Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment—modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

Circulating tumor cells (CTCs) represent a distinct cancer biomarker established in clinical settings for early cancer detection, metastasis progression, and minimal residual disease (MRD) monitoring. Despite numerous advances, the comprehensive...

Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment - modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three P. gingivalis strains in nutrient-rich broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient-rich broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. In the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that the ability of the community to grow was largely due to Rgp gingipain. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex serum substrates. Whereas they are constitutively expressed by P. gingivalis in nutrient-rich broth, gingipain expression in the model periodontal pocket environment (serum) appears to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which can then promote growth of the whole community.

Comprehensive CtDNA and CTC profiling of treatment naïve early-stage head and neck cancer patients reveals early signatures of disease progressionBackground: We performed comprehensive ctDNA analysis on early-stage HNC patients in a pilot study to determine the mutational landscape in HNC patients with a known tobacco history. Methods: We analysed ctDNA of 18 early-stage HNC patients for genomic landscapes using the illumina NextSeq 2000 NGS. A custom-designed OncoIndex gene panel was used for the hybrid capture target-enrichment of critical cancer genes. Panel was designed to detect cancer targeting exonic sequence of 600 genes reporting SNVs and indels along fusions and copy number amplification. The gene panel detected genome-wide signatures including bTMB, MSI (microsatellite instability), HRD (homologous recombination deficiency) prediction and calculate cfDNA tumor fraction. Results: 80 % patients showed presence of at least one CTC in peripheral blood, possibly indicating the progressive disease at the time of presentation. Comprehensive genomic profile obtained from plasma cfDNA of early-stage HNC cancer patients predominantly had low bTMB and MSI Scores (99 % patients). However, HRD and LOH matrix was high for 60 % patients indicating highly dysregulated DNA repair activities. Concurring to these observations, 98 % patients had mutations in key tumor suppressor and DNA damage response (DDR) genes possibly resulting in their loss of function. Besides DNA damage pathway, 60% patients harboured alterations in RTK genes including FGFR, EGFR and PDEGFR family and 32% patients showed activating mutations in Erk1 and its upstream regulators. MSH2 was the most prominently mutated gene (37%) followed by FGFR (32%). Surprisingly, unlike HPV positive advanced HNC cases, TP53 mutations were not detected in any patient, though alterations in TS genes were most prevalent in the study population. 51% alterations resulted into truncated proteins possibly impairing their functions, while 42% alterations were point mutations, 6 % were frameshift and 1 % indels. Presence of mutations in BRAF, PDGFR, FGFR and KIT genes suggested for the potential therapy resistance. Tumor fraction representing ctDNA showed elevated range from 20 % to 45 % with a corresponding ploidy between 2 to 4. Conclusions: Comprehensive ctDNA profile showed major gene alterations in TS and DDR response pathway genes besides mutations in proliferative signaling members. TP53 mutation was not detected, although critical tumor suppressor and DDR genes were predominantly mutated, suggesting for a unique mutation pattern associated with early-stage HNC due to tobacco etiology. Our results suggest that comprehensive ctDNA analysis along CTC profiling can predict the disease progression beforehand and may offer new treatment options to early-stage HNC patients. Citation Format: Gowhar Shafi, Atul Bharde, Fauzul Moubeen, Kanchan Hariramani, Alain D’Souza, Trupti Kad, Bhagwat Jadhav, Sangita Prajapati, Aditi Rani, Madhura Basavalingegowda, Mohan Uttarwar, Gourishankar Aland, Sreeja Jayant, Aravindan Vasudevan, Pankaj Chaturvedi, Jayant Khandare. Comprehensive circulating tumor DNA and CTC profiling of treatment naïve early-stage head and neck cancer patients reveals early signature of disease progression [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 3379.

e14047 Background: Advanced brain tumors comprise an aggressive class of malignancies and are characterized by poor patient survival. In general, the 5-yr survival rate for people ≥40 years is about 21%. These tumors also appear to be recalcitrant to available SOC, hence the need for deeper insights to recognize actionable genetic alterations in order to make reliable assessments of diagnosis, prognosis, therapy response, and resistance. Comprehensive targeted Next Generation Sequencing (NGS) has allowed significant advances in precision oncology by pinpointing actionable alterations in oncogenes, tumor suppressor genes, and cellular signaling pathways. Methods: Retrospective NGS-based genome profiling was performed on ccfDNA (circulating cell-free DNA) and genomic DNA (FFPE/fresh tissue) samples from 8 brain tumor patients OncoIndx proprietary gene panel that targets exonic regions of 600 cancer related genes. Illumina compatible libraries were prepared by target hybridization method and sequenced on Nextseq 2000 platform in pair-end fashion. Variant calling was performed using an in-house developed bioinformatics pipeline. The 8 patients comprised 1 each for glioma, anaplastic astrocytoma, pilocytic astrocytoma and parietal SOL (Space Occupying Lesion) and 2 each for GBM and ependymoma. Results: Novel mutations such as GNASA366S, BRAFM590K and RAD54LD592V were observed in glioblastoma. Additionally, novel SNVs such as STK11G215V, CREBBPG1465E, GNA11G208C and SF3B1R549S were observed in ependymoma along with as yet unreported amplification of KIT, KDR and PDGFRA. NOTCH3 double mutation C428S and C455S was identified for the first time in this study in anaplastic astrocytoma. EGFRH835L, as yet unreported in glioma or ependymoma, was also identified here. Strikingly, some of these alterations are actionable: preclinical data links GNAS to MAPK and Wnt signaling creating a potential workspace for these targeted agents. Similarly, loss of STK11 sensitizes 3D gliomaspheres to metformin and mutations in SF3B1 are known to cause cellular sensitivity to PRMT5i. SF3B1 R549S mutation has not been reported yet and it will be crucial to assess the effect this alteration has on protein function. Furthermore, amplification of KIT, KDR and PDGFRA is known to create a sensitive niche for TKI in adenoid cystic carcinoma and may also have potential in ependymoma where these amplifications have been reported for the first time in this study. The EGFR H835L finding has clinical implication since L833V/H835L co-mutation has shown remarkable efficacy to Afatinib in LUAD. Conclusions: We report, for the first-time actionable mutations in key genes related to critical signaling pathways in a mixed population of brain tumor patients. The data highlights that comprehensive NGS-based deep assessment can lead to clinically viable insights in this population of patients with brain tumors.