Wild-type Cohort Research Articles

DPYD genotype-guided dose personalisation for fluoropyrimidine-based chemotherapy prescribing in solid organ cancer patients in Australia: GeneScreen 5-FU study protocol

BackgroundFluoropyrimidine (FP) chemotherapies are commonly prescribed for upper and lower gastrointestinal, breast and head and neck malignancies. Over 16,000 people with cancer require FP chemotherapies per annum in Australia. Between 10 and 40% patients experience grade 3–4 (≥ G3) toxicities that require hospital-based management ± intensive care admission. Approximately 1% of patients die secondary to FP toxicities. Prospective screening for DPYD gene variants (encoding the key enzyme for FP catabolism) can identify patients at risk of ≥ G3 toxicity and allow for dose adjustment prior to first FP exposure. Evidence supports this as a cost-effective method of improving patient safety and reducing healthcare burden internationally; however, no Australian data confirms its feasibility on a large scale.MethodThis investigator-led, single-arm study will determine large scale feasibility of prospective DPYD genotyping, confirming patient safety and cost-effectiveness within the Australian health care system. 5000 patients aged 18 years and older with solid organ cancers requiring FP chemotherapy will be consented and genotyped prior to commencing treatment, and early toxicity (within 60 days) post-FP exposure will be determined. Toxicity data for DPYD variant carriers who have dose adjustments will be compared to the wild-type cohort and historical cohorts of carriers who did not undergo genotyping prior to FP exposure, and prospective variant carriers who do not undergo dose-adjustment. Prevalence of the four standard DPYD gene variants will be confirmed in an Australian population. Additionally, health economic analysis, implementation research via semi-structured interviews of patients and clinicians, and feasibility of UGT1A1 genotyping will be conducted.DiscussionThis study will determine the prevalence of DPYD gene variant status in Australia and its impact on FP-induced toxicity among Australians with cancer. Feasibility and cost-effectiveness for Australian health care system will be estimated to support national roll-out of prospective DPYD genotyping prior to FP administration. Additionally, feasibility will be confirmed with the intention of including UGT1A1 in future pharmacogenomic panels to aid chemotherapy prescribing.Trial registrationThis trial was registered with the Australian and New Zealand Cancer Trials Registry on 13th Dec 2023, ACTRN12623001301651.

USP20 deletion promotes eccentric cardiac remodeling in response to pressure overload and increases mortality.

Left ventricular hypertrophy (LVH) caused by chronic pressure overload with subsequent pathological remodeling is a major cardiovascular risk factor for heart failure and mortality. The role of deubiquitinases in LVH has not been well characterized. To define whether the deubiquitinase ubiquitin-specific peptidase 20 (USP20) regulates LVH, we subjected USP20 knockout (KO) and cognate wild-type (WT) mice to chronic pressure overload by transverse aortic constriction (TAC) and measured changes in cardiac function by serial echocardiography followed by histological and biochemical evaluations. USP20-KO mice showed severe deterioration of systolic function within 4 wk of TAC compared with WT cohorts. Both USP20-KO TAC and WT-TAC cohorts presented cardiac hypertrophy following pressure overload. However, USP20-KO-TAC mice showed an increase in cardiomyocyte length and developed maladaptive eccentric hypertrophy, a phenotype generally observed with volume overload states and decompensated heart failure. In contrast, WT-TAC mice displayed an increase in cardiomyocyte width, producing concentric remodeling that is characteristic of pressure overload. In addition, cardiomyocyte apoptosis, interstitial fibrosis, and mouse mortality were augmented in USP20-KO-TAC compared with WT-TAC mice. Quantitative mass spectrometry of LV tissue revealed that the expression of sarcomeric myosin heavy chain 7 (MYH7), a fetal gene normally upregulated during cardiac remodeling, was significantly reduced in USP20-KO after TAC. Mechanistically, we identified increased degradative lysine-48 polyubiquitination of MYH7 in USP20-KO hearts, indicating that USP20-mediated deubiquitination likely prevents protein degradation of MYH7 during pressure overload. Our findings suggest that USP20-dependent signaling pathways regulate the layering pattern of sarcomeres to suppress maladaptive remodeling during chronic pressure overload and prevent cardiac failure.NEW & NOTEWORTHY We identify ubiquitin-specific peptidase 20 (USP20) as an important enzyme that is required for cardiac homeostasis and function, particularly during myocardial pressure overload. USP20 regulates protein stability of cardiac MYH7, an essential molecular motor protein expressed in sarcomeres; loss-of-function mutations of MYH7 are associated with human hypertrophic cardiomyopathy, cardiac failure, and sudden death. Enhancing USP20 activity could be a potential therapeutic approach to prevent the development of maladaptive state of eccentric hypertrophy and heart failure.

Aging reveals a sex-dependent susceptibility of sarcospan-deficient mice to cardiometabolic disease.

Numerous genes including sarcospan (SSPN) have been designated as obesity-susceptibility genes by human genome-wide association studies. Variants in the SSPN locus have been linked with sex-dependent obesity-associated traits; however, this association has not been investigated in vivo. To delineate the role SSPN plays in regulating metabolism with potential to impact cardiac function, we subjected young and aged global SSPN-deficient (SSPN-/-) male and female mice to obesogenic conditions (60% fat diet). We hypothesized that loss of SSPN combined with metabolic stress would increase susceptibility of mice to cardiometabolic disease. Baseline and end-point assessments of several anthropometric parameters were performed including weight, glucose tolerance, and fat distribution of mice fed control (CD) and high-fat (HFD) diet. Doppler echocardiography was used to monitor cardiac function. White adipose and cardiac tissues were assessed for inflammation by histological, gene expression, and cytokine analysis. Overall, SSPN deficiency protected both sexes and ages from diet-induced obesity, with a greater effect in females. SSPN-/- HFD mice gained less weight than wild-type (WT) cohorts, while SSPN-/- CD groups increased weight. Furthermore, aged SSPN-/- mice developed glucose intolerance regardless of diet. Echocardiography showed preserved systolic function for all groups; however, aged SSPN-/- males exhibited significant increases in left ventricular mass (CD) and signs of diastolic dysfunction (HFD). Cytokine analysis revealed significantly increased IL-1α and IL-17Α in white adipose tissue from young SSPN-/- male mice, which may be protective from diet-induced obesity. Overall, these studies suggest that several sex-dependent mechanisms influence the role SSPN plays in metabolic responses that become evident with age.NEW & NOTEWORTHY Young and aged sarcospan (SSPN)-deficient mice were examined to assess the role of SSPN in obesity and cardiometabolic disease. Both sexes displayed a "leaner" phenotype in response to high-fat diet (HFD). Notably, several sex differences were identified in aged SSPN-deficient mice: 1) females developed glucose intolerance (control and HFD) and 2) males exhibited increased left ventricular mass (control) and diastolic dysfunction (HFD). Therefore, we conclude that SSPN exerts a sex-dependent influence on obesity-associated diseases.

Abstract A069: Development of an AAV-mediated GEM model to investigate the impact of age on pancreatic tumorigenesis

Abstract Much of our understanding of PDAC initiation comes from Genetically Engineered Mouse (GEM) models where a pancreas-specific Cre (such as Pdx- or p48-Cre) drives expression of specific mutations. While these GEM models generally recapitulate the key histopathological features of human PDAC, they have limitations when it comes to studying the impact of age on PDAC initiation since the Cre drivers induce recombination at the embryonic stage. This is an important consideration since in humans the disease manifests later in life, with the median age at diagnosis being 70 years old. Here, we describe the development of an intrapancreatic injection model using adeno-associated virus (AAV) to study how age influences PDAC initiation and the dynamics of tumor progression. This new viral transduction-based mouse model employs AAV-Cre to induce expression of an oncogenic KRasG12D allele together with a Trp53R172H tumor suppressor mutation within the pancreata of mice at different ages. Our approach utilizes mice with the genotype LSL-KrasG12D; LSL-Trp53R172H that are injected with different AAV-Cre directly into the parenchyma of the pancreas. In our initial studies, we used rAAV vectors expressing Cre recombinase and EGFP (AAV-Cre-EGFP) into young mice (10-14 weeks old) and older mice (32 weeks old) and analyzed EGFP expression using IVIS imaging and immunohistochemistry (IHC) at various timepoints post-injection. To evaluate tumor initiation, we assessed the extent of PanIN lesion formation using H&E staining. Our preliminary findings indicate that AAV-Cre-EGFP effectively transduces cells in vivo and triggers tumor development in the pancreas. To account for potential immunogenicity of the fluorescent protein, we then used rAAV vectors lacking EGFP expression. As controls, we administered identical rAAV vectors to wild-type cohorts matched for age and sex, along with mock injections. Transduction efficiency within the pancreas was determined by evaluating Cre and p53 expression levels, given that the R172H mutation stabilizes the protein. Notably, we found that EGFP is not necessary for either successful transduction or tumor initiation. We are currently evaluating PDAC initiation using a wide spectrum of ages, but our preliminary data using this model suggest that increased age may impact tumor progression, and that this effect may be sex-specific. Our novel AAV-Cre injection model holds promise for investigating age-related aspects of PDAC initiation, and in the future, such approaches could be leveraged to investigate how age impacts the effectiveness of different therapeutic modalities. Citation Format: Karen XD Duong-Polk, Madelaine Neff, Cosimo Commisso. Development of an AAV-mediated GEM model to investigate the impact of age on pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A069.

Left Atrial Strain in Omicron-Type COVID-19 Patients

BackgroundInformation about left atrial (LA) 2-dimensional (2D) strain parameters in patients with the Omicron variant of COVID-19 is limited. The aim of this study is to evaluate LA strain (LAS) in COVID-19 patients with the Omicron variant and compare it to that of propensity-matched patients with the wild-type (WT) variant. MethodsA total of 148 consecutive patients who were hospitalized with Omicron COVID-19 underwent an echocardiographic evaluation within the first day after hospital admission and were compared to propensity-matched patients (1:1) with the WT variant. LA 2D speckle tracking echocardiography parameters included the following: LAS, reservoir (LASr); LAS, conduit (LAScd); LAS, contraction (LASct); and LASr to E/e' ratio. The values for the parameters that occurred during acute Omicron-type infection were compared with those found on historic examinations in 36 patients. ResultsCompared to the matched WT cohort, patients with acute Omicron-type infection had similar LASr (31.3% ± 13.3% vs 33.0% ± 14.2%), LAScd (–18.7% ± 9.8% vs –18.6% ± 10.8%), and LASct (–12.5% ± 8.6% vs –13.6% ± 8.2%) values (P > 0.2 for all), but a higher E/e' ratio (11.8 ± 6 vs 10.1 ± 7; P = 0.03). Surprisingly, LASr (31.9% ± 13.7% vs 22.6% ± 13.9%; P = 0.04) and LAScd (–18.7% ± 9.7% vs –10.7% ± 6.6%; P < 0.001) improved in patients during acute infection. LASr, LAScd, and LASr/(E/e') were significantly associated with an increased risk of either in-hospital mortality, need for mechanical ventilation, or the combined event. ConclusionsIn hospitalized patients with an Omicron COVID-19 infection, LAS parameters are similar to those of matched patients with WT variant and are associated with mortality and respiratory deterioration. These abnormalities were recorded previously in the 36 patients with historical echocardiograms, suggesting that they are related to background cardiac disease.

Routine, prospective DPYD genotyping guided dose-individualisation for patients receiving fluoropyrimidines: Implementation, prevalence and patient safety outcomes from a multi-institutional clinical trial.

12121 Background: Prospective data regarding the prevalence of DPYD variant alleles and the impact of genotype guided dosing on safety and efficacy of fluoropyrimidines (FP) remains limited. We assessed the feasibility and safety impact of routine, prospective, DPYD genotype guided FP dosing. Methods: Adult patients (&gt;18 years), commencing FP for the first time were enrolled. Sanger PCR sequencing was designed to target DPYD variants NM_000110.4:c.1905+1G&gt;A (DPYD*2A), NM_000110.4:c.1679T&gt;G (DPYD*13), NM_000110.4:c.1236G&gt;A and NM_000110.4:c.2846A&gt;T. Dose reductions were implemented as per current international pharmacogenomic guidelines. Gr3/4 toxicity, dose reductions/delays and hospital admissions were reviewed for the first 60 days after commencement. For comparison purposes, a retrospective review of patients receiving FP without testing in a prior year was completed. Results: Between July 2021 – May 2023, 334 patients were enrolled for testing. 95% ECOG 0-1. 57% curative treatment intent. Mean time from phlebotomy to result 6.9 days (range 2-15). Clinician/patient acceptance was high (&gt;95% of eligible patients starting FP were enrolled). 20 (6.0%) variant DPYD allele carriers were detected. 11 heterozygous for c.1236G&gt;A, 5 heterozygous for C.1905+1G&gt;A, 3 heterozygous for c.2846A&gt;T and 1 homozygous for c.1236G&gt;A. 295 of patients tested proceeded to receive FP. 62% received Infusional-FP, 38% Capecitabine. 39 patients commenced treatment prior to DPYD result being available (treating clinician decision). Dose modification recommendations were followed in all but two patients. 24% experienced FP related Grade 3/4 toxicity within 60 days. (23% DPYD wild type, 37% DPYD variant) versus 35% in the retrospective cohort. 16% had FP related dose delays (16% DPYD wild type, 12% DPYD variant) versus 25% retrospective cohort. 14% had FP related dose reductions (13% DPYD wild type, 25% DPYD variant) versus 12% retrospective cohort. 13% had FP related hospital admissions (13% DPYD wild type, 19% DPYD variant) versus 17% retrospective cohort. 1 death in the DPYD wild type cohort was deemed potentially related to FP. Conclusions: Prospective DPYD genotype guided dosing was feasible in clinical practice, with prevalence of variant alleles comparable to published literature. There was a modest reduction in risk of FP related severe toxicity, dose delays, and hospital admissions with DPYD guided dosing compared to a historical, untested cohort. Clinical trial information: ACTRN12621001117808.

Combining KRAS gene status with preoperative D‑dimer levels as a predictive marker of venous thromboembolism risk in patients with resectable colorectal cancer: A prospective cohort study.

Colorectal cancer (CRC), one of the most prevalent types of cancer, is accompanied by a notably high incidence of thrombotic complications. The present study aimed to elucidate the association between KRAS mutations and hypercoagulability in operable CRC. The prognostic value of preoperative D-dimer levels was also investigated, thus providing novel insights into the development of therapeutic strategies to enhance patient survival and diminish morbidity. Therefore, a prospective analysis of 333 CRC cases post-surgery at Yan'an Hospital Affiliated to Kunming Medical University, between May 2019 and October 2022 was performed. Data on demographics, tumor characteristics and D-dimer levels were compiled from the electronic health records. Venous thromboembolism (VTE) was diagnosed by doppler or computed tomography angiography, with D-dimer thresholds set at 550 and 1,650 µg/l. KRAS mutations at codons 12 and 13 were assessed in a subset of 56 cases. Subsequently, the factors affecting the hypercoagulable state in these patients were prospectively analyzed, focusing on the pivotal role of KRAS. The results showed that KRAS mutations were associated with elevated preoperative D-dimer levels, with 1,076 µg/l compared with 485 µg/l in the wild-type cohort, indicative of a hypercoagulable state. Increased D-dimer levels were also associated with vascular invasion, distant metastases and a heightened risk of postoperative VTE. Furthermore, multivariate analyses identified KRAS mutations, distant metastases and vascular invasion as independent predictors of elevated D-dimer levels, with relative risk values of 2.912, 1.884 and 1.525, respectively. Conversely, sex, age, tumor location, differentiation grade, Ki67 index and tumor stage could not significantly affect D-dimer levels, thus indicating a complex interplay between tumor genetics and coagulation dysfunction in CRC. The current study suggested that the KRAS mutation status, distant metastasis and vascular invasion could be considered as independent risk factors of blood hypercoagulability in patients with CRC, potentially serving as prognostic factors for VTE risk.

Abstract 1746: KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles

Abstract Background: Lung cancer is the 3rd most common cancer in the UK and leading cause of cancer mortality worldwide. Somatic RAS mutations are the most common oncogenes in human cancer, and KRAS mutations are the largest molecular subset of lung adenocarcinoma (LUAD). Each cancer genome accumulates a unique combination of somatic mutations, and these mutational signatures provide a genetic imprint of mutational processes that have occurred during tumorigenesis. Mutational signatures provide information on tumor aetiology/maintenance and could highlight potential therapeutic targets. This is the largest study to date to profile KRAS mutant LUAD mutational signatures using whole genome sequencing (WGS) data. Methods: Paired tumor and germline WGS data were obtained through the 100 000 Genomes Project (Genomics England). KRAS mutations were profiled using Ensembl Variant Effect Predictor (VEP) v109.0. Single base substitution (SBS), doublet base substitution (DBS), small insertion and deletions (ID) and copy number (CN) mutational signature extraction was performed using SigProfiler (COSMIC version 3.3). Statistical analysis and plotting were performed in R; a two-tailed p-value &amp;lt;0.05 was considered statistically significant. Results: 680 LUAD patients were included; 301 KRAS mutant (44%), 379 KRAS wild type (63%). Common KRAS variants were present, including G12C (38.9%), G12V (15.9%) and G12D (14.0%). 17 SBS, 9 DBS, 10 ID and 9 CN signatures were extracted and present in both KRAS mutant and wild type cohorts. KRAS mutant samples were enriched in smoking related signatures (SBS4, DBS2, ID3; odds (OR) ratio 5.54, 3.80, 4.97 respectively, p &amp;lt;0.001) and diploid CN signature (CN1, OR 1.41, p = 0.03). Conversely, KRAS mutant samples were less likely to have clocklike (ID5, OR 0.61, p = 0.012; ID8, OR 0.44, p &amp;lt;0.001), APOBEC (SBS2, OR 0.49, p &amp;lt;0.001; SBS13; 0.50, p &amp;lt;0.001), chromosomal instability (CN9, OR 0.63, p = 0.019), loss of heterozygosity (CN14, OR 0.47, p = 0.015), or homologous recombination deficiency (CN17, OR 0.58, p &amp;lt;0.001) signatures. Discussion: These results show distinct mutational signature profiles between KRAS mutant and wild type LUAD. As previously described, KRAS mutant LUAD is dominated by smoking related signatures; however, the majority of remaining signatures were more common in the KRAS wild type cohort where there was a statistically significant difference. This data suggests different mutational processes are observed in KRAS mutant compared to wild type LUAD. KRAS mutations are commonly clonal and thought to initiate tumorigenesis: timing signatures relative to KRAS mutations is necessary to further our understanding of KRAS cancer evolution and the subject of future work. Combined with clinical data integration, mutational signatures could provide prognostic information and inform predictive models. Citation Format: Laura Woodhouse, Aliah Hawari, Andreas Gruber, Colin Lindsay, David Wedge. KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1746.

Abstract 6832: Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC

Abstract Background: Lemur tail kinase 3 (LMTK3) is a serine/threonine protein kinase with roles in multiple cellular pathways, including Wnt signaling, KIT modulation, and the estrogen receptor pathway; identifying it as a potential target in various cancers. LMTK3 is highly expressed within the central nervous system (CNS) and has been demonstrated to influence neuronal signaling. This study aimed to investigate the potential connection between LMTK3 and neuronal signaling pathways in colorectal cancer (CRC). Methods: Using the Cre/loxP system, we generated a C57BL/6 mouse strain that lacks the expression of LMTK3. 1 × 106 MC38 cells were subcutaneously implanted into 8 weeks old male and female wildtype (WT) and knockout (KO) mice. Tumor growth was measured until endpoint was reached. Tumors were then processed for RNA sequencing. Differentially expressed genes (DEGs) for each comparison group: WT Female vs WT Male, WT Female vs KO Female, WT Male vs KO Male, and KO Female vs KO Male were analyzed. Ingenuity pathway analysis (IPA) was done for identification of upstream regulators involved and affected cellular pathways. Expression levels of DEGs for glutamate receptor subunits were validated using qRT-PCR. Results: Both male and female LMTK3 KO mice showed highly significant (p&amp;lt;0.01) reduced tumor size when compared to WT mice of the same sex. Additionally, the KO female tumors were significantly (p&amp;lt;0.05) smaller when compared to the KO males. RNA sequencing revealed 761 DEGs in KO and WT female and 133 DEGs in KO female and male cohort comparisons (q&amp;lt;0.05). IPA results showed significant (p&amp;lt;0.01, Z-score←2) alterations in pathways including glutaminergic receptor signaling, neurovascular coupling, CREB signaling in neurons, and synaptic long-term depression in the WT vs KO female tumor cohort comparison. Within these pathways DEGs: CACNA1E, CACNA2DA, CACNG2, GRIA1, GRIA2, GRIN1, and GRIN2B, were downregulated in KO females. Upstream regulator analysis identified transcription regulators such as OTX2 and NEUROD1 as well as beta-estradiol to be significantly inhibited (p&amp;lt;0.01, Z-score←2) in KO females. qRT-PCR analysis showed significant (p&amp;lt;0.05) decreased expression in GRIA1, GRIN1, and GRIN2B as well as GRIN2A and GRIN2C in KO females when compared to WT females or KO males, validating RNA seq results. Conclusions: These results showed the involvement of LMTK3 in the gut-brain axis and its effect on CRC tumor growth. Downregulation of multiple neuronal pathways and genes suggest that LMTK3 may influence neuron signaling and neurotransmitter release to produce stimulatory or inhibitory effects within the tumor environment. The concurrent inhibition of beta-estradiol in LMTK3 KO tumors allude to possible sex differences the kinase’s effect on the observed tumor growth. Further studies are needed to confirm the mechanism of interaction of LMTK3 within these pathways. Citation Format: Lesly Torres-Gonzalez, Shivani Soni, Yan Yang, Goar Smbatyan, Jae Ho Lo, Pooja Mittal, Francesca Battaglin, Indrakant K. Singh, Priya Jayachandran, Sandra Algaze, Alexandra Wong, Karam Ashouri, Wu Zhang, Georgios Giamas, Joshua Millstein, Heinz-Josef Lenz. Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6832.

Abstract 6928: Expressed mitochondrial variants capture patterns of tumor evolution at single-cell level

Abstract Single-cell lineage tracing (scLT) has recently emerged as a promising tool to investigate cancer evolution. Among other scLT markers, mitochondrial variants (MT-SNVs) have been successfully used to track clones in a variety of experimental and clinical settings. However, in spite of the dynamic accrual of such genetic mutations, analysis of MT-SNVs variation has often been limited to standard clustering procedures, not exploiting the information associated with these markers at full potential. Here, we benchmarked expressed MT-SNVs-based single-cell phylogenies. To this end, we generate two single-cell multi-omics datasets: i) a benchmarking dataset, with joint profiling of MT-SNVs, gene expression and ground truth (GT) clonal identity (from lentiviral barcodes cells), and ii) a clinical cohort of wild type and mutated SRFS2 Acute Myeloid Leukemia patients, at diagnosis, with joint profiling of MT-SNVs, gene expression and targeted nuclear SNVs. We first leveraged our benchmarking dataset for validation purposes, taking advantage of its different levels of clonal complexities (in vitro clonal mixtures vs in vivo xenografts) and its longitudinal nature (i.e., matched primary tumor-lung metastasis couples). Supervised analysis detected (GT) clonal-specific mutations rarer than previously shown, but significantly associated with GT clonal labels. The performance of tested feature selection methods varied wildly in terms of GT MT-SNVs recover, with higher False Positive than False Negative Rates. Accurate detection of GT-clones from MT-SNVs correlated with samples clonal complexity, with a substantial number of GT-clones sub-optimally detected in the highest complexity sample generated. Longitudinal analysis of matched primary tumor-metastasis couples revealed remarkable stability of MT-SNVs, corroborating evidence from other studies without longitudinal GT lentiviral clones. We then asked whether expressed MT-SNVs hold enough signal to reconstruct reliable single-cell phylogenies. Transfer Bootstrap (TS) supports of reconstructed trees correlated with clades size, with a significant number of deep clades showing high (&amp;gt;.70) TS values across samples and bootstrapping procedures. Moreover, GT clonal labels significantly clustered across phylogenies, suggesting high phylogenetic signal. Finally, we investigated evolutionary patterns in our clinical cohort. Here, we found independent acquisition of both nuclear- and MT-SNVs by distinct normal and malignant lineages, and characterized patterns of gene expression inheritance along recovered phylogenies. All in all, our data support the emerging role of MT-variants based lineage tracing approaches in cancer evolution studies, highlighting intrinsic limitations and potential of these endogenous markers for retrospective, phylo-phenotypic analysis. Citation Format: Andrea Cossa, Alberto Dalmasso, Andrea Tirelli, Zhan Yinxiu, Chiara Caprioli, Giulia Perticari, Pier Giuseppe Pelicci. Expressed mitochondrial variants capture patterns of tumor evolution at single-cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6928.

Focus on RAS Codon 61 Mutations in Metastatic Colorectal Cancer: A Retrospective Analysis.

RAS mutations involving codon 61 are rare in metastatic colorectal cancer (mCRC), accounting for only 1-4%, but they have recently been identified with high frequency in the circulating tumor DNA (ctDNA) of patients with secondary resistance to anti-EGFRs. This retrospective monocentric study aimed to investigate the clinical phenotype and prognostic performance of codon 61 RAS-mutated mCRC. Fifty patients with codon 61 RAS-mutated mCRC treated at our institution between January 2013 and December 2021 were enrolled. Additional datasets of codon 61 RAS wild-type mCRCs (648 patients) were used as comparators. The endpoint for prognostic assessment was overall survival (OS). Metastatic involvement of the peritoneum or ovary was significantly more frequent in codon 61 RAS-mutated mCRC compared to codon 61 RAS wild-type (54 vs. 28.5%), non-codon 61 RAS-mutated (35.6%), BRAF V600E-mutated (25%), and RAS/BRAF wild-type (20.5%) cohorts. At a median follow up of 96.2 months, the median OS for codon 61 RAS-mutated patients was significantly shorter compared to RAS/BRAF wild-type (26.9 vs. 36.0 months, HR 0.56) patients, while no significant difference was observed compared to non-codon 61 RAS-mutated and BRAF V600E-mutated patients. We showed a negative prognostic impact and a statistically significant correlation between codon 61 RAS mutations and metastatic involvement of the peritoneum and ovary.

Abstract TP233: Cerebral Microbleeds Are More Frequent With BRCA Mutations: A Single-Center Retrospective Study

Introduction: Breast cancer susceptibility gene (BRCA) mutations are well-known causes of systemic cancers. We have previously identified that those with BRCA mutations have a higher likelihood of cerebral microbleeds (CMBs) on brain MRI compared to BRCA wildtype. We aimed to confirm our prior findings in a subsequent cohort. Methods: We performed a single-center retrospective review of all individuals with a confirmed diagnosis of BRCA 1 or 2 mutation and a standard-of-care brain MRI. All individuals were ≥ 18 years old, without intracranial radiation, cerebral metastases, or other cerebral processes which may impact interpretation. Our comparative cohort were individuals with breast cancer who have a genetically confirmed BRCA wildtype status. The primary outcome was the presence of CMBs, defined by consensus criteria. Standard descriptive techniques were used. Likelihood ratios were calculated by binary logistic regression and adjusted for significant variables. Results: We identified 49 individuals who met inclusion criteria: 15 with BRCA mutation and 34 with BRCA wildtype. Only one patient was with aspirin use in the BRCA mutation cohort. All cancers were of breast origin with 4 BRCA mutations patients with cancer staging ≥ 3 compared with 14 in the BRCA wildtype cohort. Chemotherapy rates did not differ. Patients with BRCA were younger, less often with hyperlipidemia, and more commonly had CMBs, Table 1. The presence of BRCA mutation trended towards higher odds of having a CMB (OR 5.8, 95%CI 0.93 - 36.16, p = 0.059) after adjustment for age and hyperlipidemia. Conclusion: We found that CMBs were more common in the BRCA mutation cohort, with a trend towards a 5-fold higher likelihood in the regression analysis. While underpowered, our findings support prior data highlighting this association. Further studies with larger sample sizes are warranted.

Prevalence and prognosis of PIK3CA mutations in Bulgarian patients with metastatic breast cancer receiving endocrine therapy in first-line setting.

In approximately 40% of patients with HER2-negative/HR-positive breast cancer tumors, the PIK3CA gene is mutated. Despite this, clinical outcomes vary between studies in this cohort. We aimed to ascertain the prevalence of PIK3CA mutations in patients with metastatic HR+/HER2- breast in Bulgaria, as well the evaluation and comparison of progression free survival (PFS) between wild-type (WT) and mutation-positive groups in the real-world setting. Three oncology centers in Bulgaria collected 250 tissue samples between 2016 and 2022 for this multicentric retrospective study. PIK3CA mutations were identified using Real-Time qPCR. The median follow-up period was 35 months. The mean age of the mutant cohort was 57.6 ± 11.6 years, compared to 56.5 ± 12.2 years for the WT cohort (p = .52). The percentage of patients with visceral metastasis was 58.8% (n = 147). Approximately 84.3% (n = 210) of the patients had reached postmenopause. 29.2% (n = 73) of the patients had PIK3CA mutations. The predominant mutation was present in exon 20, H1047R (46.5%). We found a significant correlation only between the presence of a mutation and the metastatic diseases at diagnosis (p = .002). As first-line therapy, 67.1% of patients received endocrine therapy (ET) plus cyclin dependent kinase (CDK4/6) inhibitor, while the remainder received ET alone. The median PFS of patients in the group with the mutation was 32 months (95%, CI: 22-40) compared to 24 months in the WT cohort ((95%, CI: 21-36) (p = .45)); HR = 0.86 (95%, CI: 0.5-1.3) (p = .46). We corroborated our conclusion using propensity matching score analysis, (36 months [95% CI: 20-40] vs. 26 months [95% CI: 21-38], [p = .69]). We found that the prevalence of PIK3CA mutations in our patients was comparable to what has been reported in other nations. Our results suggest that PIK3CA mutational status has no bearing to ET efficacy in first-line setting.

Anthracycline-related cardiotoxicity in patients with breast cancer harboring mutational signature of homologous recombination deficiency (HRD)

The BRCA proteins play a key role in the homologous recombination (HR) pathway. Beyond BRCA1/2, other genes are involved in the HR repair (HRR). Due to the prominent role in the cellular repair process, pathogenic or likely pathogenic variants (PV/LPVs) in HRR genes may cause inadequate DNA damage repair in cardiomyocytes. This was a multicenter, hospital-based, retrospective cohort study to investigate the heart toxicity from anthracycline-containing regimens (ACRs) in the adjuvant setting of breast cancer (BC) patients carrying germline BRCA PV/LPVs and no-BRCA HRR pathway genes. The left ventricular ejection fraction (LVEF) was assessed using cardiac ultrasound before starting ACR therapy and at subsequent time points according to clinical indications. Five hundred and three BC patients were included in the study. We predefined three groups: (i) BRCA cohort; (ii) no-BRCA cohort; (iii) variant of uncertain significance (VUS)/wild-type (WT) cohort. When baseline (T0) and post-ACR (T1) LVEFs between the three cohorts were compared, pre-treatment LVEF values were not different (BRCA1/2 versus HRR-no-BRCA versus VUS/WT cohort). Notably, during monitoring (T1, median 3.4 months), patients carrying BRCA or HRR no-BRCA germline pathogenic or likely pathogenic variants showed a statistically significant reduction of LVEF compared to baseline (T0). To assess the relevance of HRR on the results, we included the analysis of the subgroup of 20 BC patients carrying PV/LPVs in other genes not involved in HRR, such as mismatch repair genes (MUTYH, PMS2, MSH6). Unlike HRR genes, no significant differences in T0-T1 were found in this subgroup of patients. Our data suggest that deleterious variants in HRR genes, leading to impaired HR, could increase the sensitivity of cardiomyocytes to ACR in early BC patients. In this subgroup of patients, other measurements, such as the global longitudinal strain, and a more in-depth assessment of risk factors may be proposed in the future to optimize cardiovascular risk management and improve long-term survival.

Outcome Prediction in DDX41-Mutant Myelodysplastic Syndromes Is Not Possible with General Disease Schemes and Requires a Dedicated Risk Scoring System

Hypomorphic DDX41 mutations are prototypic lesions for germline predisposition to late onset myeloid neoplasia (MN), chiefly MDS. In a recent study 1, we demonstrated that germline mutations in this gene are found in up to 5% of MDS cases and explain nearly 80% of the etiologic fraction of known MN inherited predisposition. DDX41 mutation carriers have a ~50% lifetime risk of developing MN, which commonly accompanies the acquisition of a secondary somatic DDX41 mutation in the contralateral allele (most commonly p.R525H) with a strong male predominance. Because of these peculiarities, risk classification assigned by currently available schemes such as the revised/molecular international prognostic scoring system (IPSS-R/M) has also been questioned, differentiating DDX41-mutant MDS as a distinct nosologic subentity, whereby current prognostic tools are not able to capture invariant biologic features crucial for outcome prediction. Particularly, the impact of somatic DDX41 mutations on disease progression has not been evaluated. In this study, we explored the applicability of current schemes used for MDS outcome prediction leveraging an international collaboration across 6 academic centers. We accrued a total of 238 MDS cases from a cohort of 409 MN patients with DDX41 mutations with clinical-molecular annotations and relevant follow up data. For relevant comparisons, a cohort of 1212 DDX41 wild-type (WT) MDS cases with matching annotations and follow up was used. Overall, the median age at MDS diagnosis was 69 years (IQR 61-76) with a strong male predominance (4.4 M:F ratio). According to IPSS-R, patients clustered in very low (10%), low (17%), intermediate (34%), high (20%) and very high (18%) risk groups, whereas by IPSS-M the redistribution of risk categories was as follows: very low (7%), low (27%), moderate low (18%), moderate high (16%), high (15%), and very high (17%). After merging the moderate low/high IPSS-M categories for comparison purposes, the overall shift was 51%, with 38% up- and 62% down-staged. Notably, pairwise Log-Rank tests across IPSS-R/M risk categories resulted in non-significant survival differences in majority of subgroups, pinpointing how these commonly used schemes fail in separating DDX41-mutant MDS into groups of diverse prognoses. Conversely, the same analysis in the WT cohort led to a significant separation and identification of distinct prognostic categories. ( Fig1A) Next, we examined the reasons why DDX41-mutant MDS cases defy accuracy of current prognostic schemes. When looking at DDX41 specific genomic configuration, cases with germline plus p.R525H somatic mutations showed higher bone marrow blast percentages (median 11%) vs those acquiring other secondary somatic hits (4%, p&amp;lt;.001) or harboring only 1 germline alteration (7%, p=.002). By dividing patients according to IPSS-R scores (lower &amp;lt;3.5, higher-risk &amp;gt;3.5), we noticed a twice higher frequency of adverse risk MDS among patients with germline plus p.R525H mutations (p=.009). Thus, we explored whether DDX41 specific mutational configuration may have a role in driving MDS outcomes. While no survival difference was found between cases with germline alone vs germline plus somatic configuration, the dissection of the genomic architecture of double mutants showed worse survival outcomes and faster leukemia evolution in cases carrying truncating and/or p.R525H mutations ( Fig1B). These features still held significance in a DDX41-specific multivariable model for leukemia progression considering age and gender, with the best goodness of fit (Akaike Information Criterion-AIC: 131; c-index: 0.81) as compared to IPSS-R (AIC: 150, c-index: 0.71) and IPSS-M (AIC: 144, c-index: 0.73), whereas the acquisition of p.R525H mutation was the only independent predictor of survival (HR=3.9, 95% CI 1.5-9.9, p=.005). We show that IPSS-R/M are not able to adequately assess leukemic evolution and survival outcomes in DDX41-mutant MDS. Indeed, germline truncating and somatic p.R525H mutations are better predictors of faster leukemic evolution and survival. Additional risk factors inherent to this MDS subentity hold a prognostic significance beyond the consideration of traditional disease-specific variables, substantiating the need for a dedicated risk scoring system. This refined prognostication approach is also a consideration for other known germline mutations with predisposition to MN.

TNF- α from the Proximal Nephron Exacerbates Aristolochic Acid Nephropathy.

Aristolochic acid nephropathy (AAN) presents with tubular epithelial cell (TEC) damage and tubulointerstitial inflammation. Although tumor necrosis factor-α (TNF) regulates cell apoptosis and inflammatory responses, the effects of tubular TNF in the progression of AAN require elucidation. Floxed TNF mice on the 129/SvEv background were crossed with PEPCK-Cre mice to generate PEPCK-Cre+TNFflox/flox (TNF PTKO) mice or bred with Ksp-Cre mice to generate KSP-Cre+TNFflox/flox (TNF DNKO) mice. TNF PTKO, TNF DNKO, and WT controls (Cre negative littermates) were subjected to acute and chronic AAN. Deletion of TNF in the proximal but not distal nephron attenuated kidney injury, renal inflammation, and tubulointerstitial fibrosis after acute or chronic aristolochic acid exposure. The TNF PTKO mice did not have altered numbers of infiltrating myeloid cells in AAN kidneys. Nevertheless, kidneys from aristolochic acid-treated TNF PTKO mice had reduced levels of proteins involved in regulated cell death, higher proportions of TECs in the G0/G1 phase, and reduced TEC proportions in the G2/M phase. Pifithrin-α, which restores the cell cycle, abrogated differences between the wild-type and PTKO cohorts in G2/M phase arrest of TECs and kidney fibrosis after aristolochic acid exposure. TNF from the proximal but not the distal nephron propagates kidney injury and fibrogenesis in aristolochic acid nephropathy in part by inducing G2/M cell cycle arrest of TECs.

Clinical outcomes during and beyond different COVID-19 critical illness variant periods compared with other lower respiratory tract infections

BackgroundIt is yet to be better understood how outcomes during and after the critical illness potentially differ between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from other lower respiratory tract infections (LRTIs). We aimed to compare outcomes in adults admitted to an intensive care unit (ICU) with coronavirus disease 2019 (COVID-19) during the Wild-type, Alpha, Delta, and Omicron periods with individuals admitted with other LRTI.MethodsPopulation-based cohort study in Stockholm, Sweden, using health registries with high coverage, including ICU-admitted adults from 1 January 2016 to 15 September 2022. Outcomes were in-hospital mortality, 180-day post-discharge mortality, 180-day hospital readmission, 180-day days alive and at home (DAAH), and incident diagnoses registered during follow-up.ResultsThe number of ICU admitted individuals were 1421 Wild-type, 551 Alpha, 190 Delta, 223 Omicron, and 2380 LRTI. In-hospital mortality ranged from 28% (n = 665) in the LRTI cohort to 35% (n = 77) in the Delta cohort. The adjusted cause-specific hazard ratio (CSHR) compared with the LRTI cohort was 1.33 (95% confidence interval [CI] 1.16–1.53) in the Wild-type cohort, 1.53 (1.28–1.82) in the Alpha cohort, 1.70 (1.30–2.24) in the Delta cohort, and 1.59 (1.24–2.02) in the Omicron cohort. Among patients discharged alive from their COVID-19 hospitalization, the post-discharge mortality rates were lower (1–3%) compared with the LRTI cohort (9%), and the risk of hospital readmission was lower (CSHRs ranging from 0.42 to 0.68). Moreover, all COVID-19 cohorts had compared with the LRTI cohort more DAAH after compared with before the critical illness.ConclusionOverall, COVID-19 critical was associated with an increased hazard of in-hospital mortality, but among those discharged alive from the hospital, less severe long-term outcomes were observed compared with other LRTIs.

Prevalence of PIK3CA mutations in Taiwanese patients with breast cancer: a retrospective next-generation sequencing database analysis.

Breast cancer is the most common cancer type that affects women. In hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is the most frequently mutated gene associated with poor prognosis. This study evaluated the frequency of PIK3CA mutations in the Taiwanese breast cancer population. This is a retrospective study; patient data were collected for 2 years from a next-generation sequencing database linked to electronic health records (EHRs). The primary endpoint was the regional prevalence of PIK3CA mutation. The secondary endpoints were to decipher the mutation types across breast cancer subtype, menopausal status, and time to treatment failure after everolimus (an mTOR inhibitor) or cyclin-dependent kinase 4/6 (CDK4/6) inhibitor treatment. PIK3CA mutations were identified in 278 of 728 patients (38%). PIK3CA mutations were reported in 43% of patients with HR-/HER2+ subtype and 42% of patients with HR+/HER2- postmenopausal status. A lower prevalence of PIK3CA mutations was observed in triple-negative (27%) and HR+/HER2- premenopausal patients (29%). The most common mutation was at exon 20 (H1047R mutation, 41.6%), followed by exon 9 (E545K mutation, 18.9% and E542K mutation, 10.3%). Among patients treated with CDK4/6 inhibitors, the median time to treatment failure was 12 months (95% CI: 7-21 months) in the PIK3CA mutation cohort and 16 months (95% CI: 11-23 months) in the PIK3CA wild-type cohort, whereas patients receiving an mTOR inhibitor reported a median time to treatment failure of 20.5 months (95% CI: 8-33 months) in the PIK3CA mutation cohort and 6 months (95% CI: 2-9 months) in the PIK3CA wild-type cohort. A high frequency of PIK3CA mutations was detected in Taiwanese patients with breast cancer, which was consistent with previous studies. Early detection of PIK3CA mutations might influence therapeutic decisions, leading to better treatment outcomes.

Characterization of POT1 tumor predisposition syndrome: Tumor prevalence in a clinically diverse hereditary cancer cohort

PurposeGermline variants in POT1 have been implicated in predisposition to melanoma, sarcoma, and glioma in limited studies. Here, we determine the prevalence of cancer types in individuals with POT1 pathogenic variants (PVs) undergoing multigene panel testing (MGPT) for a broad variety of cancer indications. MethodsWe performed a retrospective review of data provided on clinical documents from individuals with POT1 PVs identified via MGPT over a 5-year period. Tumor prevalence in POT1 PV heterozygotes was compared with MGPT-negative wild-type (WT) controls using χ2 test. ResultsPOT1 PVs were identified in 227 individuals. POT1 PV and WT (n = 13,315) cohorts had a similar proportion of reported tumors (69.6% and 69.2%, respectively); however, POT1 PV heterozygotes were more likely to be diagnosed with multiple tumors (18.9% vs 8.7%; P < .001). Compared with POT1 WT, we identified a significant increase in melanoma (odds ratio 7.03; 95% CI 4.7-10.5; P < .001) and sarcoma (odds ratio 6.6; 95% CI 3.1-13.9; P < .001). ConclusionThis analysis of the largest POT1 PV cohort to date validates the inclusion of POT1 in hereditary cancer MGPT and has the potential to impact clinical management recommendations, particularly for patients and families at risk for melanoma and sarcoma.

Atezolizumab, vemurafenib, and cobimetinib in patients with melanoma with CNS metastases (TRICOTEL): a multicentre, open-label, single-arm, phase 2 study

Targeted therapy and immunotherapy have shown intracranial activity in melanoma with CNS metastases, but there remains an unmet need, particularly for patients with symptomatic CNS metastases. We aimed to evaluate atezolizumab in combination with cobimetinib or vemurafenib plus cobimetinib in patients with melanoma with CNS metastases. TRICOTEL was a multicentre, open-label, single-arm, phase 2 study done in two cohorts: a BRAFV600 wild-type cohort and a BRAFV600 mutation-positive cohort, recruited at 21 hospitals and oncology centres in Brazil, France, Germany, Hungary, Italy, Spain, and Switzerland. Eligible patients were aged 18 years or older with previously untreated metastatic melanoma, brain metastases of 5 mm or larger in at least one dimension, and an Eastern Cooperative Oncology Group performance status of 2 or less. Patients in the BRAFV600 wild-type cohort received intravenous atezolizumab (840 mg, days 1 and 15 of each 28-day cycle) plus oral cobimetinib (60 mg once daily, days 1-21). Patients in the BRAFV600 mutation-positive cohort received intravenous atezolizumab (840 mg, days 1 and 15 of each 28-day cycle) plus oral vemurafenib (720 mg twice daily) plus oral cobimetinib (60 mg once daily, days 1-21); atezolizumab was withheld in cycle 1. Treatment was continued until progression, toxicity, or death. The primary outcome was intracranial objective response rate confirmed by assessments at least 4 weeks apart, as assessed by independent review committee (IRC) using modified Response Evaluation Criteria in Solid Tumours version 1.1. Because of early closure of the BRAFV600 wild-type cohort, the primary endpoint of intracranial objective response rate by IRC assessment was not done in this cohort; intracranial objective response rate by investigator assessment was reported instead. Efficacy and safety were analysed in all patients who received at least one dose of study medication. This trial is closed to enrolment and is registered with ClinicalTrials.gov, NCT03625141. Between Dec 13, 2018, and Dec 7, 2020, 65 patients were enrolled in the BRAFV600 mutation-positive cohort; the BRAFV600 wild-type cohort was closed early after enrolment of 15 patients. Median follow-up was 9·7 months (IQR 6·3-15·0) for the BRAFV600 mutation-positive cohort and 6·2 months (3·5-23·0) for the BRAFV600 wild-type cohort. Intracranial objective response rate was 42% (95% CI 29-54) by IRC assessment in the BRAFV600 mutation-positive cohort and 27% (95% CI 8-55) by investigator assessment in the BRAFV600 wild-type cohort. Treatment-related grade 3 or worse adverse events occurred in 41 (68%) of 60 patients who received atezolizumab plus vemurafenib plus cobimetinib in the BRAFV600 mutation-positive cohort, the most common of which were lipase increased (15 [25%] of 60 patients) and blood creatine phosphokinase increased (11 [18%]). Eight (53%) of 15 patients treated with atezolizumab plus cobimetinib in the BRAFV600 wild-type cohort had treatment-related grade 3 or worse adverse events, most commonly anaemia (two [13%]) and dermatitis acneiform (two [13%]). Treatment-related serious adverse events occurred in 14 (23%) of 60 patients who received triplet therapy in the BRAFV600 mutation-positive cohort and two (13%) of 15 in the BRAFV600 wild-type cohort. No treatment-related deaths occurred. Atezolizumab plus vemurafenib and cobimetinib provided intracranial activity in patients with BRAFV600-mutated melanoma with CNS metastases. F Hoffmann-La Roche.