Suspected Germline TP53 Variants and Clonal Hematopoiesis of Indeterminate Potential: Lessons Learned From a Molecular Tumor Board.

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.

Oral shedding of herpesviruses and clinical outcomes in hematopoietic stem cell transplant patients.

OBJECTIVES: To characterize the oral shedding of herpes viruses in patients who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) and investigate its relationship with clinical outcomes. MATERIALS AND METHODS: Polymerase chain reaction and enzymatic digestion were performed to identify the oral shedding of the members of the Herpesviridae family in 31 patients. The samples were collected from the oral cavity at five timestamps. RESULTS: The presence of each herpesvirus in the oral cavity was observed in 3.2%, 12.9%, 19.3%, 32.2%, 54.8% and 93.5% patients for human herpesvirus (HHV)-6A, herpes simplex virus-1, HHV-6B, cytomegalovirus (CMV), Epstein-Barr virus (EBV) and HHV-7, respectively. Oral shedding of herpes virus was not uncommon after alloHSCT. There was a statistically significant association between the EBV and CMV oral shedding at C1 and the cumulative incidence of acute graft-versus-host disease (aGVHD). The results suggested that the presence of HSV-1 at C2 was related to a relapse. The HHV-7 oral shedding at C2 suggests a possible link between relapse, progression-free survival and overall survival of the patients. CONCLUSIONS: Patients who developed aGVHD showed higher CMV and EBV shedding in the oral cavity at aplasia, suggesting modifications to the pattern of immune cell response and inflammatory microenvironment.

Natural killer cells 56bright16- have higher counts in the umbilical cord blood than in the adult peripheral blood.

INTRODUCTION AND HYPOTHESIS: Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for allogeneic hematopoietic stem cell transplantation in the absence of a compatible donor. The UCB transplantation has a lower incidence of chronic graft versus host disease (GvHD), but is associated with slower engraftment and slower immune reconstitution, compared to other sources. Dendritic cells (DCs) and Natural Killer cells (NKs) play a central role in the development of GvHD and the graft versus leukemia (GvL) effect, as well as in the control of infectious complications. METHOD: We quantified by multiparametric flow cytometry monocytes, lymphocytes, NK cells, and DCs, including their subsets, in UCB samples from 54 healthy newborns and peripheral blood (PB) from 25 healthy adult volunteers. RESULTS: In the UCB samples, there were higher counts of NK cells 56bright16- (median 0.024 × 109/L), compared to the PB samples (0.012 × 109/L, p < 0.0001), NK 56dim16bright (median 0.446 × 109/L vs. 0.259 × 109/L for PB samples, p = 0.001) and plasmacytoid dendritic cells (pDCs, median 0.008 × 109/L for UCB samples vs. 0.006 × 109/L for PB samples, p = 0.03). Moreover, non-classic monocyte counts were lower in UCB than in PB (median 0.024 × 109/L vs. 0.051 × 109/L, respectively, p < 0.0001). CONCLUSION: In conclusion, there were higher counts of NK cells and pDCs and lower counts of non-classic monocytes in UCB than in PB from healthy individuals. These findings might explain the lower incidence and severity of chronic GvHD, although maintaining the GvL effect, in UCB transplant recipients, compared to other stem cell sources.

Longitudinal analysis at three oral sites links oral microbiota to clinical outcomes in allogeneic hematopoietic stem-cell transplant.

IMPORTANCE: The oral cavity is the ultimate doorway for microbes entering the human body. We analyzed oral microbiota dynamics in allogeneic hematopoietic stem-cell transplant recipients and showed that microbiota injury and recovery patterns were highly informative on transplant complications and outcomes. Our results highlight the importance of tracking the recipient's microbiota changes during allogeneic hematopoietic stem-cell transplant to improve our understanding of its biology, safety, and efficacy.

Commensal oral microbiota impacts ulcerative oral mucositis clinical course in allogeneic stem cell transplant recipients.

Oral mucositis (OM) is a complex acute cytotoxicity of antineoplastic treatment that affects 40-85% of patients undergoing hematopoietic stem-cell transplantation. OM is associated with prolonged hospitalization, increased extensive pharmacotherapy, need for parenteral nutrition, and elevated treatment costs. As OM onset relates to the mucosal microenvironment status, with a particular role for microbiota-driven inflammation, we aimed to investigate whether the oral mucosa microbiota was associated with the clinical course of OM in patients undergoing allogeneic hematopoietic stem-cell transplantation. We collected oral mucosa samples from 30 patients and analyzed the oral mucosa microbiota by 16S rRNA sequencing. A total of 13 patients (43%) developed ulcerative OM. We observed that specific taxa were associated with oral mucositis grade and time to oral mucositis healing. Porphyromonas relative abundance at preconditioning was positively correlated with ulcerative OM grade (Spearman ρ = 0.61, P = 0.028) and higher Lactobacillus relative abundance at ulcerative OM onset was associated with shortened ulcerative OM duration (P = 0.032). Additionally, we generated a machine-learning-based bacterial signature that uses pre-treatment microbial profiles to predict whether a patient will develop OM during treatment. Our findings suggest that further research should focus on host-microbiome interactions to better prevent and treat OM.

Dental Biofilm Microbiota Dysbiosis Is Associated With the Risk of Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation.

Acute graft-versus-host disease (aGVHD) is one of the major causes of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recently, aGVHD onset was linked to intestinal microbiota (IM) dysbiosis. However, other bacterial-rich gastrointestinal sites, such as the mouth, which hosts several distinctive microbiotas, may also impact the risk of GVHD. The dental biofilm microbiota (DBM) is highly diverse and, like the IM, interacts with host cells and modulates immune homeostasis. We characterized changes in the DBM of patients during allo-HSCT and evaluated whether the DBM could be associated with the risk of aGVHD. DBM dysbiosis during allo-HSCT was marked by a gradual loss of bacterial diversity and changes in DBM genera composition, with commensal genera reductions and potentially pathogenic bacteria overgrowths. High Streptococcus and high Corynebacterium relative abundance at preconditioning were associated with a higher risk of aGVHD (67% vs. 33%; HR = 2.89, P = 0.04 and 73% vs. 37%; HR = 2.74, P = 0.04, respectively), while high Veillonella relative abundance was associated with a lower risk of aGVHD (27% vs. 73%; HR = 0.24, P < 0.01). Enterococcus faecalis bloom during allo-HSCT was observed in 17% of allo-HSCT recipients and was associated with a higher risk of aGVHD (100% vs. 40%; HR = 4.07, P < 0.001) and severe aGVHD (60% vs. 12%; HR = 6.82, P = 0.01). To the best of our knowledge, this is the first study demonstrating that DBM dysbiosis is associated with the aGVHD risk after allo-HSCT.

Disruption of the oral microbiota is associated with a higher risk of relapse after allogeneic hematopoietic stem cell transplantation.

Intestinal microbiota (IM) diversity and composition regulates host immunity and affects outcomes after allogeneic stem cell transplantation (allo-HSCT). We evaluated if the oral mucosa microbiota (OM) could impact the outcomes in patients who underwent allo-HSCT. Samples from the oral mucosa of 30 patients were collected at three time points: before the conditioning regimen, at aplasia, and at engraftment. We analyzed the associations of OM diversity and composition with allo-HSCT outcomes. Lower OM diversity at preconditioning was associated with a higher risk of relapse at 3 years (68% versus 33%, respectively; P = 0.04). Dominance (relative abundance ≥ 30%) by a single genus at preconditioning was also associated with a higher risk of relapse (63% versus 36% at 3 years, respectively; P = 0.04), as well as worse progression-free survival (PFS; 19% versus 55%, respectively; P = 0.01), and overall survival (OS) at 3 years (38% versus 81%, respectively; P = 0.02). In our study we observed that OM dysbiosis is associated with a higher risk of relapse and worse survival after allo-HSCT.

The impact of pretransplant malnutrition on allogeneic hematopoietic stem cell transplantation outcomes.

BACKGROUND: Malnutrition is a common finding in allogeneic hematopoietic stem cell transplantation (alloHSCT) patients, and there is some evidence that malnutrition might negatively affect the transplant outcomes. METHOD: We performed a retrospective study with 148 patients aged 18-75 years, who underwent alloHSCT between 2011 and 2017. Patients were classified according to the body mass index (BMI) and the Subjective Global Assessment (SGA). The SGA was assessed on the day of hospitalization for the transplant, and classifies patients into three groups: A (well-nourished), B (moderately malnourished) and C (severely malnourished). RESULTS: The SGA classified 49 (33%) patients as well-nourished, 54 (37%) as moderately malnourished, and 45 (30%) as severely malnourished. SGA-C was also associated with severe acute graft versus host disease (aGVHD) with a cumulative incidence (CI) of 31% vs. a CI of 14% for combined well-nourished or moderately malnourished group (SGA-A or -B, P = 0.017). In multivariate analysis, SGA-C compared to SGA-A or -B, remained as an independent risk factor for aGVHD (hazard ratio - HR 1.68, 95% confidence interval - 95% CI 1.02-2.74), and nonrelapse mortality (NRM - HR 3.63, 95% CI 1.76-7.46), worse progression free survival (HR 2.12, 95% CI 1.25-3.60), and worse overall survival (HR 3.27, 95% CI 1.90-5.64). CONCLUSION: Malnutrition increases the risk of aGVHD and NRM and has a negative impact on survival.