Pretreatment Blood Samples Research Articles

Clonal Hematopoiesis and Clinical Outcomes in Metastatic Castration-Resistant Prostate Cancer Patients Given Androgen Receptor Pathway Inhibitors (Alliance A031201).

Mutations in hematopoietic progenitor cells accumulate with age leading to clonal expansion, termed clonal hematopoiesis (CH). CH in the general population is associated with hematopoietic neoplasms and reduced overall survival (OS), predominantly through cardiovascular adverse events (CVAE). Because androgen receptor pathway inhibitors (ARPI) used in metastatic castration-resistant prostate cancer (mCRPC) are also associated with CVAEs and because CH negatively impacted survival in an advanced solid tumor cohort, we hypothesized that CH in mCRPC may be associated with increased CVAEs and inferior survival. A targeted DNA sequencing panel captured common CH mutations in pretreatment blood samples from 957 patients enrolled in Alliance A031201: a randomized trial of enzalutamide ± abiraterone/prednisone in the first-line mCRPC setting. The primary outcome was the impact of CH on OS; the secondary outcomes were progression-free survival (PFS) and CVAEs. Baseline comorbidities were similar by CH status. No differences in OS/progression-free survival were detected regardless of treatment arm or the variant allele frequency threshold used to define CH [primary: 2% (normal-CH, N-CH); exploratory: 0.5% (low-CH) and 10% (high-CH, H-CH)]. Patients with H-CH (7.2%) and TET2-mutated N-CH (6.0%) had greater odds of any CVAE (14.5% vs. 4.0%; P = 0.0004 and 12.3% vs. 4.2%; P = 0.010, respectively). More major CVAEs were observed in patients with H-CH (5.8% vs. 1.9%; P = 0.042) and N-CH (3.4% vs. 1.8%; P = 0.147). CH did not affect survival in patients with mCRPC treated with ARPIs in A031201. H-CH and TET2-mutated CH were associated with more CVAEs. These findings inform the risk/benefit discussion about ARPIs in mCRPC.

Baseline sLAG-3 levels in Caucasian and African-American breast cancer patients.

Worse survival persists for African-Americans (AA) with breast cancer compared to other race/ethnic groups despite recent improvements for all. Unstudied in outcomes disparities to date is soluble LAG-3 (sLAG-3), cleaved from the LAG-3 immune checkpoint receptor which is a proposed target for deactivation in emerging immunotherapies due to its prominent immunosuppressive function in the tumoral microenvironment. A prior study has found that lower sLAG-3 baseline level was associated with poor outcomes. In a cross-sectional study of 95 patients with primary breast cancer (n = 58 Caucasian, n = 37 AA), we measured sLAG-3 (ELISA pg/ml) in pre-treatment blood samples using the non-parametric Mann-Whitney u-Test for independent samples, and, calculated Pearson r correlation coefficients of sLAG-3 with circulating cytokines by race. Mean sLAG-3 level was lower in AA compared to Caucasian patients (1377.6 vs 3690.3, P = .002), and in patients with triple-negative breast cancer (TNBC) compared to those with non-TNBC malignancies (P = .02). When patients with TNBC tumors were excluded from analyses, the difference in sLAG-3 level between AA (n = 21) and Caucasian patients (n = 40) substantially remained (1937.4 vs 4182.4, P = .06). Among Caucasian patients, sLAG-3 was correlated with IL-6, IL-8 and IL-10 (r = .69, P < .001; r = .70, P < .001; and, r = .46, P = .01; respectively). For AA patients, sLAG-3 was correlated only with IL-6 (r = .37, P = .03). We present the first report that African-American breast cancer patients might have comparatively low pre-treatment sLAG-3 levels, independent of TNBC status, along with reduced co-expression with circulating cytokines. The mechanistic and prognostic role of cleaved LAG-3, particularly in disparate outcomes, remains to be elucidated.

PNI is useful for predicting the prognosis of patients with soft tissue sarcoma: A retrospective study

BackgroundIt is known that preoperative Prognostic Nutritional Index (PNI) is useful in predicting prognosis in gastrointestinal diseases and that preoperative improvement of nutritional status improves prognosis. However, there have been few large-scale reports examining the prognostic value of PNI in soft tissue sarcomas. Therefore, the aim of this study is to investigate whether the PNI can be useful for predicting overall survival in soft tissue sarcoma. MethodsBetween January 2006 and March 2022 at our hospital, 111 patients with pathologically diagnosed soft tissue sarcoma were included, retrospectively. Several nutritional or inflammatory biomarkers such as PNI were calculated from the pretreatment blood sample results. The patients were classified into two groups (low and high groups) based on the median value of each parameter. Overall survival was analyzed by the Kaplan‒Meier method and log-rank test. Univariate and multivariate analyses using the Cox proportional hazards model were used to investigate prognostic factors for overall survival. ResultsThe median overall survival was 24.3 months (mean 37.3 months), and the high PNI group had a significantly longer overall survival than the low PNI group (p < 0.0001). PNI was the most significant univariate factor for overall survival among other nutritional and inflammatory parameters (HR: 5.64, 95% CI: 2.26–14.12, p = 0.0002). The multivariate proportional hazards model was built using variables with prognostic potential as suggested by previous analysis with respect to patient characteristics and PNI. As potential confounding factors, we included PNI, stage, age, and tumor location. PNI was also an independent prognostic factor in multivariate analysis (HR: 7.02, CI: 2.52–19.40, p = 0.0002). ConclusionPNI is a useful prognostic factor among various parameters for overall survival in patients with soft tissue sarcoma.

Elevated allostatic load is associated with poorer response to repetitive transcranial magnetic stimulation in treatment-resistant depression

This cohort study investigated whether allostatic load (AL) is associated with treatment response to repetitive transcranial magnetic stimulation (rTMS) in treatment-resistant depression (TRD). Pre-treatment blood samples measured AL across multiple systems. Pre- and post-treatment mood changes were assessed using the Montgomery-Åsberg Depression Rating Scale (MADRS). Associations between AL and treatment outcomes were explored. Higher pre-treatment AL was significantly associated with poorer post-treatment response status but was not significantly associated with smaller reduction in MADRS score after 4 weeks of treatment. Identifying biomarker profiles informed by the AL model could enhance treatment decisions in TRD, reducing risks associated with prolonged, ineffective rTMS trials and emphasizing the need for reliable predictive biomarkers.

Integrative prediction model for radiation pneumonitis incorporating genetic and clinical-pathological factors using machine learning

PurposeWe aimed to develop a machine learning-based prediction model for severe radiation pneumonitis (RP) by integrating relevant clinicopathological and genetic factors, considering the associations of clinical, dosimetric parameters, and single nucleotide polymorphisms (SNPs) of genes in the TGF-β1 pathway with RP. MethodsWe prospectively enrolled 59 primary lung cancer patients undergoing radiotherapy and analyzed pretreatment blood samples, clinicopathological/dosimetric variables, and 11 functional SNPs in TGFβ pathway genes. Using the Synthetic Minority Over-sampling Technique (SMOTE) and nested cross-validation, we developed a machine learning-based prediction model for severe RP (grade ≥ 2). Feature selection was conducted using four methods (filtered-based, wrapper-based, embedded, and logistic regression), and performance was evaluated using three machine learning models. ResultsSevere RP occurred in 20.3 % of patients with a median follow-up of 39.7 months. In our final model, age (>66 years), smoking history, PTV volume (>300 cc), and AG/GG genotype in BMP2 rs1979855 were identified as the most significant predictors. Additionally, incorporating genomic variables for prediction alongside clinicopathological variables significantly improved the AUC compared to using clinicopathological variables alone (0.822 vs. 0.741, p = 0.029). The same feature set was selected using both the wrapper-based method and logistic model, demonstrating the best performance across all machine learning models (AUC: XGBoost 0.815, RF 0.805, SVM 0.712, respectively). ConclusionWe successfully developed a machine learning-based prediction model for RP, demonstrating age, smoking history, PTV volume, and BMP2 rs1979855 genotype as significant predictors. Notably, incorporating SNP data significantly enhanced predictive performance compared to clinicopathological factors alone.

Prognostic Implications of Blood Immune-Cell Composition in Metastatic Castration-Resistant Prostate Cancer.

The prognosis for patients with metastatic castration-resistant prostate cancer (mCRPC) varies, being influenced by blood-related factors such as transcriptional profiling and immune cell ratios. We aimed to address the contribution of distinct whole blood immune cell components to the prognosis of these patients. This study analyzed pre-treatment blood samples from 152 chemotherapy-naive mCRPC patients participating in a phase 2 clinical trial (NCT02288936) and a validation cohort. We used CIBERSORT-X to quantify 22 immune cell types and assessed their prognostic significance using Kaplan-Meier and Cox regression analyses. Reduced CD8 T-cell proportions and elevated monocyte levels were substantially connected with a worse survival. High monocyte counts correlated with a median survival of 32.2 months versus 40.3 months for lower counts (HR: 1.96, 95% CI 1.11-3.45). Low CD8 T-cell levels were associated with a median survival of 31.8 months compared to 40.3 months for higher levels (HR: 1.97, 95% CI 1.11-3.5). These findings were consistent in both the trial and validation cohorts. Multivariate analysis further confirmed the independent prognostic value of CD8 T-cell counts. This study highlights the prognostic implications of specific blood immune cells, suggesting they could serve as biomarkers in mCRPC patient management and should be further explored in clinical trials.

A phase 2 study of amivantamab plus lazertinib in patients with EGFR-mutant lung cancer and active central nervous system disease.

8517 Background: Patients (pts) with EGFR-mutant lung cancer (EGFR+LC) with active brain metastases (BrM) or leptomeningeal disease (LM) are often excluded from clinical trials. Despite central nervous system (CNS) disease control with osimertinib, &gt; 50% of pts with EGFR-mutant lung cancer have involvement of the brain (BrM) or leptomeninges (LM). Amivantamab and lazertinib have demonstrated systemic activity in pts with EGFR-mutant lung cancer whose disease progressed after osimertinib and/or platinum-based chemotherapy. This trial evaluated amivantamab +lazertinib in the setting of progressive CNS metastases (NCT04965090). Methods: We evaluated amivantamab and lazertinib in 2 cohorts: pts with 1) progressive or new BrM or 2) LM. All pts with EGFR exon 19 del/L858R/atypical mutations had prior osimertinib. All pts with EGFRexon 20 insertions (ex20ins) had prior chemotherapy. Each cohort was to enroll 20 pts powered to differentiate overall response rate (ORR) of 0.05 vs 0.25. Positive CSF assessment (cytology or circulating tumor cells (CTC)) +/- BrM allocated a pt to the LM cohort. Pts received standard dosing of amivantamab and lazertinib 240 mg daily. Co-primary endpoints were systemic ORR by RECIST v1.1 and CNS ORR by RANO-BM or LM. Paired pre-treatment tumor, blood, and CSF samples had targeted DNA sequencing and cell-free RNA (cfRNA) sequencing. Results: Trial enrolled 20 pts in BrM cohort and 22 pts in LM cohort. Median age 55 (range 31-80), 69% women,. 40% had L858R (n = 17), 40% del19 (n = 17), 12% ex20 ins (n = 5), and 8% atypical EGFR mutations (n = 3). Median lines of prior therapy: 2 (1-7). Except patients with ex20ins, all patients had prior osimertinib, and 55% had chemo. Efficacy Data: . 14 LM pts (64%) had decrease in CSF CTCs and 7 (32%) had improvement in neurologic symptoms. Most frequent treatment-related adverse events (TRAEs) (≥30% in overall population) were rash (71%), infusion-related reaction (59%), paronychia (43%), fatigue (40%), edema (40%), mucositis (33%), and nausea (33%). Most frequent (≥5%) grade ≥3 TRAEs were infusion related reactions (7%), thromboembolic event (5%), elevated AST/ALT (5%), and rash (5%). Three pts (7%) discontinued treatment due to TRAEs. Genomic data comparing concurrent systemic tumor, plasma and CSF samples, and ctDNA and cfRNA dynamics will be presented. Conclusions: The combination of amivantamab+lazertinib is a promising treatment for pts with EGFR-mutant lung cancer and active CNS disease, with clinically meaningful time on treatment and radiographic response. This is the first clinical trial successfully completed in pts with EGFR-mutant lung cancer and progressive CNS metastases. Clinical trial information: NCT04965090 .[Table: see text]

Hypertension as predictive factor for bevacizumab-containing first-line therapy in metastatic breast and colorectal cancer in BRECOL (GEICAM/2011-04) study

BackgroundRetrospective data suggest an association between bevacizumab efficacy and the incidence of arterial hypertension (AHT). Additionally, epigenetic mechanisms have been related to AHT.MethodsThis prospective observational study conducted by GEICAM Spanish Breast Cancer Research Group included metastatic breast (MBC) or colorectal (mCRC) cancer patients treated with bevacizumab-containing chemotherapy as first-line treatment. Blood pressure (BP) levels were measured (conventional and 24-h Holter monitoring) at baseline and up to cycle 3. Primary endpoint assessed BP levels increase as predictive factor for progression-free survival (PFS). Germline DNA methylation profile was explored in pre-treatment blood samples; principal component analysis was used to define an epigenetic predictive score for increased BP levels.ResultsFrom Oct-2012 to Jul-2016, 143 (78 MBC and 65 mCRC) patients were included. The incidence of AHT according to guidelines was neither predictive of PFS nor of best overall tumor response (BOR). No statistically significant association was observed with systolic BP nor diastolic BP increment for PFS or BOR. Grade 3 and 4 adverse events were observed in 37 and 5% of patients, respectively. We identified 27 sites which baseline methylation status was significantly associated to BP levels increase secondary to bevacizumab-containing chemotherapy.ConclusionsNeither the frequency of AHT nor the increase of BP levels were predictive of efficacy in MBC and mCRC patients treated with bevacizumab-containing chemotherapy.Clinical trial registryClinicalTrials.gov Identifier: NCT01733628.

Trends of pre-treatment drug resistance in antiretroviral-naïve people with HIV-1 in the era of second-generation integrase strand-transfer inhibitors in Taiwan.

Monitoring the trends of pre-treatment drug resistance (PDR) and resistance-associated mutations (RAMs) among antiretroviral-naïve people with HIV (PWH) is important for the implementation of HIV treatment and control programmes. We analysed the trends of HIV-1 PDR after the introduction of second-generation integrase strand-transfer inhibitors (INSTIs) in 2016 in Taiwan, when single-tablet regimens of non-nucleoside reverse-transcriptase inhibitor (NNRTI-) and INSTI-based antiretroviral therapy became the preferred treatments. In this multicentre study, we included newly diagnosed, antiretroviral-naïve PWH who underwent tests for RAMs between 2016 and 2022. Pre-treatment genotypic resistance testing was performed, along with HIV-1 subtyping and determinations of plasma HIV RNA load and CD4 lymphocyte counts. RAMs were analysed using the Stanford University HIV Drug Resistance Database and only RAMs conferring at least low-level resistance were included. From 2016 to 2022, pre-treatment blood samples from 3001 newly diagnosed PWH, which constituted 24.3% of newly diagnosed PWH in Taiwan during the study period, were tested. Of the PWH with analysable gene sequences, the HIV-1 PDR prevalence to NNRTIs, nucleoside reverse-transcriptase inhibitors (NRTIs), first- and second-generation INSTIs and PIs was 10.0%, 2.1%, 2.5%, 0.6% and 0.4%, respectively. While the trends of PDR remained stable for NRTIs, INSTIs and PIs, there was a significantly increasing trend of PDR to NNRTIs from 6.0% in 2016% to 13.1% in 2022 (P = 0.001). After the introduction of second-generation INSTIs in Taiwan, the trends of HIV-1 PDR to NRTIs and INSTIs remained low. Furthermore, there was no significant decrease of the prevalence of PDR toward NNRTIs between 2016 and 2022.

Abstract 4025: A phase II trial of neoadjuvant gemcitabine, cisplatin plus tislelizumab followed by concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma: Clinical efficacy, safety and the biomarker analysis

Abstract Immune checkpoint inhibitors (ICIs) have been widely used in recurrent or metastatic nasopharyngeal carcinoma (NPC), but the addition of ICIs to chemoradiotherapy in locoregionally advanced (LA) NPC requires further investigation. We conducted a phase II, single-arm trial to evaluate the efficacy and safety of neoadjuvant gemcitabine, cisplatin (GP) plus tislelizumab followed by concurrent chemoradiotherapy (CCRT) in LA-NPC. All participants received 3 cycles of GP chemotherapy plus tislelizumab (200 mg) followed by 3 cycles of cisplatin-based CCRT. The primary endpoint was the clinical complete response rate (CR) after neoadjuvant chemoimmunotherapy (NAT). The secondary endpoints include pathological CR rate, progression-free survival (PFS), overall survival (OS), locoregional relapse-free survival (LRRFS), distant metastasis-free survival (DMFS), etc. Pretreatment blood samples and paired (pre-/post-NAT) tissue biopsies were collected to perform full-spectrum peripheral T-cell profiling, PD-L1 and tertiary lymphoid structures (TLS) staining. Among all 63 participants, the CR rate after NAT reached 41.3% (95% CI, 28.8%-53.8%), far exceeding our hypothesized CR rate of 22%. The objective response rate (ORR) and pCR rate after NAT were 88.9% (95% CI, 80.9% to 96.9%) and 74.6% (95% CI, 62.1% to 84.7%), respectively. As of July 15th, 2023, after the median follow-up of 25 months, the 2-year PFS and OS were 98.4% and 100%. The incidence of grade 3-4 immunotherapy-related adverse events was 1.6%. The compliance of CCRT was not compromised. Patients who achieved CR had significantly more circulating CD4+ and CD8+ effector memory T cells (Tem), regulatory T cells (Treg) and follicular helper T cells (Tfh). The peripheral abundance of LAG3+ Tem and Tfh were independently predictive of NAT response. The number and maturation of TLS significantly increased after NAT in the CR group, but not in the Non-CR group. GP chemotherapy plus tislelizumab followed by CCRT was highly therapeutic and safe in treating LA-NPC, further randomized phase III studies are awaited. Citation Format: Xiao-Yun Li, Si-Yi Xie, Qiu-Yan Chen, Hai-Qiang Mai, Lin-Quan Tang. A phase II trial of neoadjuvant gemcitabine, cisplatin plus tislelizumab followed by concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma: Clinical efficacy, safety and the biomarker analysis [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 4025.

Abstract 1167: Prospective analysis of pretreatment circulating CD4 memory T cell features associated with severe immunotherapy toxicity

Abstract Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized melanoma treatment, significantly improving outcomes. Nevertheless, too many melanoma patients undergoing ICI treatment experience severe and often debilitating immune-related adverse events (irAEs). In a retrospective melanoma cohort treated with ICIs, we previously found that elevated baseline levels of circulating CD4 effector memory T (TEM) cells were associated with the development of severe irAEs, irrespective of the affected organ system (Lozano et al., Nature Medicine, 2022). Here, we present data from an extended prospective validation cohort from 47 patients across two academic medical centers. Methods: Of 47 patients, 26 received combination anti-PD1/anti-CTLA4 ICIs, 3 combination anti-PD1/anti-LAG3 ICIs, 17 anti-PD1 ICI, and 1 anti-CTLA4 ICI. Peripheral blood mononuclear cells (PBMCs) were prepared from pretreatment (Cycle 1 Day 1) blood samples obtained from all patients. We performed mass cytometry (CyTOF) on PBMCs using a 38-marker panel to deeply profile immune subsets. We further leveraged bulk RNA-seq from 24 patients to (i) deconvolve CD4 memory T cell abundance using CIBERSORTx and (ii) determine T cell receptor (TCR) diversity using Shannon entropy following MiXCR clonotype assembly. irAEs were graded on a 5-point scale using the Common Terminology Criteria for Adverse Events v5. Results: After treatment initiation, 14 patients experienced severe irAEs (grade 3+) at a median of 12.1 weeks (range 4-54), including 4 with life-threatening (grade 4) irAEs. CyTOF revealed elevated circulating CD4 TEM cells in patients that developed severe irAEs (P&amp;lt;0.0001; AUC=0.87), including those treated with combination ICIs (P=0.0001; AUC=0.89). We further observed a continuous relationship between increasing CD4 TEM levels and irAE grade (P=3e-6, Jonckheere-Terpstra test). We then combined CD4 memory T cell abundance and TCR diversity derived from bulk RNA-seq on 24 of these patients using logistic regression (trained on data from Lozano et al.) to yield a composite model score. The pretreatment composite model score was strongly associated with severe irAE development (P=0.0009, AUC=0.88), and in a median split, identified patients with shorter time to severe irAE development (3 months vs. not reached; P=0.006; HR=10.5). Notably, composite model features and the overall score had no significant association with immunotherapy benefit. Conclusion: In this prospective study, both circulating CD4 TEM levels determined by CyTOF and a composite model score measured by RNA-seq were strongly associated with severe irAE development, independent of response status. These findings hold promise for future pretreatment risk stratification of ICI therapy. Citation Format: Abul Usmani, Noah Earland, Wubing Zhang, Peter K. Harris, Antonietta Bacchiocchi, Chloe M. Sachs, Aishwarya Nene, David Y. Chen, Mario Sznol, Ruth Halaban, Aaron M. Newman, Aadel A. Chaudhuri. Prospective analysis of pretreatment circulating CD4 memory T cell features associated with severe immunotherapy toxicity [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 1167.

Abstract 3848: Comprehensive protein analysis to identify the proteins responsible for sarcopenia and their impact on antitumor immunotherapy in non-small cell lung cancer

Abstract Background: Previous retrospective studies have shown that sarcopenia (Sar) is associated with poor outcome in cancer patients (pts) receiving immune checkpoint inhibitors (ICIs). We conducted a prospective study to determine whether Sar-related proteins induce Sar and reduce the efficacy of ICI. Method: Non-small cell lung cancer (NSCLC) pts receiving ICI as first-line treatment at Niigata University Medical and Dental Hospital were eligible. Sar was diagnosed with physical performance and skeletal muscle mass. Comprehensive protein analysis of pre-treatment blood samples was performed to identify factors that correlate with muscle loss and ICI effects. Result: 41 patients were enrolled from October 2019 through August 2022. PFS for Sar was 4.6 months, 6.7 months for Pre-Sar, and 16.7 months for Non-Sar (P=0.0028). OS for Sar was not reached, 15.7 months for Pre-Sar, and not reached for Non-Sar (P=0.0014). Leptin, IL-1β, IL-12, and Pentraxin 3 were significantly correlated with loss of muscle mass. In contrast, leptin, insulin, IGF-1, GDF-8, Activin A, IL-2Ra, IL-8, MMP-3, osteopontin, and Pentraxin-3 were significantly associated with poor OS. Pentraxin 3 and leptin were associated with both the effectiveness of immunotherapy and sarcopenia. A multivariate analysis that considered factors commonly associated with the effectiveness of immunotherapy, including age, PD-L1 expression rate, and neutrophil-lymphocyte ratio, Pentraxin 3 was determined to be an independent prognostic factor. Conclusion: This study indicated that Sar-related proteins and inflammatory cytokines are involved in the development of Sar and ICI efficacy. Citation Format: Takaaki Masuda, Satoshi Watanabe, Tomoya Wakabayashi, Susumu Tanaka, Kunihiro Shono, Ryo Suzuki, Tomoki Sekiya, Naohiro Yanagimura, Masashi Arita, Aya Ohtsubo, Tomohiro Tanaka, Koichiro Nozaki, Yu Saida, Satoshi Hokari, Rie Kondo, Kenjiro Shima, Yosuke Kimura, Nobumasa Aoki, Yasuyoshi Ohshima, Toshiyuki Koya, Kazuko Sakai, Kazuto Nishio, Motohiko Yamazaki, Hiroyuki Ishikawa, Toshiaki Kikuchi. Comprehensive protein analysis to identify the proteins responsible for sarcopenia and their impact on antitumor immunotherapy in non-small cell lung cancer [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 3848.

Physiologic responses of Cownose Rays following freshwater dips for treatment of capsalid monogeneans.

Two Cownose Rays Rhinoptera bonasus were presented for management of a severe capsalid monogenean infection Benedeniella posterocolpa in a mixed species habitat at an aquarium. A series of freshwater (FW) dips were elected to mitigate parasite resistance to praziquantel due to the endemic monogenean population in the system. A pretreatment blood sample was opportunistically obtained, and subsequent samples were processed due to development of clinical signs. While the first FW dip was tolerated well and did not induce abnormal behavior, the second led to one individual presenting with tachypnea, pallor, and lethargy. Marked hematological abnormalities requiring medical intervention occurred in both individuals after the second dip. After treatment with elasmobranch Ringer's fluid therapy, sodium bicarbonate, prednisolone acetate, and vitamin E/selenium in the more severely affected ray, and elasmobranch Ringer's alone in the second ray, hematologic derangements and symptoms resolved. Hemoconcentration, hyperproteinemia, hyperlactatemia, and hyperglycemia were attributed to a stress response. A selective loss of urea after exposure to FW resulted in decreased plasma osmolality. Given the severity of the stress response and associated complications observed, hyposalinity treatments should be utilized with caution in this species. If this therapy is employed, the clinician is advised to be prepared to administer supportive care coinciding with the FW dip if necessary.

Clonal hematopoiesis of indeterminate potential (CHIP) and cardiovascular toxicity from androgen deprivation therapy in men with prostate cancer.

312 Background: Androgen deprivation therapy (ADT) with agonists of the gonadotropin releasing hormone (GnRH) receptor (e.g. leuprolide) is associated with coronary artery disease and major adverse cardiovascular (CV) events. CHIP, an age-related expanded somatic blood cell clone without other hematologic abnormalities, is associated with inflammation and accelerated atherosclerosis. Whether CHIP is associated with adverse CV outcomes in patients receiving ADT is unknown. Methods: Twenty-five men with localized prostate cancer pursuing radiotherapy plus ≥ 6 months of leuprolide were enrolled on a prospective biorepository. Using pre-treatment blood samples, whole exome sequencing libraries were generated; identified variants were filtered to retain &lt;1% MAF variants found amongst the three most common CHIP genes ( DNMT3A, TET2, ASXL1). Pre-treatment and 6-month blood samples from each patient subsequently were analyzed using a commercially available, validated 4-protein risk score that is predictive of 4-year major adverse CV events (Prevencio Inc HART CVE, Seattle WA). Multivariable logistic regression measured the association of CHIP with increased CV risk after initiation of leuprolide, defined as a clinically significant rise in 4-protein risk score at six months compared with baseline. Results: Median age was 70 years (IQR 65-76), 10 men (40%) were Black, and 4 men (16%) had a history of myocardial infarction. CHIP gene variants were found in 7 men (28%). Amongst the men with CHIP variants, 6 (85.7%) were Black. Amongst the entire cohort, 11/25 (44%) men had a clinically significant rise in CV risk score after six months of ADT. These men, compared with those who did not experience a rise in CV risk, were more often Black (72.7% versus 14.3%), on anti-hypertensive medication (72.7% versus 14.3%), and had higher baseline LDL cholesterol level (median 98 versus 81 mg/dL). On univariate analysis, harboring ≥1 CHIP variant was associated with a significantly increased odds of having an elevated CV risk score after 6 months of ADT. On multivariable analysis, after adjusting for race, anti-hypertensive medication use, and baseline LDL cholesterol, harboring ≥1 CHIP variant was associated with increased odds of having an elevated CV risk score after 6 months of ADT (adjusted OR 1.44, 95% CI 0.88-1.83), which did not meet statistical significance but limited by small sample size. Conclusions: Six months of GnRH-agonist leuprolide resulted in a rise in CV risk score in 44% of this cohort. CHIP variants, identified in 28% of this cohort, was associated with increased odds of experiencing a rise in CV risk score. Notably, Black men were more likely to harbor CHIP and experience increased CV risk after 6 months of leuprolide, respectively. Larger studies are underway to determine if CHIP variants may serve as a biomarker of ADT-induced CV risk, especially in older Black men.

Predicting disease recurrence in limited disease small cell lung cancer using cell-free DNA-based mutation and fragmentome analyses.

Limited disease (LD) small cell lung cancer (SCLC) treated with definitive concurrent chemoradiotherapy (CCRT) potentially experience disease recurrence. We investigated the feasibility of circulating-tumor DNA (ctDNA)-based genomic and fragmentome analyses to assess the risk of recurrence. Targeted sequencing was conducted using pre-treatment and on-treatment blood samples from definitive CCRT-treated patients with LD-SCLC (n=50). Based on 12-month recurrence-free survival (RFS), patients were categorized into persistent-response (PeR, n=29) and non-PeR (n=21) groups. Fragmentome analysis was conducted using ctDNA fragments of different lengths: P1 (100-155 bp) and P2 (160-180 bp). Patients with TP53 (n=15) and RB1 (n=11) mutation in on-treatment samples demonstrated significantly shorter RFS than patients with wild-type (WT) (P=0.05, P=0.0014, respectively). Fragmentome analysis of all available on-treatment samples (n=26) revealed that the non-PeR group (n=10) had a significantly higher P1 range (P=0.003) and lower P2 range (P=0.002). The areas under the curves for P1, P2, and the fragmentation ratio (P1/P2) in distinguishing the PeR and non-PeR were 0.850, 0.725, and 0.900, respectively. Using optimal cut-off, longer RFSs were found with the low-fragmentation-ratio group than with the high-fragmentation-ratio group (not reached vs. 7.6 months, P=0.002). Patients with both WT RB1 and a low-fragmentation-ratio (n=10) showed better outcomes than patients with both mutated RB1 and a high-fragmentation-ratio (n=10; hazard ratio, 7.55; 95% confidence interval: 2.14-26.6; P=0.002). RB1 mutations and high fragmentation ratios correlated with early disease recurrence. Analyzing ctDNA could help in predicting early treatment failure and making clinical decisions for high-risk patients.

Circulating tumor DNA (ctDNA) positivity and its association with clinicopathological characteristics by novel blood-based test for colorectal cancer (CRC) screening from a multi-center large cohort: COSMOS-CRC-01.

69 Background: Blood-based CRC screening has yielded performance comparable to stool-based screening tests and may be a modality that addresses the adherence gap in CRC screening. To investigate the diagnostic performance and its clinical relevance, we evaluated the test performance of a blood-based CRC screening test in a large cohort of individuals enriched for advanced colorectal neoplasia and early CRCs. Methods: 501 individuals presenting to ten institutions in Japan from 2020 to 2021 for treatment of advanced adenomas (AAs) or CRCs were consented and provided a pre-treatment blood sample (40mL whole blood in Streck cfDNA blood collection tubes). All eligible individuals underwent surgery or endoscopic resection within 30 days after sample collection and the pathological specimens were reviewed to determine cancer histology and stage, as well as AA histology. Pre-treatment blood samples were analyzed with a multi-modal (genomic and epigenomic) cfDNA CRC detection assay (Shield, Guardant Health, USA). Final cfDNA results were correlated with clinical data to determine the association of clinicopathological characteristics with test sensitivity. This study is registered as a clinical trial under UMIN000037765. Results: 451/501 (90%) individuals met inclusion/exclusion criteria and provided adequate blood samples that passed testing quality control criteria. Median age at consent was 67.4 years (range 31 – 92). 44% were female. 214/451 (47%) had a prior positive fecal occult blood test (FOBT). Of individuals with a negative or unknown FOBT testing, 161/237 (68%) had GI symptoms (e.g. abdominal pain, bloody/narrowing stool, constipation/diarrhea). The test identified 88% (268/305) of CRCs, including 71% (79/111) Stage I, 98% (87/89) Stage II, 97% (90/93) Stage III, and 100% (12/12) Stage IV. Overall sensitivity in detecting Stage I-III was 87%. The test identified 37% (54/146) of AAs, including 49% (33/68) of carcinoma in situs and 24% (9/37) of AAs with high-grade dysplasia. Sensitivity for right-sided CRC was 84% (61/73), 87% (59/68) for left-sided CRC, and 90% (148/164) for rectal tumors. Stage I test sensitivity was significantly associated with tumor size (&gt;20mm; p&lt;0.01), left-sided or rectal tumor location (p&lt;0.05), and tumor depth (p&lt;0.01) in univariate analysis, whereas the tumor depth (T2) was the sole factor associated with test positivity (p&lt;0.05) in multivariate analysis. Specificity on this assay is 90% as determined in the ECLIPSE clinical study (NCT04136002). Conclusions: This study evaluates the performance of a blood-based test for CRC and its clinicopathological relevance in a large-scale cohort. In this population enriched for early colorectal neoplasia, the sensitivity was 88% for CRC and 37% for AAs. This performance is consistent with previous reports. Clinical trial information: UMIN000037765 .

Lenvatinib in recurrent hepatocellular carcinoma after liver transplantation.

TPS578 Background: Liver transplantation is a curative treatment option in hepatocellular carcinoma (HCC) however; up to 20% of patients develop recurrent disease. HCC recurrence post-transplant is usually extrahepatic (up to 67%) hence requires effective systemic therapy options. Available data are limited and restricted to small non-randomized studies and case series. Lack of prospective clinical trial data and limited data indicates an area of unmet need in treatment of recurrent HCC after liver transplantation. This phase II study aims evaluate the safety and efficacy of lenvatinib in patients with recurrent HCC after liver transplantation. Methods: This is a multi-institutional phase II trial with lenvatinib in patients with recurrent HCC after transplantation. Lenvatinib is administered 12 mg daily orally in patients ≥60 kg, and 8 mg daily orally in patients &lt;60 kg. Each cycle is 28 days. Restaging scans will be performed every 8 weeks. Eligible patients must have histologically proven HCC that has recurred after liver transplantation and not amenable for surgical resection, age ≥18 years, ECOG PS 0-1, Child Pugh Class A, measurable disease per RECIST version 1.1, adequate organ function, no prior systemic therapy with lenvatinib or another systemic therapy in the post-transplant setting. Prior liver directed therapy is allowed, should be at least &gt;28 days prior to the study enrollment, and should have at least one measurable untreated lesion by RECIST 1.1 Primary endpoint is overall response rate (ORR). Secondary endpoints are safety/tolerability, progression free survival, and overall survival. Pre-treatment and on-treatment peripheral blood samples will be collected for correlative research. A Simon`s two stage Minimax design is employed (H0: ORR = 5%; H1: ORR = 24%; Type I error = 0.05; power = 80%). In the first stage, 11 patients will be accrued, and if there is no objective response among them, the study will be stopped for futility. Otherwise, additional 6 patients will be accrued for a total of 17 patients. This study is currently enrolling patients. Clinical trial information: NCT05103904 .

A Phase 1 Study of Adding Pitavastatin to Venetoclax-Based Therapy in AML and CLL/SLL

Statins (HMG-CoA-reductase inhibitors) are drugs used widely for the treatment of high cholesterol and are generally well-tolerated. There has been increasing interest in repurposing statins for oncology based on the recognition that many cancer cells are “addicted” to the mevalonate biosynthetic pathway (Iannelli et al, 2017; Juarez and Fruman 2021). We have shown that statins increase expression of the pro-apoptotic BCL2 family member PUMA (Lee at al, 2018). We have strong pre-clinical evidence that statins improve responses to the BCL2 inhibitor venetoclax (VEN) in cell line models and ex vivo primary patient specimens from patients with both chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) (Lee et al, 2018). Additionally, retrospective review of patients enrolled into the early clinical trials of VEN in CLL revealed improved responses to VEN (complete remission [CR] and progression-free survival) in the patients who were concurrently on a statin medication (Lee et al, 2018). These retrospective analyses may underestimate the potential of statins for leukemia therapy, as the statin drugs most commonly prescribed for hypercholesterolemia are not pharmacologically optimized for oncology (Abdullah et al., 2018). We developed a phase 1 study of adding pitavastatin (PIT) to VEN-based therapy in patients with AML and CLL/small lymphocytic lymphoma (SLL) (NCT04512105). AML patients were eligible if receiving induction therapy with azacitidine (AZA) and VEN per standard of care. CLL/SLL patients could receive either VEN with obinutuzumab (O) or rituximab. A 3+3 design was employed. Dose level 1 was PIT 2 mg daily, and dose level 2 (DL2) was 4 mg daily. These doses are consistent with the doses used for the treatment of hypercholesterolemia. Those enrolling at DL2 had a creatinine clearance of 60 mL/min or higher based on the prescribing information for pitavastatin. Patients already on statins for the treatment for hypercholesterolemia were allowed; these patients stopped their prescribed statin for 72 hours prior to initiation of PIT. PIT started after patients completed VEN ramp-up and had been on a stable VEN dose for at least 5 days. Primary objectives were assessing safety and tolerability and determining the recommended phase 2 dose of PIT in combination with VEN-based therapies. Secondary objectives included CR rates. Exploratory objectives included performing BH3 profiling on pretreatment and post-treatment blood samples to assess whether add-on treatment with PIT increases apoptotic priming, as seen in preclinical models (Lee et al, 2018). 14 patients signed informed consent. 6 were deemed ineligible. 2 withdrew consent prior to starting PIT therapy. 6 patients received PIT on study. 2 patients had AML and received AZA+VEN. 2 patients had CLL, and 2 patients had SLL. The CLL/SLL patients all received VEN+O. Grade 1 myalgia was reported in one subject on the 4mg PIT dose. 1 patient experienced a grade 1 increase in AST on the 4mg dose. Another patient had grade 1 elevation in bilirubin and alkaline phosphatase at the 4 mg dose. Grade 3-4 toxicities included leukopenia, neutropenia (1 case of febrile neutropenia, grade 3), anemia, and thrombocytopenia. Cycle 1 was defined as the dose-limiting toxicity (DLT) period, and no hematologic toxicities occurred during the DLT period. 1 patient had grade 3 pancreatitis, but this was felt to be unrelated to therapy or disease (also outside the DLT period). No grade 5 toxicities occurred while on study therapy. Toxicities were overall felt to be similar to patients receiving VEN-based therapy in standard clinical practice. All evaluable patients (n=5) achieved clinical CR. 1 patient with SLL is awaiting re-staging at the time of this abstract. Correlative studies are ongoing. In 2 patient peripheral blood samples analyzed, the % blasts (for an AML sample) and % CD5+ B-cells (for a CLL sample) were reduced 24 hours after PIT dosing compared to prior to any treatment and before PIT/after VEN ramp-up. Based on the tolerability of PIT and encouraging clinical outcomes, a phase 2 study is being planned to better assess the efficacy of adding PIT to VEN-based therapies in AML and CLL/SLL.

Immunomodulatory Effects of Chemo-Immunotherapy ± Idelalisib in Chronic Lymphocytic Leukaemia (CLL)

Introduction: Chronic lymphocytic leukaemia (CLL) is characterised by a dysfunctional immune profile. However, the precise impact of therapy on this altered immune state, both short and long term, remains unclear. The collection of pre-treatment and longitudinal blood samples in the NCRI phase III RIAltO trial (NCT01678430) offered a unique opportunity to address this question in the context of frontline chemo-immunotherapy (CIT) containing bendamustine or chlorambucil. The factorial randomisation to idelalisib versus placebo also provided a framework for investigating the immunomodulatory effects of PI3Kδ inhibition in this setting. Methods: We conducted mass cytometry analysis on samples obtained from CLL patients enrolled in the RIAltO trial. Patients were randomly assigned to receive ofatumumab plus either bendamustine or chlorambucil, with or without idelalisib. Samples were collected before treatment and at early (median 5.9 [IQR 5.7 - 6.4] months) and late (18.5 [16.1 - 22.6] months) post-treatment timepoints. Three healthy controls (HC) were included for comparison. CD3 + T cells were analysed using a customised CyTOF panel comprising 36 markers. Uniform manifold approximation and projection (UMAP) and manual gating analysis were utilised to evaluate global and specific T-cell changes. Results: In all, 177 samples from 79 CLL patients, obtained before or after CIT, were analysed. Baseline characteristics were similar between the different treatment arms. Compared to HC, treatment-naïve CLL patients exhibited a distinct T-cell profile with increased regulatory T cells, decreased naïve and central memory (CM) cells, elevated effector memory (EM) cells, reduced non-senescent cells, and increased expression of HLA-DR, PD-1, TOX, T-bet, and Eomes. Following CIT, the T-cell profile was significantly altered but remained distinct from that of HCs. CIT-induced changes persisted at the late post-treatment timepoint, indicating a sustained effect. Analysis of specific T-cell populations showed that, although some CLL-associated features became less prominent following CIT (expression of PD-1, TOX and TIGIT), most were enhanced (shift from naïve and CM cells towards a terminal effector phenotype; shift from a non-senescent to a terminally senescent phenotype; increased HLA-DR and T-bet expression). The post-treatment immune profiles were similar in patients receiving bendamustine or chlorambucil. Despite its immunomodulatory effects when used as a single agent, idelalisib also induced minimal additional changes. Furthermore, the post-treatment immune profile was similar in patients achieving minimal residual disease (MRD) negativity versus those who remained MRD positive, suggesting a direct pharmacodynamic effect. Finally, non-metric multidimensional scaling (NMDS) analysis demonstrated that CIT shifted the overall CLL-associated immune profile further away from that of HC, and that the shift was independent of chemotherapy allocation, idelalisib, or the level of CLL cytoreduction achieved. Conclusion: CIT results in profound immune changes that persists up to 18.5 [16.1 - 22.6] months following treatment. Furthermore, the overall effect of these changes further differentiates the CLL-associated immune profile from that of HC, deepening the existing T-cell dysfunction irrespective of chemotherapy choice, or the addition of idelalisib. Our study has important potential implications for patients receiving CIT in a range of clinical settings and supports the move towards more targeted agents that are less likely to cause indiscriminate immune perturbation.

Deep Immune Profiling Identifies Novel T-Cell Subpopulations That Influence Specific Clinical Outcomes in Chronic Lymphocytic Leukaemia (CLL)

Introduction: In chronic lymphocytic leukaemia (CLL), a dynamic interplay exists whereby CLL cells shape the immune microenvironment and perturb normal immune function while at the same time relying on non-malignant immune cells for survival and proliferation signals. However, it is unclear to what extent different T-cell populations vary among individual CLL patients and whether such variation influences the occurrence of specific clinical events. To address this knowledge gap, pre-treatment blood samples obtained from patients enrolled in a large clinical trial of frontline therapy were subjected to deep immune phenotyping by mass cytometry and the results related with clinical outcomes. Methods: The discovery cohort comprised pre-treatment samples from 79 CLL patients enrolled in the NCRI RIAltO trial (NCT01678430) who underwent factorial randomisation to ofatumumab and either bendamustine or chlorambucil, with or without idelalisib. The validation cohort included a randomly selected group of 59 patients who received bendamustine and ofatumumab only. Thirty pre-treatment samples from the discovery cohort were further selected for ex vivo T-cell stimulation using CD3/CD28 microbeads. All samples were analysed using mass cytometry and customised antibody panels. Results: FlowSOM clustering was applied to all CD3 + T cells in the discovery cohort. Generated clusters were merged and annotated based on their median marker expression. 17 distinct CD4 + clusters were identified and categorised as cytotoxic cells (C1-3), helper cells (Th; H1-11), and regulatory cells (R1-3), whereas 23 CD8 + clusters were identified and categorised as NK T-cells (NKT1-4) and cytotoxic T-cells (T1-19). Cox proportional hazard regression analysis considering baseline characteristics and treatment allocation revealed associations between pre-treatment cluster frequencies and clinical endpoints. Cluster H1 correlated with a lower risk of grade ≥3 infections (HR 0.007 [95% CI: 0.001 - 0.782], P = 0.039), cluster T10 with a lower risk of grade ≥3 second primary malignancy (SPM; HR 0.811 [95% CI: 0.660 - 0.997], P = 0.047), and cluster T12 with longer overall survival (OS; HR 0.844 [95% CI: 0.719 - 0.990], P = 0.037). Based on the expression of non-redundant markers, corresponding T-cell subpopulations with distinct immune signatures were prospectively defined which closely resembled the three clinically significant clusters and were associated with the same clinical endpoints (ICOS +HLA-DR +PD1 +TIGIT +Tbet +CD4 + helper (Th) cells and H1; CD27 +CD28 -PD1 +Tbet +Eomes +CD8 + cells and T10; CD27 +CD28 -GrymB +Tbet +Eomes +CD8 + terminal effector (TE) cells and T12). Using the same gating strategies and cut-off values, the association between ICOS +HLA-DR +PD1 +TIGIT +Tbet +CD4 + Th cells and infections, as well as CD27 +CD28 -GrymB +Tbet + Eomes +CD8 + TE cells and OS were confirmed in the validation cohort (it was not possible to confirm the association between CD27 +CD28 -GrymB +Tbet +Eomes +CD8 + TE cells and SPM due to a paucity of events in the validation cohort). Following stimulation by CD3/CD28 microbeads, enrichment of primed and polyfunctional cells was observed in all clinically significant T-cell subpopulations compared to their parent CD4 + or CD8 + T cells. Conclusion: The present study has identified three previously undescribed T-cell subpopulations that appear to influence the risk of infection, SPM, and death in patients with CLL receiving frontline chemo-immunotherapy. Further studies are warranted to validate these findings in the setting of targeted agents and in patients receiving CIT for other haematological malignancies.