Female Oncofertility: Current Understandings, Therapeutic Approaches, Controversies, and Future Perspectives

Recent advances in early detection and oncological therapies have ameliorated the survival rate of young cancer patients. Yet, ovarian impairment induced by chemotherapy and radiotherapy is still a challenging issue. This review, based on clinical and lab-based studies, summarizes the evidence of gonadotoxicity of chemoradiotherapy, the recent approaches, ongoing controversies, and future perspectives of fertility preservation (FP) in female patients who have experienced chemo- or radio-therapy. Existing data indicate that chemotherapeutic agents induce DNA alterations and massive follicle activation via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Meanwhile, the radiation causes ionizing damage, leading to germ cell loss. In addition to the well-established methods, numerous therapeutic approaches have been suggested, including minimizing the follicle loss in cryopreserved ovarian grafts after transplantation, in vitro activation or in vitro growing of follicles, artificial ovarian development, or fertoprotective adjuvant to prevent ovarian damage from chemotherapy. Some reports have revealed positive outcomes from these therapies, whereas others have demonstrated conflictions. Future perspectives are improving the live birth rate of FP, especially in patients with adverse ovarian reserve, eliminating the risk of malignancy reintroducing, and increasing society’s awareness of FP importance.

Clinical Utility of Circulating Tumor DNA in Advanced Rare Cancers

PurposePatients with advanced/relapsed rare cancers have few treatment options. Analysis of circulating tumor DNA in plasma may identify actionable genomic biomarkers using a non-invasive approach.Patients and MethodsRare cancer patients underwent prospective plasma-based NGS testing. Tissue NGS to test concordance was also conducted. Plasma DNA alterations were assessed for incidence, functional impact, therapeutic implications, correlation to survival, and comparison with tissue NGS.ResultsNinety-eight patients were analyzed. Diseases included soft-tissue sarcoma, ovarian carcinoma, and others. Mean turn-around-time for results was 9.5 days. Seventy-six patients had detectable gene alterations in plasma, with a median of 2.8 alterations/patient. Sixty patients had a likely pathogenic alteration. Five received matched-therapy based on plasma NGS results. Two developed known resistance mutations while on targeted therapy. Patients with an alteration having VAF ≥5% had a significantly shorter survival compared to those of lower VAF. Tissue NGS results from eleven of 22 patients showed complete or partial concordance with plasma NGS.ConclusionPlasma NGS testing is less invasive and capable of identifying alterations in advanced rare cancers in a clinically meaningful timeframe. It should be further studied as a prospective enrollment assay in interventional studies for patients with rare advanced stage cancers.Clinical Registration[https://www.umin.ac.jp/ctr/index-j.htm], identifier UMIN000034394.

TP53 Alterations Enrich in Ezb/C3-like Subclassified Relapsed or Refractory DLBCL and Are Susceptible to BRD4 Protac Inhibition

Introduction: New Diffuse Large B-cell (DLBCL) treatments remain a clinical need despite the success of rituximab combined with CHOP chemotherapy (RCHOP), which results in durable responses in 60-70% of patients. Those refractory to or who relapse following first-line therapy (rrDLBCL) have a very poor outcome, with only 20% surviving beyond 5 years, although CAR-T cell therapy is making a significant impact. Genomic insights can inform mechanism and treatment studies within this population, specifically the precision application of CAR-T. Rushton et. al. recently analyzed a large rrDLBCL patient cohort, noting significant rises in specific genes during the transition to advanced disease (MS4A1, TP53, NFKBIE, FOXO1, and CREBBP), building on the innovations of pre-treatment classifications. Addressing the heterogeneous activation of oncogenic pathways remains a challenge. Inhibitors targeting genes responsible for epigenetic regulation of rrDLBCL represent an attractive option, namely the transcriptional regulator BRD4, with PROTAC inhibition resulting in downregulation of several key DLBCL oncogenes. Herein, we report the first genomic subclassifications of rrDLBCL, the enrichment of TP53 alterations in the EZB-like subclassification, and data supporting the application of BRD4-targeting PROTAC therapies. Methods: Non-negative matrix factorization (NMF) was applied to a publicly available cohort of 127 RCHOP-treated rrDLBCL patients with matching sequencing data. Marker selection was applied to the 2 emergent groups to detect differential presence of DNA alterations. A Pearson similarity matrix was applied to the rrDLBCL cohort and 3 pretreatment cohorts to detect patterns of DNA alteration association and exclusion. Next, CRISPR-KO gene scores were integrated against gene expression fold change values before and after BRD4 PROTAC treatment. Lasty, 4 DLBCL cell lines were assayed against the BRD4 PROTAC ARV-825, Venetoclax, and inhibitors of epigenetic regulation. Results: NMF produced 2 clusters based on DNA alterations from the rrDLBCL cohort: RR1 and RR2. Significantly associated genes (FDR ≤ 0.05) included MYD88, PIM1, IGLL5, HIST1HC, SOCS1, HIST1H1E, and CD79B in RR1 , and CREBBP, EZH2, STAT6, BCL2, TP53, TNFRSF14, CARD11, and double hit translocations in RR2. Notably, TP53 alterations significantly enriched in the EZB subclassification during rrDLBCL transition in comparison to pre-treatment distribution (P = 0.0016). Significant associations between TP53 and EZB genes were observed alongside significant exclusion from MCD and BN2-associated genes. Integrating significant gene expression changes following BRD4-PROTAC treatment and CRISPR-KO scores revealed the loss of several key oncogenes in cell lines, including BCL2, BCL6, CD79B, POU2F2, and MDM4. Markedly, reductions in 9 established GCB and 4 ABC gene signatures were observed alongside the reduction of the Reactome signature of TP53 Regulators of Expression and Degradation (FDR = 0.0377). In vitro inhibition assays measuring ARV-825 (BRD4 PROTAC) response revealed significantly lower rates of cell viability compared to Venetoclax across 4 cell lines. Conclusions: Our results indicate that distinct genomic groups exist within rrDLBCL, that TP53 alterations strongly associate with the EZB-enriched group, and that if TP53 alterations continue to predict poor CAR-T response, these tumors may be susceptible to BRD4 inhibition. Our observations indicate a significant climb in TP53 association with EZB tumors and their matched alterations after rrDLBCL transition in comparison to the pre-treatment landscape. Enrichment may indicate that EZB-like tumors lack some of the tools necessary to make the transition to advanced disease but provide an ideal environment for the acquisition of TP53 alterations. In contrast, the disassociation between TP53 alterations and MCD/BN2-like alterations may indicate that these tumors have more inherent capability to reject therapy. The emergence of BRD4-targeting PROTACs provides an additional clinical tool, one perhaps more suitable than Venetoclax against specific rrDLBCL cases. In closing, This study provides insights towards the rrDLBCL landscape and reinforces the necessity of evaluating tissue biopsies for TP53 alterations throughout treatment to guide precision application of therapy, noting susceptible pathways of these particular tumors. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Virulent Bacteria as Inflammatory and Immune Co-Factor in Colon Carcinogenesis: Evidence From Two Monozygotic Patients and Validation in CRC Patient and Healthy Cohorts

Colorectal carcinoma (CRC) is a common disease, the incidence of which is increasing according to Western lifestyle; it remains to have a poor prognosis. Western nutriments are presumed to induce mild inflammation within the colonic mucosa, resulting in the accumulation of DNA alterations in colonocytes through a multistage carcinogenesis process. This suggests that most CRCs are related to the environment. Of interest, fecal microbiota composition has been shown yielding a novel approach regarding how environment changes may impact health and disease. Here, we compare whole shotgun metagenomic gut microbiota of two monozygotic twin sisters, one of whom is suffering from an advance colorectal tumor with a profound disequilibrium of the composition of the gut microbiota due to the overexpression of virulent bacteria such as E. coli, Shigella, and Clostridium species in the colon cancer patient’s feces contrasting with low levels of bacterial species such as Faecalibacterium and Akkermansia usually enriched in the healthy adults’ microbial flora. The disequilibrium in microbiota of the CRC patient’s feces as compared to her monozygotic twin sister is linked to inflammatory and immune cell infiltrates in the patient’s colonic tissue. We speculate on the role of microbiota disequilibrium on the immune-tolerant cell infiltrate within CRCs.

Therapeutic Actionability of Circulating Cell-Free DNA Alterations in Carcinoma of Unknown Primary

PURPOSE Cancer of unknown primary (CUP) is a metastatic disease with unidentifiable primary tumor. Somatic alterations can be assessed noninvasively via liquid biopsies interrogating cell-free DNA (cfDNA). METHODS We evaluated 1,931 patients with CUP with a cfDNA next-generation sequencing panel (73-74 genes). RESULTS Overall, 1,739 patients (90%) had ≥ 1 cfDNA alteration. We then explored alteration actionability (per the levels of evidence from the OncoKB database); 825 patients (47.4% of 1,739) had level 1, level 2, or resistance/R1 alterations. Among 40 clinically annotated patients with CUP who had cfDNA evaluated, higher degrees of matching treatment to alterations (Matching Score > 50% v ≤ 50%) was the only variable predicting improved outcome: longer median progression-free survival (10.4 v 2.5 months; P = .002), overall survival (13.4 v 5.7 months; P = .07, trend), and higher clinical benefit rate (stable disease ≥ 6 months/partial response/complete response; 83% v 25%; P = .003). CONCLUSION In summary, cfDNA frequently reveals strong level-of-evidence actionable alterations in CUP, and high degrees of matching to therapy correlates with better outcomes.

Concomitant and decoupled effects of cigarette smoke and SCAL1 upregulation on oncogenic phenotypes and ROS detoxification in lung adenocarcinoma cells

Lung cancer is the leading cause of cancer deaths worldwide, with smoking as its primary predisposing factor. Although carcinogens in cigarettes are known to cause oncogenic DNA alterations, analyses of patient cohorts revealed heterogeneous genetic aberrations with no clear driver mutations. The contribution of noncoding RNAs (ncRNAs) in the pathogenesis of lung cancer has since been demonstrated. Their dysregulation has been linked to cancer initiation and progression. A novel long noncoding RNA (lncRNA) called smoke and cancer-associated lncRNA 1 (SCAL1) was recently found upregulated in smoke-exposed adenocarcinomic alveolar epithelial cells. The present study characterized the phenotypic consequences of SCAL1 overexpression and knockdown using A549 cells as model system, with or without prior exposure to cigarette smoke extract (CSE). Increase in SCAL1 levels either by CSE treatment or SCAL1 overexpression led to increased cell migration, extensive cytoskeletal remodeling, and resistance to apoptosis. Further, SCAL1 levels were negatively correlated with intracellular levels of reactive oxygen species (ROS). In contrast, SCAL1 knockdown showed converse results for these assays. These results confirm the oncogenic function of SCAL1 and its role as a CSE-activated lncRNA that mediates ROS detoxification in A549 cells, thereby allowing them to develop resistance to and survive smoke-induced toxicity.

OMRT-8. Precision targeting of cellular pathways with complementary diagnostics

Precision medicine tailors treatment for each patient by identifying the molecular drivers of their disease. This can allow more effective tumour targeting, avoid harmful standard chemotherapeutic side-effects, and offer savings to the healthcare system through not treating patients who are unlikely to respond to a specific agent. Treatment regimes are usually designed by identifying DNA-level alterations and selecting drugs tailored to that mutation. However, cancer is not a one-pathway disease and not all patients with particular mutations will respond to treatment, while patients without canonical pathway-activating mutations are excluded from potentially life-saving treatment. To address this, we have developed a NanoString assay combining proteomic and transcriptomic profiles of 4 key actionable, cancer-related pathways (MAPK, PI3K, NFκB and JAK/STAT). We used RNA-Seq data from gold standard cell lines with defined pathway changes to identify minimal gene sets indicative of pathway activation, and integrated them with phospho protein measurements to generate a pathway activation score. The combined panel was run on isogenic cell lines as well as glioma samples with both known and unknown driving alterations. We found pathway activation to be more variable than expected based on DNA alterations alone, implying that consideration of proteomic and/or transcriptomic-level information is important for future therapeutic decision-making.