a number of factors including choice of immunogenic cancer rejection antigens, optimization of vaccine platforms and immune adjuvants to effectively polarize the immune response, and incorporation of strategies to reverse cancer mediated immune suppression by utilization of effective adjuvant/immune modulators. WT-1 (Wilms' tumor gene 1) is a cancer antigen that is required for tumorigenesis, expressed in a high percentage of tumor cells and rarely expressed in adult normal cells. Moreover spontaneous immunity to WT-1 is seen in cancer patients and can be augmented with various therapeutic vaccine approaches. IRX-2 is an immune modulator with demonstrated preclinical and clinical pleiotropic immune activities including enhancement of the immune response to potential tumor antigens. This paper presents the rationale and preclinical data for utilizing the WT-1 tumor antigen in a novel vaccine platform consisting of a synthetic long peptide containing multiple class I and class II epitopes in combination with the IRX-2 immunomodulatory regimen to overcome immuno-suppressive pathways and enhance the anti-tumor response.