Sequential Biopsies Research Articles

Abstract 3703: Utility of circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) for prognosis of patients with recurrent high grade glioma

Abstract High-Grade Gliomas (HGGs) are the most aggressive primary brain tumors in adults and molecular characterization is crucial for diagnosis and optimal disease treatment. Due to the eloquent location of many HGGs, upfront or repeat tumor sampling may be sub-optimal. Hence, there is an urgent need to develop alternative, minimally-invasive means to obtain diagnostic information and determine the expected clinical behavior. Here we report that circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) can be used to identify disease defining genomic alterations and to track clonal evolution. Additionally, we demonstrate that detection of CSF ctDNA is positively correlated with leptomeningeal disease and overall survival suggesting that CSF ctDNA should be integrated into clinical decision making in the clinic. Our study includes 315 samples from 254 patients with recurrent glioma treated at Memorial Sloan Kettering Cancer Center who underwent CSF collection for routine clinical care and were sequenced using the MSK-IMPACT targeted clinical sequencing assay (468 and 505 genes). Alterations were classified as oncogenic based on OncoKB. 23% of genomic alterations detected were identified using a secondary bioinformatics analysis, following an informed approach with less stringent mutation calling criteria and Gaussian Mixture Model to call copy number events. CSF ctDNA positivity was determined by the presence of at least one oncogenic disease defining alteration or any shared alteration with the tumor. Within this cohort, we found 198 CSF ctDNA positive (63%) and 117 CSF negative (37%) samples. Of note, CSF ctDNA positivity was the highest amongst histone mutant tumors with 94% CSF ctDNA positivity compared to 56% CSF ctDNA positivity for IDH-WT tumors. We noticed considerable evolution of the cancer genome in patients with sequential biopsies with only 35% of alterations shared between the tumor and the CSF, in particular within the growth signaling pathways (e.g. PDGFRA). The majority of the shared alterations were clonal, and we noticed the emergence of new clonal events in the CSF. Our cohort demonstrated that patients with positive CSF ctDNA had a significantly shorter overall survival compared to those who were CSF ctDNA negative (4.83 months vs 11.83 months, HR 2.1, p < 0.001). In tandem with secondary clinical analysis, radiographic findings also correlated with CSF ctDNA positivity. Specifically, the presence of enhancing disease and contact of the tumor with the ventricular space were positively associated with detection of CSF ctDNA. Additionally, CSF ctDNA was associated with positive cytology in 21/21 cases (100%). With our improved bioinformatics pipeline we hypothesize ctDNA from CSF may be used as a prognostic biomarker for survival, but confirmation requires further validation in a prospective study. Citation Format: Bridget M. Holle, Subhiksha Nandakumar, Christoph Kreitzer, Timothy Song, Marlene Tartaro, Charli Ann J. Hertz, Johnathan Rafailov, Carl Campos, Anne S. Reiner, Robert Young, Alicia Meng, Luca Pasquini, A. Rose Brannon, Nicholas D. Socci, Maria E. Arcila, David B. Solit, Michael F. Berger, Nikolaus Schultz, Ingo K. Mellinghoff, Alexandra M. Miller. Utility of circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) for prognosis of patients with recurrent high grade glioma [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 3703.

Longitudinal Muscle Biopsies Reveal Inter- and Intra-Subject Variability in Cancer Cachexia: Paving the Way for Biopsy-Guided Tailored Treatment.

In the rapidly evolving landscape of cancer cachexia research, the development and refinement of diagnostic and predictive biomarkers constitute an ongoing challenge. This study aims to introduce longitudinal muscle biopsies as a potential framework for disease monitoring and treatment. The initial feasibility and safety assessment was performed for healthy mice and rats that received two consecutive muscle biopsies. The assessment was performed by utilizing three different tools. Subsequently, the protocol was also applied in leiomyosarcoma tumor-bearing rats. Longitudinal muscle biopsies proved to be a safe and feasible technique, especially in rat models. The application of this protocol to tumor-bearing rats further affirmed its tolerability and feasibility, while microscopic evaluation of the biopsies demonstrated varying levels of muscle atrophy with or without leukocyte infiltration. In this tumor model, sequential muscle biopsies confirmed the variability of the cancer cachexia evolution among subjects and at different time-points. Despite the abundance of promising cancer cachexia data during the past decade, the full potential of muscle biopsies is not being leveraged. Sequential muscle biopsies throughout the disease course represent a feasible and safe tool that can be utilized to guide precision treatment and monitor the response in cancer cachexia research.

Kidney Fibrosis and Oxidative Stress: From Molecular Pathways to New Pharmacological Opportunities.

Kidney fibrosis, diffused into the interstitium, vessels, and glomerulus, is the main pathologic feature associated with loss of renal function and chronic kidney disease (CKD). Fibrosis may be triggered in kidney diseases by different genetic and molecular insults. However, several studies have shown that fibrosis can be linked to oxidative stress and mitochondrial dysfunction in CKD. In this review, we will focus on three pathways that link oxidative stress and kidney fibrosis, namely: (i) hyperglycemia and mitochondrial energy imbalance, (ii) the mineralocorticoid signaling pathway, and (iii) the hypoxia-inducible factor (HIF) pathway. We selected these pathways because they are targeted by available medications capable of reducing kidney fibrosis, such as sodium-glucose cotransporter-2 (SGLT2) inhibitors, non-steroidal mineralocorticoid receptor antagonists (MRAs), and HIF-1alpha-prolyl hydroxylase inhibitors. These drugs have shown a reduction in oxidative stress in the kidney and a reduced collagen deposition across different CKD subtypes. However, there is still a long and winding road to a clear understanding of the anti-fibrotic effects of these compounds in humans, due to the inherent practical and ethical difficulties in obtaining sequential kidney biopsies and the lack of specific fibrosis biomarkers measurable in easily accessible matrices like urine. In this narrative review, we will describe these three pathways, their interconnections, and their link to and activity in oxidative stress and kidney fibrosis.

Wound healing of experimental equine skin wounds and concurrent microbiota in wound dressings following topical propylene glycol gel treatment.

Topical wound treatments rely on carrier formulations with little to no biological impact. The potential for a common vehicle, a propylene glycol (PG) gel, to affect wound healing measures including microbiota is not known. Microbiome characterization, based on next generation sequencing methods is typically performed on tissue or directly obtained wound fluid samples. The utility for primary wound dressings to characterize equine wound microbiota in the context of topical treatments is currently unknown. This investigation reports the topical effect of an 80% PG based gel on wound healing and microbiota in wound dressings. Experiments were performed in six mature horses utilizing a surgical, distal limb wound model, histology of sequential wound biopsies, photographic wound measurements and microbiota profiling via 16s rRNA sequencing of wound dressing samples. Experimental wounds were surveyed for 42 days and either treated (Day 7, 14, 21 and 28; at 0.03 ml/cm2) or unexposed to the PG gel. Wound surface area, relative and absolute microbial abundances, diversity indices and histologic parameters were analyzed in the context of the experimental group (treatment; control) using qualitative or quantitative methods depending on data characteristics. Compared to controls, treatment slowed the wound healing rate (17.17 ± 4.27 vs. 18.56 ± 6.3 mm2/day), delayed the temporal decline of polymorphonucleated cells in wound beds and operational taxonomic units (OTU) in wound dressings and lowered alpha-diversity indices for microbiota in primary wound dressing. Relative abundances of OTUs were in line with those previously reported for equine wounds. Clinical outcomes 42 days post wounding were considered similar irrespective of PG gel exposure. Results highlight the potential for vehicle exposure to alter relevant wound outcome measures, imposing the need for stringent experimental control measures. Primary wound dressings may represent an alternate sample source for characterization of the wound microbiome alleviating the need for additional interventions. Further studies are warranted to contrast the microbiome in wound dressings against that present on wound surfaces to conclude on the validity of this approach.

Early Identification of TP53 mutation in Myelodysplastic Neoplasms and Acute Myeloid Leukemia Via Point-of-Care p53 Immunohistochemistry

Background: Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) are heterogeneous diseases that often carry a poor prognosis. Once MDS or AML is diagnosed, first-line therapy choices include hypomethylating agent/venetoclax, induction chemotherapy, and/or targeted therapeutics as appropriate. Traditional “7+3” induction therapy has limited efficacy in in patients with TP53 disruption. Mutations in TP53 occur in at least 8% of de novo AML genomes, but turnaround time for next-generation sequencing (NGS) can be 10-14 days. Hence, it is possible that patients with TP53 mutation(s) will receive a regimen that will not be effective as it will not be known whether a patient has a TP53 mutation soon after diagnosis. Furthermore, health care disparities are amplified by the lack of readily available precision diagnostics in low- and middle-income countries (LMIC), leading to worse outcomes. Early identification of TP53 mutation through p53 immunohistochemistry (IHC) testing might allow for more timely prognostication and intervention. Methods: The UMass Chan Leukemia Registry and UMass Hematopathology archives identified patients with MDS or AML with TP53 disruption between 2011 and 2023. Immunohistochemical staining for p53 was performed on sections of bone marrow core biopsies by VENTANA CONFIRM anti-p53 (DO-7) primary antibody. Images were scored by manual counting by at least 2 separate hematopathologists. TP53 mutational analysis was performed as previously described (Patel SA , ..., Gerber JM. Leuk Lymphoma 2021; 62: 3348-60). For TP53 exome sequencing, the entire coding region from codons 1-393 of isoform A were covered, with coverage depth > 500X. The concordance between p53 IHC and the referent test ( TP53 mutational testing as the gold standard) was assessed. Results: Query of theUMass Leukemia Registry and UMass Hematopathology archives yielded 86 clinically annotated patients with MDS and AML with TP53 disruption. Among 42 patients with MDS (or its subentities) with TP53 disruption, 61 bone marrow biopsies had concurrently available data for p53 IHC and TP53 mutational testing. (Some cases were sequential biopsies from the same patient). Among 44 patients with AML (or its subentities) with TP53 disruption, 36 bone marrow biopsies had concurrently available data for p53 IHC and TP53 mutational testing. For patients with MDS with TP53 disruption, the sensitivity of p53 IHC testing was 68.1% and specificity was 93.3% (Panel A). For patients with AML with TP53 disruption, the sensitivity of p53 IHC testing was 75.8% and specificity was 100% (Panel B). Merged MDS and AML analysis showed sensitivity of 71.3% and specificity of 93.8%. When using a cutoff of ≥ 5% for p53 IHC testing, positive predictive value (PPV) was 100%. Conclusion: Our data show that p53 IHC testingdemonstrates high specificity and PPV but low sensitivity and negative predictive value (NPV), suggesting that IHC may be a valuable “rule-in” test. A cutoff value of ≥ 5% for IHC confers 100% PPV. This suggests that a positive IHC result may justify p53 IHC-adapted first-line choice of therapy, but a negative result does not exclude the possibility of TP53 mutation. IHC testing is a more cost-effective measure and is more readily available than NGS, which would make it an attractive alternative especially across global markets and in geographic regions marked by health care inequities, due to inability to access genomic testing. Limitations to our study include the relatively small sample size, as well as presence of TP53 mutations that prevent protein expression and thus prevent IHC positivity (i.e. stop codons and/or post-transcriptional modifications). Furthermore, results from our study may be distinct from other centers' studies given different inclusion criteria and non-uniform criteria for p53 staining analysis. However, our study retains value in that the use of a high-fidelity point-of-care p53 IHC staining method may lead to better outcomes with reduced financial toxicity and may inform first-line therapeutics if the IHC is positive. Further large-scale studies will be needed to confidently establish the p53 IHC staining method as the preferred surrogate for TP53 mutational testing. Additional work needs to be performed to assess the link between p53 staining pattern and TP53 allelic state. Earlier precision treatment may improve outcomes in this exceptionally high-risk population.

Selinexor Disrupts Epigenetic Programing and Modulates Immunogenicity in Multiple Myeloma

Introduction: Multiple myeloma (MM), remains an incurable, but treatable, malignancy that has benefitted from the integration of novel immunologic agents in clinical use, such as monoclonal antibody, bispecific T cell engager (TCE) and chimeric antigen receptor T (CAR-T) cell and antibody-drug conjugate based therapies. While these agents have shown great promise in treating relapsed/refractory MM patients, there is heterogeneity in response to therapy due to differences in patients' immune tumor microenvironments (iTME) and immunogenicity of the tumor cells. Previously (Sudalagunta et. al., ASH 2021), we used functional genomic screening of 53 MM samples to characterize the transcriptomic profile associated with sensitivity to the selective nuclear export inhibitor, selinexor (SELI), which was shown to be inversely correlated with the transcriptomic profile associated with sensitivity to an anti-CD38 monoclonal antibody in 106 MM samples. In this study, we investigate the cellular mechanisms of SELI resistance leading to an immunogenic cell state. Methods: Primary cells from an additional set of MM patients (n=65) were used for paired bulk RNAseq and an organotypic ex vivo drug sensitivity assay. Cellular viability was assessed using digital analysis of brightfield longitudinal images, and drug sensitivity was assessed as area under the dose-time-response curve (AUC) for each sample. We conducted gene set enrichment analysis (GSEA) on paired RNAseq data and ex vivo AUC of SELI. Individual genes and their respective correlations with ex vivo AUC of SELI were ranked from highest to lowest. We used RNAseq to examine the role of acute exposure to SELI on CD138-selected cells from paired pre/post treatment bone marrow specimens of five MM patients enrolled in the NCT02186834 clinical trial, taken prior to treatment with SELI and dexamethasone (DEX) and one/two weeks post-induction. Paired t-tests were carried out to quantify the changes. Results: In our previous analysis of 844 MM samples, a subset of the genes whose expression is positively correlated with SELI AUC are controlled by H3K27me3, which is catalyzed by nuclear accumulation of EZH2. Tripathi et. al. (AACR Annual Meeting 2022 & 2023) have demonstrated that XPO1 and EZH2 form a complex, and inhibition of XPO1 activity blocks export of EZH2. GSEA of a new cohort of 65 MM patient samples demonstrated increased ex vivo SELI sensitivity (lower AUC, hence negative correlation) in samples with higher expression of known epigenetic regulators of the polycomb repressive complex 2 (PRC2), such as EZH2, SUZ12, and EED; as shown in Figure a, which led to lowering of expression of genes bound by these proteins due to gene silencing via H3K27me3, shown in red in Figure a, according to GSEA of ChIPseq databases. Furthermore, gene expression profiles of paired pre/post SELI/DEX treatment samples showed reduced expression of genes associated with H3K27me3 histone modification (paired t-test p=2.06e-83, 1.1061e-81, 2.56e-80, 5.15e-12, 6.22e-4) in all five patients. These observations suggest that XPO1 inhibition leads to nuclear accumulation of epigenetic modulators like EZH2, SUZ12, and EED, which catalyze H3K27me3 activity leading to silencing of H3K27me3-regulated genes. GSEA of paired ex vivo AUC and RNAseq data for an EZH2 inhibitor (EPZ-6438) resulted in negative correlation between expression of genes in PRC2 and ex vivo sensitivity, similar to SELI. Interestingly, we observed that this epigenetic mechanism of resistance to SELI is inversely associated with key immunological targets in MM such as CD74, SLAMF7, CD38, CD46, TNFRSF13B, and LY9 as shown in Figure a. In Figure b, we present a graphical visualization of intracellular changes due to exposure to SELI in a naïve and a SELI-resistant MM cell. Conclusions: Here we propose that SELI disrupts EZH2 subcellular localization, dysregulating H3K27me3-gene suppression. Sequential BM biopsies from SELI treated patients demonstrate decreased expression of H3K27me3-regulated genes, suggesting increased EZH2 activity by nuclear accumulation. Finally, SELI-resistant primary cells have increased expression of EZH2 regulated immunotherapy target genes (e.g. CD38, SLAMF7), suggesting a possible therapeutic strategy of sequential treatment of SELI followed by immunotherapy. Using multiparameter flow cytometry of samples pre/post SELI/DEX induction, we will study SELI's effect on iTME.

Validation of mitochondrial function measurements in permeabilized myocardial tissue from porcine left ventricular specimens with ischemia/reperfusion

Abstract Background Mitochondrial function is critical to myocardial ischemia/reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, permeabilized myocardial tissue can be used for quantification of mitochondrial respiration in small myocardial specimens of about only 2-5 mg, which can be retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. Whether or not mitochondrial dysfunction during ischemia/reperfusion is reflected to the same extent in saponin-permeabilized myocardial tissue specimens (PMT) and in isolated mitochondria is still unclear. Purpose We attempted to validate measurements of mitochondrial respiration from PMT by comparison to those from isolated mitochondria in left ventricular myocardium from anesthetized pigs undergoing coronary occlusion/reperfusion. Methods Göttingen open-chest, anesthetized minipigs (n=10) were subjected to 60 min distal left anterior descending artery occlusion and 180 min reperfusion. At the end of reperfusion, PMT and isolated mitochondria were prepared from remote and ischemic/reperfused myocardium. Baseline, adenosine diphosphate-stimulated complex I (without/with cytochrome c, to account for absence or presence of the outer mitochondrial membrane integrity) and complex IV respiration was measured at 37°C during magnetic stirring using a high-resolution respirometer. Mitochondrial respiration in PMT and isolated mitochondria was normalized to protein content and the content of the mitochondrial marker protein cytochrome c oxidase 4. Results Mitochondrial respiration measurements in PMT and isolated mitochondria correlated well (Figure 1A) and agreed well in Bland-Altman plot (Figure 1B). While baseline respiration in PMT was somewhat higher than that in isolated mitochondria (Figure 2A), mitochondrial dysfunction during ischemia/reperfusion was equally reflected in PMT and isolated mitochondria (Figure 2B & 2C). Complex IV respiration was not different between remote myocardium and ischemic/reperfused myocardium, independently of the preparation, indicating a comparable amount of viable mitochondria (Figure 2D). Conclusion Mitochondrial function measurements in PMT can substitute for that in isolated mitochondria to reflect their dysfunction in ischemia/reperfusion. Thus, mitoprotective strategies targeting ischemia/reperfusion injury in small tissue samples and also in human myocardial biopsies can be followed in PMT.Figure 1Figure 2

Whole exome sequencing uncovers HRAS mutations as potential mediators of resistance to metronomic chemotherapy

ObjectivesThe aim of this pilot study is to identify the genetic factors that contribute to the response of metronomic chemotherapy in head and neck squamous cell carcinoma (HNSCC) patients using whole-exome sequencing (WES). This study would facilitate the identification of predictive biomarkers, which would enable personalized treatment strategies and improve treatment outcomes for patients with HNSCC. Materials and MethodsWe have selected patients with recurrent head and neck cancer who underwent metronomic chemotherapy. Sequential tumor biopsies were collected from the patients at different stages of treatment to capture the genomic alterations and tumor evolution during metronomic chemotherapy and sequenced using WES. ResultsWe identified several known HNSCC hallmark genes reported in COSMIC, including KMT2B, NOTCH1, FAT1, TP53, HRAS, CASP8, and CDKN2A. Copy number alteration analysis revealed amplifications and deletions in several oncogenic and tumor suppressor genes. COSMIC Mutational Signature 15 associated with defective DNA mismatch repair was enriched in 73% of HNSCC samples. Further, the comparison of genomic alterations between responders and non-responders identified HRAS gene uniquely mutated in non-responders that could potentially contribute to resistance against metronomic chemotherapy. DiscussionOur findings corroborate the molecular heterogeneity of recurrent HNSCC tumors and establish an association between HRAS mutations and resistance to metronomic chemotherapy, suggesting HRAS as a potential therapeutic target. Combining HRAS inhibitors with metronomic regimens could improve treatment sensitivity in HRAS-mutated HNSCC patients. Further studies are needed to fully elucidate the genomic mechanisms underlying the response to metronomic chemotherapy.

Preliminary Analysis Demonstrates Durvalumab (Anti-PD-L1) & Lenalidomide Is Superior to Single-Agent Durvalumab (anti-PD-L1) in Refractory/Advanced Cutaneous T Cell Lymphoma in a Randomized Phase 2 Trial

Introduction: Mycosis fungoides (MF) and the leukemic variant Sézary syndrome (SS), commonly referred to as cutaneous T cell lymphoma (CTCL), are characterized by clonal expansion of malignant T cells in the skin within a background of chronic inflammation. Advanced stages have an unfavorable prognosis. The skin microenvironment plays an important role in the development and progression of MF and SS. We have shown that the malignant T cells escape immune surveillance via immune checkpoint signaling such as the PD-1/PD-L1 axis. In our phase I dose-escalating study anti-PD-L1 (durvalumab) & lenalidomide demonstrated a tolerable safety profile with significant clinical activity in refractory/advanced CTCL. We initiated the randomized Phase 2 portion to compare single agent durvalumab to durvalumab combined with lenalidomide in relapsed/advanced CTCL (NCT03011814). The primary end point was objective response rate (ORR) using the global composite response (based on skin, blood, nodes, and viscera) according to consensus guidelines. Secondary end points included duration of response, progression-free survival, and toxicity. Relationships between gene expression profile, tumor-microenvironment, and antitumor activity were exploratory end points. Methods:Adult patients with histologically confirmed MF or SS, who had failed ≥2 systemic therapies were enrolled and randomized 1:1 to single agent durvalumab (1500 mg (day 1 of 28-day cycle) or durvalumab (same dose) & lenalidomide (10 mg for cycle 1, 15 mg for cycle 2, then 20 mg for subsequent cycles daily for 21 days of each 28-day cycle). The study used a “pick a winner” design based on ORR. Serial skin and blood samples were collected to assess the impact on the tumor microenvironment (TME) and anti-tumor activity. Results:A total of 23 patients were randomized and had the following characteristics (11 durvalumab vs 12 durvalumab/lenalidomide): median age 57 yrs (35-79) vs 63 yrs (32-88); stage IB, 2 (18%) vs 4 (33%); stage IIB, 4 (36%) vs 3 (25%); stage III/IV, 5 (45%) vs 5 (42%); MF, 8 (73%) vs 8 (67%); e-MF/SS, 4 (36%) vs 2 (17%); large cell transformation 3 (27%) vs 5 (42%). The median number of prior systemic treatments for both single and combo arm was 3. Global best ORR was 58% for durvalumab/ lenalidomide vs 36% for durvalumab alone.Median follow up time was 7.9 (range, 0.9-27.6) months. Analysis is ongoing. One patient died one month after discontinuation from study due to PD. No serious AEs were observed. The most common treatment-emergent adverse events (shown below for combo arm) were more frequent in the durvalumab/lenalidomide arm vs durvalumab arm and included fatigue (n=9), diarrhea (n=4), decreased platelets (n=4), leg edema (n=3), constipation (n=3), hyperglycemia (n=3), anemia (n=3), and leukopenia & neutropenia (n=3). The majority of AEs with both treatment arms were mild to moderate in severity (grade I/II, 91%; grade III, 8 %). No grade IV event except 1 neutropenia on combo arm was observed. Median cycles of treatment were 4 (range, 1-12) and 5 (range 1-7) for single and combo arms.) Five pts remain on treatment on single agent durvalumab vs 3 pts on combo arm. Molecular profiling using CIBERSORT and single cell analysis revealed distinct changes of immune cell signatures and cellular signaling interactions within the TME that occurred in each treatment arm when compared to baseline. Conclusions: This randomized phase 2 trial of durvalumab (anti-PD-L1) +/- lenalidomide evaluating anti-tumor activity demonstrated superior clinical activity of combinatorial durvalumab/lenalidomide vs single-agent durvalumab in refractory/advanced CTCL. Responses were durable and ongoing, and treatment was well tolerated. Our correlative results from sequential skin biopsies demonstrated immune signatures for enhanced anti-tumor responses on skin biopsies at baseline may be predictive of response to checkpoint blockade and yield insights into mechanisms of therapeutic resistance.

Multi-omics network analysis on samples from sequential biopsies reveals vital role of proliferation arrest for Macrosteatosis related graft failure in rats after liver transplantation

To investigate the molecular impact of graft MaS on post-transplant prognosis, based on multi-omics integrative analysis. Rats were fed by methionine-choline deficient diet (MCD) for MaS grafts. Samples were collected from grafts by sequential biopsies. Transcriptomic and metabolomic profilings were assayed. Post-transplant MaS status showed a close association with graft failure. Differentially expressed genes (DEGs) for in-vivo MaS were mainly enriched on pathways of cell cycle and DNA replication. Post-transplant MaS caused arrests of graft regeneration via inhibiting the E2F1 centered network, which was confirmed by an in vitro experiment. Data from metabolomics assays found insufficient serine/creatine which is located on one‑carbon metabolism was responsible for MaS-related GF. Pre-transplant MaS caused severe fibrosis in long-term survivors. DEGs for grafts from long-term survivors with pre-transplant MaS were mainly enriched in pathways of ECM-receptor interaction and focal adhesion. Transcriptional regulatory network analysis confirmed SOX9 as a key transcription factor (TF) for MaS-related fibrosis. Metabolomic assays found elevation of aromatic amino acid (AAA) was a major feature of fibrosis in long-term survivors. Graft MaS in vivo increased post-transplant GF via negative regulations on graft regeneration. Pre-transplant MaS induced severe fibrosis in long-term survivors via activations on ECM-receptor interaction and AAA metabolism.

EP12.01-65 Prognostic Value of Sequential Liquid Biopsy (LB) after Osimertinib Among Hispanic Patients with Advanced EGFR-Mutant NSCLC

EP12.01-65 Prognostic Value of Sequential Liquid Biopsy (LB) after Osimertinib Among Hispanic Patients with Advanced EGFR-Mutant NSCLC

Deep molecular tracking over the 12-yr development of endometrial cancer from hyperplasia in a single patient.

Although the progressive histologic steps leading to endometrial cancer (EndoCA), the most common female reproductive tract malignancy, from endometrial hyperplasia are well-established, the molecular changes accompanying this malignant transformation in a single patient have never been described. We had the unique opportunity to investigate the paired histologic and molecular features associated with the 12-yr development of EndoCA in a postmenopausal female who could not undergo hysterectomy and instead underwent progesterone treatment. Using a specially designed 58-gene next-generation sequencing panel, we analyzed a total of 10 sequential biopsy samples collected over this time frame. A total of eight pathogenic/likely pathogenic mutations in seven genes, APC, ARID1A, CTNNB1, CDKN2A, KRAS, PTEN, and TP53, were identified. A PTEN nonsense mutation p.W111* was present in all samples analyzed except histologically normal endometrium. Apart from this PTEN mutation, the only other recurrent mutation was KRAS G12D, which was present in six biopsy samplings, including histologically normal tissue obtained at the patient's first visit but not detectable in the cancer. The PTEN p.W111* mutant allele fractions were lowest in benign, inactive endometrial glands (0.7%), highest in adenocarcinoma (36.9%), and, notably, were always markedly reduced following progesterone treatment. To our knowledge, this report provides the first molecular characterization of EndoCA development in a single patient. A single PTEN mutation was present throughout the 12 years of cancer development. Importantly, and with potential significance toward medical and nonsurgical management of EndoCA, progesterone treatments were consistently noted to markedly decrease PTEN mutant allele fractions to precancerous levels.

Targeted NGS on sequential bone marrow biopsies aids in the evaluation of cytopenias and monocytosis and documents clonal evolution—a proof of principle study

Differential diagnosis of clonal versus reactive cytopenia and monocytosis, respectively, frequently presents a diagnostic challenge. With the two recent classifications of myeloid disorders, mutational analysis has gained importance as a diagnostic tool. However, reports on its utility on trephine bone marrow biopsies (BMB) are sparse. The aim of our proof of principle study was to determine the suitability of targeted sequencing for the longitudinal evaluation of cytopenia and monocytosis and demonstration of clonal evolution on sequential BMB. Seventy-seven EDTA-decalcified BMB of 33 patients with peripheral cytopenia and/or monocytosis, including at least one follow-up biopsy/patient, were included. Initial morphological diagnoses were idiopathic cytopenia of undetermined significance (ICUS, 8 cases), MDS (without blast increase, 7 cases), MDS with increased blasts/excess blasts (MDS-IB/EB) (11 cases), and CMML (7 cases). Thirty-one genes relevant for myeloid disorders were examined using two custom AmpliSeq NGS panels. Mutations were found in the initial BMB of 5/8 cases of ICUS, thus changing the diagnosis to clonal cytopenia of unknown significance (CCUS), 5/7 MDS, 10/11 MDS-IB/EB, and 7/7 CMML. Clonal evolution was observed in 14/33 (42%) cases, mostly associated with disease progression. None of the wild-type patients acquired mutations during follow-up. NGS-based mutation profiling is a robust diagnostic tool for BMB and provides valuable additional information, especially for cases with no/minimal dysplasia, and for better risk stratification of MDS. Tracking variant allele frequency and appearance of mutations over time allows for observing clonal evolution or relapse.

Development and validation of a nomogram for predicting internal mammary sentinel node metastasis in breast cancer patients

ObjectiveInternal mammary nodes are important in breast cancer prognosis, but their diagnosis is often missed in clinical practice, leading to inaccurate staging and treatment. We developed a validated nomogram to predict the presence of internal mammary sentinel nodes (IMSN) metastasis. MethodsA total of 864 sequential IMSN biopsy procedures from a prospective studies database of 1505 cases were used for model development and validation. Multivariable logistic regression was performed on 519 sequential IMSN biopsy procedures from multi-center data between August 2018 and July 2022 to predict the presence of IMSN metastasis. A nomogram was developed based on the logistic regression model and subsequently applied to 345 sequential IMSN biopsy procedures from single-center data between November 2011 and July 2018. The model's discrimination was assessed using the area under the receiver operating characteristic curve. ResultsThe overall frequency of IMSN metastasis was 17.0% in our study. A predictive model for IMSN metastasis was constructed using tumor size, tumor location, lymphovascular invasion, the number of positive axillary nodes (P < 0.05 for all variables in multivariate analysis), and histological grade (P < 0.05 only in univariate analysis). The nomogram was accurate, with a concordance index of 0.84 in the bootstrapping analysis and an area under the receiver operating characteristic curve of 0.80 in the validation population. ConclusionOur nomogram provides an accurate and validated multivariable predictive model for estimating the individual likelihood of having IMSN metastasis. This may be useful for personalized treatment decisions regarding internal mammary radiotherapy in breast cancer patients.

Variants in FGF10 cause early onset of severe childhood interstitial lung disease: A detailed description of four affected children.

Fibroblast growth factor 10 (FGF10) is a signaling molecule with a well-established role for lung branching morphogenesis. Rare heterozygous, deleterious variants in the FGF10 gene are known causes of the lacrimo-auriculo-dento-digital (LADD) syndrome and aplasia of lacrimal and salivary glands.Previous studies indicate that pathogenic variants in FGF10 can cause childhood Interstitial Lung Disease (chILD) due to severe diffuse developmental disorders of the lung, but detailed reports on clinical presentation and follow-up of affected children are lacking. We describe four children with postnatal onset of chILD and heterozygous variants in FGF10, each detected by exome or whole genome sequencing. All children presented with postnatal respiratory failure. Two children died within the first 2 days of life, one patient died at age of 12 years due to right heart failure related to severe pulmonary hypertension (PH) and one patient is alive at age of 6years, but still symptomatic. Histopathological analysis of lung biopsies from the two children with early postpartum demise revealed diffuse developmental disorder representing acinar dysplasia and interstitial fibrosis. Sequential biopsies of the child with survival until the age of 12 years revealed alveolar simplification and progressive interstitial fibrosis. Our report extends the phenotype of FGF10-related disorders to early onset chILD with progressive interstitial lung fibrosis and PH. Therefore, FGF10-related disorder should be considered even without previously described syndromic stigmata in children with postnatal respiratory distress, not only when leading to death in the neonatal period but also in case of persistent respiratory complaints and PH.

BL-MOL-AR Project, Preliminary Results about Liquid Biopsy: Molecular Approach Experience and Research Activity in Oncological Settings.

Background Liquid biopsy is mainly used to identify tumor cells in pulmonary neoplasms. It is more often used in research than in clinical practice. The BL-MOL-AR study aims to investigate the efficacy of next-generation sequencing (NGS) and clinical interpretation of the circulating free DNA (cfDNA) levels. This study reports the preliminary results from the first samples analyzed from patients affected by various neoplasms: lung, intestinal, mammary, gastric, biliary, and cutaneous. Methods The Biopsia Liquida-Molecolare-Arezzo study aims to enroll cancer patients affected by various malignancies, including pulmonary, intestinal, advanced urothelial, biliary, breast, cutaneous, and gastric malignancies. Thirty-nine patients were included in this preliminary report. At time zero, a liquid biopsy is executed, and two types of NGS panels are performed, comprising 17 genes in panel 1, which is already used in the routine tissue setting, and 52 genes in panel 2. From the 7th month after enrollment, 10 sequential liquid biopsies are performed up to the 17th month. The variant allele frequency (%) and cfDNA levels (ng/mL) are measured in every plasmatic sample. Results The NGS results obtained by different panels are similar even though the number of mutations is more concordant for lung pathologies. There are no significant differences in the actionability levels of the identified variants. Most of the molecular profiles of liquid biopsies reflect tissue data. Conclusions Preliminary data from this study confirm the need to clarify the limitations and potential of liquid biopsy beyond the lung setting. Overall, parameters related to cfDNA levels and variant allele frequency could provide important indications for prognosis and disease monitoring.

Clinical activity and exploratory resistance mechanism of milademetan, an MDM2 inhibitor, in intimal sarcoma with MDM2 amplification: an open-label phase 1b/2 study.

Intimal sarcoma is an extremely rare life-threatening malignant neoplasm. Murine double minute 2 (MDM2) amplification is observed in > 70% of intimal sarcomas. Milademetan, an MDM2 inhibitor, may provide a clinical benefit in this patient population. We conducted a phase 1b/2 study in patients with MDM2-amplified wild-type TP53 intimal sarcoma as a sub-study of a large nationwide registry for rare cancers in Japan. Milademetan (260 mg) was administered orally once daily for three days every 14 days, twice in a 28-day cycle. Of 11 patients enrolled, 10 were included in the efficacy analysis. Two patients (20%) showed durable responses for >15 months. Anti-tumor activity correlated with TWIST1 amplification (P = 0.028) and negatively with CDKN2A loss (P = 0.071). Acquired TP53 mutations were detected in sequential liquid biopsies as a novel exploratory resistance mechanism to milademetan. These results suggest that milademetan could be a potential therapeutic strategy for intimal sarcoma.

Exploratory novel biomarker and resistance mechanism of milademetan, an MDM2 inhibitor, in amplified MDM2 intimal sarcoma from an open-label phase 1b/2 trial (NCCH1806/MK004).

11544 Background: Amplified Murine double minute 2 ( MDM2) is found in &gt; 70% of intimal sarcoma, known as one of the ultra-rare sarcomas. Milademetan (DS3032, RAIN-32) is a novel, specific, small-molecule MDM2 inhibitor that disrupts MDM2 and the tumor suppressor protein p53 interactions in tumor cells. We conducted a phase 1b/2 trial (Trial registration No: JMA-IIA00402) in patients with amplified MDM2 wild-type TP53 intimal sarcoma as a sub-study under the nationwide large registry for rare cancers in Japan (MASTERKEY Project). Eleven patients were enrolled, and ten were included in the efficacy analysis. Two (20%) patients had durable responses for &gt; 15 months. Milademetan provided clinical benefits in patients with amplified MDM2 intimal sarcoma. Predictive biomarkers other than amplified MDM2 and acquired resistance mechanisms for milademetan are unknown. Methods: Whole-exome and RNA sequencing analyses of pre-treatment tissue samples were conducted to identify determinants of response. Genomic alterations were analyzed for 10 patients, and gene expression was analyzed for 9 patients using their pre-treatment tissue samples. Targeted sequencing of cell-free DNA (cfDNA) samples (liquid biopsy) was also conducted sequentially at three points [before treatment with milademetan (baseline), at Cycle 2 Day1, and at the time of disease progression] to identify determinants of response and resistance. Results: From whole-exome and RNA sequencing analyses of pre-treatment tissue samples, we could not find any molecular pathways associated with the anti-tumor activity of milademetan. Focusing on 8 genes ( CDK4, CDKN2A, CDKN2B, EGFR, ERBB3, MDM2, PDGFRA, TP53) known to be frequently affected in intimal sarcoma and 10 genes ( AKT1, ATM, BBC3, CDKN1A, CDKN1C, CHEK2, MDM4, PMAIP1, PPM1D, TWIST1) reported to be associated with MDM2 inhibitor responses; we found that anti-tumor activity correlated with amplified TWIST1 ( p-value = 0.028) and negatively with CDKN2A loss ( p-value = 0.071). Eight of the 10 patients had their cfDNA collected sequentially at baseline, at Cycle 2 Day1 and disease progression; however, one did not consent to the exploratory analysis study using cfDNA, and one had cfDNA collected at baseline but not at disease progression due to ongoing treatment. Of the eight patients, TP53 mutations in cfDNA were detected in one and five patients at baseline and disease progression, respectively. The cfDNA allele frequency of TP53 mutations increased with disease progression. Conclusions: CDKN2A loss and amplified TWIST1 could be associated with the anti-tumor activity of milademetan in patients with amplified MDM2 intimal sarcoma. Acquired TP53 mutations were detected in sequential liquid biopsies as loss-of-function mutations, and these TP53 mutations might compromise the anti-tumor activity. Clinical trial information: JMA-IIA00402 .

Initial results from the phase 2A trial of visugromab (CTL-002) + nivolumab in advanced/metastatic anti-PD1/-L1 relapsed/refractory solid tumors (The GDFATHER-TRIAL).

2501 Background: Increasing evidence has emerged that Growth and Differentiation Factor 15 (GDF-15) plays a critical role in tumoral immunosuppression. Apart from blocking immune-cell entry into the tumor microenvironment, GDF-15 also has a major impact on the formation of the immune synapse. Many tumors overexpress GDF-15 and have hijacked this mechanism to block immunotherapy success. Here we report initial results of the phase 2a part of the GDF-15 neutralizing antibody visugromab (CTL-002) in combination with nivolumab. Methods: At cut-off date (06-Feb-2023), a total of 79 pts with advanced/metastatic solid tumors have been enrolled and were treated with visugromab 10 mg/kg Q2W in combination with nivolumab 240 mg Q2W in the phase 2a expansion part of the CTL-002-001 phase 1/2 trial (NCT04725474). The primary endpoint of the phase 2a expansion is the Objective Response Rate (ORR) according to RECIST v1.1. Tumor assessments were performed every 8 weeks and patients had to be relapsed/refractory to a minimum of 12 weeks continuous prior anti-PD1/-PD-L1-containing therapy, with relapse or progression after initial response to have occurred on continued anti-PD1/PDL1 therapy. Phase 2a contained several indication-specific expansion cohorts (e.g. NSCLC, Bladder Cancer, HCC, Melanoma), following a Simon 2-stage design, and two cohorts were already expanded to stage 2 in light of initial responses observed. An additional expansion cohort is a pan-tumor basket cohort with sequential tumor biopsies for biomarker analyses. A key component of this multi-arm trial is an extensive translational research program to identify response-predictive tumor and immunologic marker and pattern. Results: Across several expansion cohorts, durable tumor regressions were observed, including several partial responses (PR) and one complete response (CR) in heavily pretreated patients with a median of 3 prior lines of systemic therapy for advanced/metastatic cancer, including at least one prior line of anti-PD1/-PD-L1 therapy and being relapsed/refractory to it. In NSCLC, so far 13 pts underwent at least one tumor assessment. Of these, two experienced a PR (one confirmed), three additional tumor shrinkage &gt; 5% (TS) and one longer-term SD (LT-SD) were seen. In bladder cancer, 7 patients underwent at least one tumor assessment with 1 confirmed CR, 1 TS and 1 LT-SD. The treatment was overall well tolerated. Conclusions: These initial phase 2a results demonstrate promising clinical activity for the combination of visugromab and nivolumab in heavily pretreated anti-PD1/-PD-L1 refractory/relapsed solid tumor patients across various indications, including induction of confirmed partial and complete responses. Extensive biomarker evaluations are ongoing to further evaluate potential response-predictive markers identified during phase 1 development. Clinical trial information: NCT04725474 .

Mitochondrial respiration analysis in permeabilized porcine left ventricular and human right atrial specimens with ischemia-reperfusion.

Mitochondrial function is critical to myocardial ischemia-reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, mitochondrial function can be measured in permeabilized myocardial tissue (PMT) specimens of about 2-5 mg, which are retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. We attempted to validate measurements of mitochondrial respiration from PMT by comparison with those from isolated mitochondria of left ventricular myocardium from anesthetized pigs undergoing 60 min coronary occlusion and 180 min reperfusion. Mitochondrial respiration was normalized to the content of mitochondrial marker proteins [cytochrome-c oxidase 4 (COX4), citrate synthase, and manganese-dependent superoxide dismutase]. When normalized to COX4, mitochondrial respiration measurements in PMT and isolated mitochondria agreed well in Bland-Altman plots (bias score, -0.03 nmol/min/COX4; 95% confidence interval: 6.31 nmol/min/COX4 and -6.37 nmol/min/COX4) and correlated well (slope of 0.77 and Pearson's R of 0.87). Mitochondrial dysfunction by ischemia-reperfusion was equally reflected in PMT and isolated mitochondria (44 and 48% reduction of ADP-stimulated complex I respiration). Also in isolated human right atrial trabeculae, simulation of ischemia-reperfusion injury by exposure to 60 min hypoxia and 10 min reoxygenation reduced mitochondrial ADP-stimulated complex I respiration by 37% in PMT. In conclusion, mitochondrial function measurements in permeabilized cardiac tissue can substitute for that in isolated mitochondria to reflect mitochondrial dysfunction following ischemia-reperfusion.NEW & NOTEWORTHY Methods to quantify mitochondrial function in translationally relevant models and in human tissue are needed to improve the translation of cardioprotection to patients. Our present approach, using PMT instead of isolated mitochondria for the quantification of mitochondrial ischemia-reperfusion damage, provides a reference for further studies in translationally relevant large animal models and in human tissue, thus possibly improving the translation of cardioprotection to the benefit of patients with acute myocardial infarction.