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Prevalence of CDKN2A gene deletion in Breast Cancer: An Indian experience Kazi Mahnaz M.1, Trivedi Pina J.2*, Patel Dharmesh M.3, Varma Priya K.4 1Junior Research Assistant, Cytogenetics Lab, Gujarat Cancer & Research Institute 2Senior Scientific Officer & Head, Cytogenetics Lab, Gujarat Cancer & Research Institute 3Research Assistant, Cytogenetics Lab, Gujarat Cancer & Research Institute 4 Junior Research Fellow, Cytogenetics Lab, Gujarat Cancer & Research Institute *Corresponding author E-mail IDs: mahnaz.kazi@gcriindia.org; pina.trivedi@gcriindia.org, dharmesh.patel@gcriindia.org, priya.varma@gcriindia.org Abstract Background: Breast cancer is the most common cancer in females, found to be associated with mutations in various genes. CDKN2A is one such tumor suppressor gene which is altered in a wide range of human neoplasms and most frequently shown to play a role in breast cancer. It may be used in the development of a targeted therapy that could result in improved patient prognostication and outcome. In the present study the frequency of CDKN2A gene deletion and its relationship with clinicopathological features were examined in breast cancer patients. Method: Histopathologically confirmed breast cancer patients were included for CDKN2A gene deletion study by Fluorescence in situhybridization (FISH) method. Formalin fixed paraffin embedded tissues were used for the assays. A total of 30 breast cancer patients were included. FISH was performed using P16 (CDKN2A) deletion probe kit. Images were captured by Epi-fluorescence microscope. Result: In breast cancer patients, CDKN2A gene was deleted in 22 patients (73%) with 17 hemizygous and 5 homozygous deletions, while 8 patients (27%) had normal intact form. CDKN2A gene deletion in breast cancer was found to be significantly inversely associated with tumor grade, presence of perineural invasion, perinodal extension and vascular permeation. Summary and Conclusion: CDKN2A gene deletion was associated with absence of known worse prognosticators indicating that the gene expression and not loss was linked to adverse tumor parameters. Hence, detection of CDKN2A gene deletion could help to stratify the specific subsets of patients into high risk and low risk groups. CDKN2A gene deletion can also help as the pre-screening tool prior to gene evaluation tests.

Additional file 1: Figure S1. A Comparative assessment of expression of HER2 CAR according to the anti-4D5 epitope idiotype antibody recognizing HER2 directly compared to the detection of the tNGFR tag. The latter, is a preferred method for sorting and phenotyping of cells since it has no direct biological effect on T cell activation/function. B Kinetics of expression of dCas9 and Q8 by qPCR. C CAR-T manufacturing process���CD3+ T cells are negatively selected from peripheral blood monocytes and stimulated with OKT3/CD28. LdCK is added for transduction a day later followed on the second day by HER2-TEV. Five days after the original stimulation the activation is removed and on the following day cells are sorted to enrich the tNGFR+/Q8+ population. The expansion is then continued till time of release between day 14 of production. Cells are also tested at that point for permanence of transduction as shown in Fig. 1A.

Additional file 4: Figure S4. A Intratumoral administration model and study design. B Experimental set up���all treatment groups included 5 or 6 mice receiving subcutaneous implantation of 1 million FaDu cells followed by adoptive transfer of CAR-T cells at day 9. Atezolizumab (10 mg/kg) was administered intravenously in the relevant groups the day before adoptive transfer (day 8) and subsequently dosed at 5 mg/kg twice a week.

Additional file 2: Figure S2. A Antigen density-dependent activation of HER2 CAR���conventional HER2 CAR cells were stimulated with beads coated with BSA, low or high densities of HER2 ectodomain (Low HER2 and High HER2) at indicated beads to CAR-T cells ratio for expression of CD69 and PD-1 at day 3 following stimulation. B Cytotoxic and expansion properties of RB-340-1 cellular components���non-transduced (NT), conventional HER2 CAR, cRB-340-1 and RB-340-1 T cells were exposed for 3 days to FaDu cell line at 1:20 effector to target ratio. Different proportion of CD4+/CD8+ T cells were tested for cytotoxic activity (upper panel) and expansion (lower panel). The numbers in the headline refer to the proportion of CD4+ T cells (%) present in each group over total number of T cells. C RB-340-1 specificity of gene regulation���primary T cells transduced with LdCK plus HER2-TEV constructs including either PD-1sg, TIM-3sg or both were tested for expression of the respective target genes 5 days after stimulation with FaDu cells to induce the expression of the two checkpoints. D Kinetics of gene-expression regulation by RB-340-1���RB-340-1 (red line) and cRB-340-1 (black line) were stimulated with HER2-coated (filled shapes) or BSA-coated (empty shapes) beads and the expression of PD-1, TIM-3 and CD69 was followed in CD8+ T cells at baseline and 48 and 72 hours after stimulation.

Additional file 5: Figure S5. A Systemic administration model and study design. B, C Experimental set up���all treatment groups included seven mice (in B for Fig. 5) or nine mice (in C for Fig. 6) receiving subcutaneous implantation of 0.5 million FaDu cells followed by adoptive transfer of CAR-T cells at day 10. Atezolizumab (10 mg/kg) was administered intravenously in the relevant groups the day before adoptive transfer (day 9) and subsequently dosed at 5 mg/kg twice a week.

Additional file 3: Figure S3. A Intratumoral administration model and study design. B Experimental set up���all treatment groups included eight mice receiving subcutaneous implantation of 0.5 million FaDu cells followed by adoptive transfer of CAR-T cells at day 10.