Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cancer among Sri Lankan females. This is the first study, to the best of our knowledge, that has focused on investigating the association between TP53 somatic DNA variants, with p53 protein expression and risk factors in a cohort of Sri Lankan patients with HNC. A total of 44 patients with cancer and 20 healthy controls were studied. In total, 36 genomic DNA sequence variants were found, including several novel variants (two deletions in exons 4 and 6, two in the 3′ untranslated region and several intronic variants). A total of 14 tumour samples carried pathogenic TP53 mutations. A random selection of 24 samples was analysed immunohistochemically for p53 protein expression. All the samples with point missense variants were strongly immuno-positive, whereas, samples with nonsense and frameshift TP53 variants were immuno-negative for p53 immunohistochemical staining. Although, the human papilloma virus is a known risk factor for HNC, results from the present study identified an absence or lower level of infection in the Sri Lankan cohort.

Background: Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. Methods: 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 functional status and compared it with the protein expression of p53. Additionally, hotspot codons of the KRAS, BRAF and PIK3CA genes were also analysed in a subset of CRC patients. Results: Twenty seven sequence variants, including several novel variants in the TP53 gene were found. Nine BC and seven CRC tumour samples carried pathogenic TP53 variants. Pathogenic point missense variants were associated with strong and diffuse positive staining for p53 by immunohistochemistry (IHC), whereas, wild type TP53 showed complete absence of positive IHC staining or rare positive cells, regardless of the type of cancer. There was no direct correlation between p21 or MDM2 expression and p53 expression in either BCs or CRCs. Four of the CRC patients had pathogenic hotspot variants in KRAS; three of them were on codon 12 and one was on codon 61. Conclusion:The prevalence of pathogenic somatic TP53 variants was 31 and 33.33% in the studied BC and CRC cohorts respectively. All of them were located in exons 5-8 and the pathogenic missense variants were associated with strong immuno-positive staining for p53.

Background Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. Methods 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 functional status and compared it with the protein expression of p53. Additionally, hotspot codons of the KRAS, BRAF and PIK3CA genes were also analysed in a subset of CRC patients. Results Twenty seven sequence variants, including several novel variants in the TP53 gene were found. Nine BC and seven CRC tumour samples carried pathogenic TP53 variants. Pathogenic point missense variants were associated with strong and diffuse positive staining for p53 by immunohistochemistry (IHC), whereas, wild type TP53 showed complete absence of positive IHC staining or rare positive cells, regardless of the type of cancer. There was no direct correlation between p21 or MDM2 expression and p53 expression in either BCs or CRCs. Four of the CRC patients had pathogenic hotspot variants in KRAS; three of them were on codon 12 and one was on codon 61. Conclusion The prevalence of pathogenic somatic TP53 variants was 31% and 33.33% in the studied BC and CRC cohorts respectively. All of them were located in exons 5 – 8 and the pathogenic missense variants were associated with strong immuno-positive staining for p53.

The 5-year survival rate for patients with metastatic prostate cancer (PCa) in the United States is 30.6%. Therefore, more effective treatment approaches are urgently required. The P2X4 purinergic receptor is well studied for its role in inflammation. Our previous studies demonstrated that the P2X4 receptor is highly expressed in PCa compared to benign regions and is associated with poor prognosis. We observed intense P2X4 expression on most macrophages and some neutrophils in the primary tumor microenvironment (TME). Our study also showed that P2X4 knock-down in mouse PCa Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in mice when compared to control cells. Allograft tumors were heavily infiltrated by P2X4-positive host immune cells. However, which components of the prostate TME express P2X4 receptors and the functional consequence of that expression is not well defined. Therefore, the objective of this study is to characterize P2X4 receptor expression and function in the prostate TME and metastatic niche. A P2X4 antagonist has completed Phase 1 clinical trials for the treatment of neuropathic pain. As such, deciphering a role for P2X4 in the prostate TME promises novel cancer therapeutic approaches against aggressive PCa. We used RNA in situ hybridization and immunohistochemistry to assess P2X4 expression on immune cells in human PCa tissues and Myc-CaP allograft primary and metastatic tumor tissues. To determine P2X4 function in macrophages in vitro, we used THP1 human monocytic cells differentiated to M0 macrophages and polarized to the M1 and M2 subtypes. M1 and M2 cells were treated with P2X4 agonist, CTP or antagonist, 5-BDBD. RNA was extracted from cells to examine the expression of macrophage subtype-specific genes, and cytokine levels in cell media were measured by ELISA. Statistical analysis was done using GraphPad Prism. Dual staining revealed abundant CD163+ M2 macrophages that express P2X4 in human PCa tissues. In Myc-CaP allograft primary and metastatic tumor tissues, we observed abundant F4/80+ macrophages and few Ly-6G+ neutrophils. Staining patterns suggest that P2X4+ immune cells likely include both cell types. CTP treatment of THP1 cell-derived M2 macrophages promoted anti-inflammatory IL-10 secretion while 5-BDBD treatment induced IL-6 secretion in M1 cells. M1 marker CD80, and M2 markers CD163 and CD206 mRNA expression were comparable across treatment groups, suggesting that treatment does not likely influence macrophage polarization. In conclusion, our study indicates that tumor-associated macrophages express P2X4 receptors both in primary and metastatic tumors. Our in vitro studies suggest a role for P2X4 receptors in macrophage cytokine secretion. Future studies will include functional assays to determine the role of P2X4 receptors on macrophages in the context of PCa. Citation Format: Vahinipriya Manoharan, Angelo M. De Marzo, Janielle P. Maynard. Characterization of P2X4 purinergic receptors in the prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2876.