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Objective Li-Fraumeni syndrome (LFS) is a pan-cancer predisposition syndrome caused by germline pathogenic variants in the gene TP53. The interpretation of TP53 variants in clinical scenarios outside the classic LFS criteria may be challenging. Here, we report a patient affected by 2 primary cancers at later ages, who harbored a likely pathogenic TP53 at low allele frequency detected in a blood sample. Methods The Molecular Tumor Board committee at our institution revisited the case of a patient who was enrolled in a research protocol for the investigation of genetic conditions associated with neuroendocrine tumors. Clinical, familial, and molecular data were reviewed. The patient received germline testing using a next generation sequencing multi-gene panel and was incidentally found to harbor a TP53 likely pathogenic variant, with 22% of variant allele fraction. Additional samples, including a second blood sample, oral swab, and saliva, were collected for DNA analysis. A new TP53 sequencing round was performed with the attempt to distinguish between a true constitutional germline variant and a somatically acquired variant due to aberrant clonal expansion of bone marrow precursors. Results Patient’s personal and familial history of cancer did not meet classic nor Chompret LFS criteria. Environmental risk factors for cancer were identified, such as alcohol abuse and tobacco exposure. The TP53 variant initially found in the next-generation sequencing was confirmed by Sanger sequencing in the previous DNA sample extracted from blood for the first analysis and in a second blood sample collected 6 years later. The TP53 variant was not detected in the DNA extracted from the oral swab and saliva samples. Conclusion Considering the low TP53 variant allele fraction in blood, absence of variant detection in oral swab and saliva samples, the lack of LFS clinical criteria, and history of exposure to environmental risk factors for cancer, the main hypothesis for this case was aberrant clonal expansion due to clonal hematopoiesis. Oncologists should interpret TP53 findings during germline testing with caution.

Supplementary Table 1 from Fibroblast Growth Factor 2 Restrains Ras-Driven Proliferation of Malignant Cells by Triggering RhoA-Mediated Senescence

Supplementary Figures 1-8 from Fibroblast Growth Factor 2 Restrains Ras-Driven Proliferation of Malignant Cells by Triggering RhoA-Mediated Senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras–dependent malignant Y1 adrenocortical cells and H-Ras V12–transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated β-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome. [Cancer Res 2008;68(15):6215–23]

Supplementary Figure Legends 1-8 from Fibroblast Growth Factor 2 Restrains Ras-Driven Proliferation of Malignant Cells by Triggering RhoA-Mediated Senescence

BackgroundAllogeneic hematopoietic stem-cell transplant (allo-HSCT) is a potentially curative therapy for several hematological disorders. Before stem-cell infusion, recipients undergo a conditioning regimen with chemo/radiotherapy and immunosuppressants, requiring the use of antibiotics to treat and prevent infections. This regimen promotes drastic alterations in the recipient’s microbiotas, including the oral microbiota, which have been associated with allo-HSCT complications and poor outcomes. However, long-term longitudinal studies on the oral microbiota of allo-HSCT recipients are scarce and disregard the existence of distinct microbiotas within the oral cavity. Here, we used 16S rRNA gene sequencing to characterize the microbiota dynamics (during and after allo-HSCT) of 31 allo-HSCT recipients at 3 oral sites (gingival crevicular fluid, oral mucosa, and supragingival biofilm).ResultsAnalysis of the oral microbiota dynamics during allo-HSCT revealed a significant decline in bacterial diversity and major shifts in microbiota composition in all oral sites, including blooms of potentially pathogenic genera. These blooms in some cases preceded respiratory infections caused by the blooming genera. We also noticed that differences in microbiota diversity and composition between oral sites were lost during allo-HSCT. Overall, oral microbiotas returned to their preconditioning state after engraftment. However, the ability to recover the initial bacterial composition varied between patients. After stratifying patients based on their ability to recover their preconditioning microbiota composition, we found that recovery of the oral mucosa microbiota composition was not associated with antibiotic usage but was associated with higher preconditioning diversity and earlier reconstitution of normal leukocyte counts. Most notably, oral mucosa microbiota composition recovery was an independent biomarker of better allo-HSCT outcomes.ConclusionWe observed clear patterns of microbiota dysbiosis in all three oral sites during allo-HSCT, however each oral site responded differently to the perturbations associated with allo-HSCT. Oral microbiota injury and recovery patterns were associated with allo-HSCT complications and outcomes. This study highlights the potential clinical impact of the oral microbiota in the allo-HSCT setting and the clinical value of tracking oral microbiota changes during allo-HSCT.

Accessibility to next-generation sequencing (NGS) technologies has enabled the profiling of microbial communities living in distinct habitats. 16S ribosomal RNA (rRNA) gene sequencing is widely used for microbiota profiling with NGS technologies. Since most used NGS platforms generate short reads, sequencing the full-length 16S rRNA gene is impractical. Therefore, choosing which 16S rRNA hypervariable region to sequence is critical in microbiota profiling studies. All nine 16S rRNA hypervariable regions are taxonomically informative, but due to variability in profiling performance for specific clades, choosing the ideal 16S rRNA hypervariable region will depend on the bacterial composition of the habitat under study. Recently, NGS allowed the identification of microbes in the urinary tract, and urinary microbiota has become an active research area. However, there is no current study evaluating the performance of different 16S rRNA hypervariable regions for urinary microbiota profiling. We collected urine samples from male volunteers and profiled their urinary microbiota by sequencing a panel of six amplicons encompassing all nine 16S rRNA hypervariable regions. After systematically comparing their performance, we show that V1V2 hypervariable regions better assess the taxa commonly present in urine samples and V1V2 amplicon sequencing is more suitable for urinary microbiota profiling. We believe our results will be helpful to guide this crucial methodological choice in future urinary microbiota studies.

Cardiovascular diseases are the main cause of death worldwide. Recent studies have revealed the influence of histone-modifying enzymes in cardiac remodeling and heart dysfunction. The Set7 methyltransferase regulates the expression of several genes through the methylation of histones and modulates the activity of non-histone proteins. However, the role of Set7 in cardiac remodeling and heart dysfunction remains unknown. To address this question, wild-type (WT) and Set7 knockout (KO) male mice were injected with isoproterenol or saline. WT mice injected with isoproterenol displayed a decrease in Set7 activity in the heart. In addition, WT and Set7 KO mice injected with isoproterenol exhibited cardiac hypertrophy. Interestingly, Set7 deletion exacerbated cardiac hypertrophy in response to isoproterenol but attenuated myocardial fibrosis. Echocardiograms revealed that WT mice injected with isoproterenol had lowered ejection fractions and fractional shortening, and increased E′-wave deceleration time and E/A ratio compared with their controls. Conversely, Set7 KO mice did not show alteration in these parameters in response to isoproterenol. However, prolonged exposure to isoproterenol induced cardiac dysfunction both in WT and Set7 KO mice. Both isoproterenol and Set7 deletion changed the transcriptional profile of the heart. Moreover, Set7 deletion increased the expression of Pgc1α and mitochondrial DNA content in the heart, and reduced the expression of cellular senescence and inflammation markers in response to isoproterenol. Taken together, our data suggest that Set7 deletion attenuates isoproterenol-induced myocardial fibrosis and delays heart dysfunction, suggesting that Set7 plays an important role in cardiac remodeling and dysfunction in response to stress.

One of the benefits of neoadjuvant chemotherapy and radiation therapy (CRT) for the management of rectal cancer is tumor downstaging that can ultimately lead to complete tumor regression (known as complete pathological response - pCR). In a previous study using PET/CT imaging to assess tumor response to CRT, 50% of rectal cancers showed a continuous decrease in metabolic activity (estimated by standard uptake value measurements) between 6 and 12 weeks from CRT completion. However the remaining 50% of the cases showed increased metabolic activity within that period. We reasoned that the increased metabolic activity observed in these later patients could be determined by the clonal expansion of a genetically distinct subpopulation of tumor cells resistant to CRT. To address this question we performed exome sequencing and mutation detection analysis on normal tissue, primary tumor and residual tumor from 7 patients with rectal cancer that exhibited an increase in metabolic activity after CRT. Overall, about 20Gb of unambiguously mapped sequences were generated for each sample resulting in an average fold-coverage of 30X. Captured sequences mapped to the reference human genome were then used for the detection of SNPs and non-synonymous somatic point mutations in all three samples from each patient. Approximately 30,000 single nucleotide variants (SNVs) were identified in each sample and most of these SNVs were common to all samples. As expected, the majority of these common inherited variants (96%) have already been described in dbSNP. To identify non-synonymous somatic mutations occurring in primary and residual tumor samples, we excluded those present in the normal sample and those already described as a known SNP in dbSNP resulting in a mean of 243 SNVs per patient. Noteworthy, a significant number of non-synonymous somatic mutations were exclusively found in the primary and residual tumor samples of each patient, 30 and 32 SNVs on average respectively, revealing a high degree of tumor genetic heterogeneity. Furthermore, we were able to identify non-synonymous somatic mutations that were presented in both samples (mean of 107 SNVs), and for these mutations we determined the mutant allele frequency (number of reads representing the mutation/total number of reads covering the mutated base) in the primary and the residual tumor. We then searched for mutations with significantly different allele frequencies in the two samples, as these mutations would likely represent genetically distinct subpopulations of tumor cells selected during CRT. We were able to identify, on average, 4 SNVs enriched in primary tumor and 37 SNVs enriched in residual tumor per patient. In conclusion, based on exome sequencing of rectal tumors that exhibit incomplete response to CRT and have increased tumor metabolism we were able to identify non-synonymous mutations that may be associated with specific mechanisms of resistance or sensitivity to CRT. Functional analysis of these mutations and mutated genes will be presented, providing new insights into the molecular events influencing response to neoadjuvant CRT in rectal cancer. Citation Format: Fabiana Bettoni, Elisa R. Donnard, Bruna R. S. Correa, Paula F. Asprino, Fernanda C. Koyama, Natália M. Felício, Bruna H. Hessel, Pedro A. F. Galante, Anamaria A. Camargo, Angelita Habr-Gama, Rodrigo O. Perez. Genetic heterogeneity in rectal cancer - Identification of subpopulations of tumor cells resistant to neoadjuvant CRT. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 05.