Tissue Biopsy Research Articles

High-purity CTC RNA sequencing identifies prostate cancer lineage phenotypes prognostic for clinical outcomes.

The development of treatment resistance remains universal for patients with metastatic prostate cancer, driven by AR alterations and lineage state transitions. Identifying the evolution of lineage transitions in treatment resistance has been limited by the challenges of collecting serial tissue biopsies on treatment, which can be overcome using blood-based liquid biopsies. Utilizing a novel circulating tumor cell (CTC) isolation approach, we collected 273 CTC samples from 117 patients with metastatic prostate cancer for RNA sequencing. 146 samples from 70 patients had tumor purity comparable to tissue biopsies. We identified four CTC transcriptional phenotypes, mirroring lineage states identified in tissue. Patients with a luminal-B-like CTC phenotype defined by persistent AR signaling and high proliferation, as well as those with a neuroendocrine CTC phenotype, had significantly shorter survival than patients with luminal-A-like and low proliferation phenotypes. In a prospective substudy, pre-treatment CTC luminal-B-like phenotype was associated with early progression on 177Lu-PSMA-617.

High and low dose radiotherapy combined with ICIs for MSS colorectal cancer patients with liver metastases: a phase I study (HaRyPOT)

IntroductionMicrosatellite stable (MSS) colorectal cancer with liver metastases (CLRM) responds poorly to immunotherapy, and various approaches to break immune tolerance have been tried. Radiotherapy in combination with immune checkpoint inhibitors is one of promising therapies, and the choice of radiotherapy and immunotherapy modalities is also a hot issue.MethodsHere, we report on a Phase I trial treating nine patients with MSS CLRM using a combination of high and low dose radiotherapy and immune checkpoint inhibitors (ICIs).ResultsThe primary endpoint of the trial was the safety and tolerability of this combination treatment modality. Secondary endpoints included the objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). The study results showed that at three dose levels—single doses of 6Gy (n=3), 8Gy (n=3), and 10Gy (n=3)—the most common treatment-related adverse events (TRAEs) were nausea, vomiting, fatigue, and abnormal liver function. At the first condition assessment, four patients were observed to have stable disease (SD) and one patient achieved partial response (PR). In exploratory endpoint analyses, tissue biopsies and paired hematologic samples from patients showed M2 macrophage reduction. Plasma cytokines IL-10, IL-17, and INF-α increased after treatment with both drugs.DiscussionIn summary, this is the first clinical trial demonstrating the safety and immunogenic activity of combined high and low dose radiotherapy with ICIs in MSS colorectal cancer liver metastases (CRLMs). The combination therapy stimulated the immune response and altered the tumour microenvironment, warranting further exploration in the future.

Unilateral hypertrophy of the tensor fascia lata muscle: a need to revisit and review.

Unilateral hypertrophy of the tensor fascia lata muscle is a rare phenomenon with a variety of aetiological causes. It may mimic soft tissue sarcoma, which can lead to misdiagnosis and unnecessary invasive procedures. A retrospective review was conducted of all adult patients referred to a statewide sarcoma service lata between January 2010 to June 2023 with confirmed unilateral hypertrophy of the tensor fascia. Their medical records and imaging were reviewed to identify the likely cause. All patients' histories, clinical findings and radiological investigations were reviewed by subspecialised radiologists and orthopaedic surgeons. We identified nine patients with unilateral hypertrophy of the tensor fascia lata muscle. Conservative treatment was recommended to all patients, however one patient who experienced hip pain pursued surgical excision of the tensor fascia lata at another centre, with ongoing pain in the region post-operatively. With ample awareness of the diagnosis and the imaging findings, this condition can be diagnosed based on history, examination and magnetic resonance imaging. Tissue biopsy and surgical excision are not indicated.

Liquid biopsies for early detection and monitoring of cancer: advances, challenges, and future directions

Abstract The article offers an extensive survey of the progressions, problems, and future trends of liquid biopsies in the early discovery and surveillance of cancer. Liquid biopsies can detect signals associated with cancer by looking at biological fluids like cerebrospinal fluid, blood, or urine, making them a less invasive alternative to traditional tissue biopsies. Methods: The review explores the molecular biology and techniques behind liquid biopsy, including circulating tumor DNA (ctDNA), circulatory tumor cells (CTCs), and exosomes. It evaluates clinical applications of liquid biopsy across different cancer types, showing their potential for early diagnosis, monitoring disease progression, and therapy response prediction. Results: The article identifies several critical issues with liquid biopsies, including achieving a balance between high sensitivity and specificity, standardizing protocols, addressing technological heterogeneity, and ensuring cost-effectiveness and accessibility. Also, ethical issues about informed consent, data privacy, incidental findings management, and equal testing access have been examined in this context. Conclusion: Finally, this article sheds light on future developments in liquid biopsies, such as enhanced specificity, sensitivity, and integration with artificial intelligence methods.

Circulating Tumor DNA as Measurable Residual Disease in Aggressive B-Cell Lymphoma: A Narrative Review.

Achieving remission is the first step toward a cure in treating aggressive B-cell lymphomas. Radiographic imaging, such as fluorodeoxyglucose-positron emission tomography/computed tomography scans are the current standard to define remission at the end of therapy but lack specificity for lymphoma and cannot detect disease at the molecular level. Identifying measurable residual disease with ultrasensitive detection of circulating tumor DNA potentially offers the possibility of improving clinical outcomes. Early studies of circulating tumor DNA in aggressive B-cell lymphomas showed a strong association with overall tumor burden, and baseline quantitative levels are associated with clinical outcomes after frontline chemotherapy. Next-generation sequencing methods that detect lymphoma-relevant genetic aberrations in circulating tumor DNA can also be used for noninvasive genotyping that strongly mirror tissue biopsies. Rapid changes in circulating tumor DNA dynamics after 1 or 2 cycles of frontline chemotherapy or within weeks of treatment with chimeric antigen receptor T-cell salvage therapy are also highly prognostic. Although serial monitoring of circulating tumor DNA can detect molecular relapse 3 to 6 months before clinical relapse, improved analytical thresholds are required to detect measurable residual disease at a singular point at the end of therapy. Modern advances in circulating tumor DNA methods now allow for the reliable detection of measurable residual disease, with an analytical detection threshold of 1 in 1 million cell-free DNA molecules. The results of this review suggest that modern ultrasensitive methods of detecting circulating tumor DNA may improve the current definition of remission in aggressive B-cell lymphomas. Incorporating circulating tumor DNA at the end of therapy assessment identifies patients who do not require surveillance monitoring and introduces paradigms of treating measurable residual disease within clinical trials. Practical barriers, including standardization of collection, availability, turnaround times, and cost, remain hurdles preventing widespread implementation into clinical practice.

Kidney Fibrosis In Vitro and In Vivo Models: Path Toward Physiologically Relevant Humanized Models.

Chronic kidney disease (CKD) affects over 10% of the global population and is a leading cause of mortality. Kidney fibrosis, a key endpoint of CKD, disrupts nephron tubule anatomy and filtration function, and disease pathomechanisms are not fully understood. Kidney fibrosis is currently investigated with in vivo models, that gradually support the identification of possible mechanisms of fibrosis, but with limited translational research, as they do not fully recapitulate human kidney physiology, metabolism, and molecular pathways. In vitro 2D cell culture models are currently used, as a starting point in disease modeling and pharmacology, however, they lack the 3D kidney architecture complexity and functions. The failure of several therapies and drugs in clinical trials highlights the urgent need for advanced 3D in vitro models. This review discusses the urinary system's anatomy, associated diseases, and diagnostic methods, including biomarker analysis and tissue biopsy. It evaluates 2D and in vivo models, highlighting their limitations. The review explores the state-of-the-art 3D-humanized in vitro models, such as 3D cell aggregates, on-chip models, biofabrication techniques, and hybrid models, which aim to mimic kidney morphogenesis and functions. These advanced models hold promise for translating new therapies and drugs for kidney fibrosis into clinics.

Unveiling the intriguing relationship: oncogenic KRAS, morphological shifts, and mutational complexity in pancreatic mucinous cystic neoplasms.

Pancreatic ductal adenocarcinoma (PDAC) often arises from preexisting cystic lesions such as intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN). This study investigated the molecular heterogeneity and mutational landscape of MCN in relation to PDAC, highlighting the significance of KRAS mutations in tumor progression. Utilizing targeted next-generation sequencing on low-grade MCN and invasive PDAC samples, we identified a substantial overlap in mutational profiles, particularly mutations in KRAS, TP53, and FBXW7. Specifically, 69.2% of MCN exhibited somatic mutations, with KRAS mutations being a predominant oncogenic driver. The characterization of mutant versus wildtype KRAS variant allele frequencies (VAF) indicated higher mutation levels in PDAC compared to MCN, suggesting an evolutionary trajectory toward malignancy. Further histological analysis of 12 additional MCN cases revealed significant intratumor heterogeneity, with variant KRAS mutation distributions correlating with distinct cellular morphologies and dysplastic features. Additionally, we explored the potential of liquid biopsies, demonstrating a concordance rate of 71.4% for KRAS mutation detection in circulating tumor DNA (ctDNA) relative to tissue biopsies across cohorts. Our findings underscore the relevance of evaluating KRAS mutations-herein referred to as VAF per microdissected region-as they relate to histopathological markers of dysplasia, contributing to improved stratification of pancreatic lesions and facilitating personalized treatment strategies. In conclusion, this comprehensive analysis of MCN highlights the importance of KRAS as a crucial biomarker for both malignant progression and therapeutic decision-making in pancreatic pathology. Ultimately, our study suggests that characterizing the mutational landscape and histological features of MCN can enhance early detection and intervention strategies for at-risk patients. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Investigating the clinical significance of E2F5 expression in circulating extracellular vesicles in prostate carcinoma.

Prostate-specific antigen (PSA) based screening strategy has caused a marked improvement in prostate cancer detection and reduction in associated mortality. However, its specificity and sensitivity is not optimal for differentiating different forms of prostate cancer, resulting in overtreatment of indolent tumors. E2F5, a member of the family of transcription factors plays essential roles during many cellular processes. E2F5 amplification is a major event in PCa. A liquid biopsy is a minimally invasive procedure to investigate the cancer-related molecules in circulating tumor cells (CTCs), cell-free DNA, and extracellular vesicles (EVs). Serum and plasma are attractive sources of EV-based biomarkers as blood sample acquisition is a minimally invasive procedure. Given, the shortcomings of PSA as a serum marker, the suitability of E2F5 expression and chemical properties of EVs can be used for the diagnosis of clinically significant prostate cancer. To technically refine the current histopathology-based diagnosis of PCa by adding it to the molecular readouts specific to disease states and biochemical fingerprints of EVs. The study included 100 TRUS-biopsied tissues, 50 representing 4 Gleason grades and another 50 representing BPH. Expression of E2F5 was studied in all the qualified tissues by Immunohistochemistry (IHC). Blood from patients was collected and EVs were isolated and characterized. E2F5 was highly upregulated through all Gleason grades, the maximum being in Gleason 10(5 + 5) and the minimum in Gleason 7(3 + 4), as compared to BPH. The EVs isolated from blood plasma were within 30-200 nm in size. E2F5, being a transcription factor, is highly overexpressed in malignant biopsy tissues, through all Gleason grades. BPH tissues served as control samples. EVs from blood plasma might serve as a potential liquid biopsy marker for predicting disease progression and better prognosis.

Circular RNA microarray expression profile and potential function of circDOCK1 in colorectal cancer

IntroductionEndoscopic tissue biopsy combined with histopathology is the gold standard for the diagnosis of colorectal cancer (CRC); however, the invasive nature of this procedure hinders its acceptance by patients. Therefore, there exists a critical need to identify novel markers facilitating early CRC detection and prognosis. Circular RNAs (circRNAs) hold promise as novel clinical diagnostic markers. This study aimed to investigate the impact of circDOCK1 on CRC metastasis and prognosis as well as its underlying molecular mechanisms.MethodsWe explored circRNA expression profiles in four pairs of CRC tissues and adjacent non-carcinoma tissues via microarray analysis. After Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and circRNA–miRNA network analyses, circDOCK1 was chosen for further investigation. We evaluated its clinical relevance in 80 CRC tissue pairs and adjacent controls, correlating circDOCK1 expression with clinical characteristics. Follow-up data from patient telephone interviews were analyzed for survival outcomes. Transfection efficiency was confirmed via qRT-PCR in HCT116 and SW480 colon cells, and the effects of circDOCK1 on cell proliferation, migration, and invasion were assessed.ResultsMicroarray data revealed 149 significantly differentially expressed circRNAs, including 71 upregulated and 78 downregulated circRNAs, in CRC tissues. CircDOCK1 exhibited elevated expression in patients with CRC and emerged as an independent prognostic factor. Kaplan–Meier curve analysis suggested that circDOCK1 expression is an unfavorable prognostic factor in patients with CRC. In vivo experiments revealed that circDOCK1 overexpression enhanced the proliferation, migration, and invasion of CRC cells, with consistent results upon circDOCK1 downregulation.ConclusionThese data indicate that circDOCK1 may play a role in promoting the proliferation, migration, and invasion of CRC cells, suggesting its potential as a CRC biomarker.

The Promise of Infrared Spectroscopy in Liquid Biopsies for Solid Cancer Detection

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy has shown significant promise in the context of liquid biopsy, offering a potential tool for cancer diagnostics. Unlike traditional tissue biopsies, which may not fully capture the clonal heterogeneity of tumors, liquid biopsy reflects the dynamic state of the disease and its progression more comprehensively. Biofluids such as serum and plasma are low-cost, minimally invasive diagnostic media with well-established clinical uses. This review assesses the use of ATR-FTIR spectroscopy to detect biochemical changes in biofluids linked to various malignancies, including breast, ovarian, endometrial, prostate, bladder, kidney, pancreatic, colorectal, hepatic, esophageal, gastric, lung, and brain cancers. While ATR-FTIR offers the advantages of rapid, minimally invasive detection and real-time disease monitoring, its integration into clinical practice faces challenges, particularly in terms of reproducibility due to variability in sample preparation, spectral acquisition, and data processing. The translation of ATR-FTIR into routine diagnostics will require validation through large-scale cohort studies and multicenter trials to ensure its clinical reliability and effectiveness.

Crohn's Disease With Latent Tuberculosis Infection or Intestinal Tuberculosis: Rapid Discrimination by Targeted Next-Generation Sequencing.

Discriminating Crohn's disease (CD) with latent tuberculosis infection (LTBI) from intestinal tuberculosis (ITB) in tuberculosis-endemic regions remains challenging. To assess whether targeted next-generation sequencing (tNGS) could be an efficient method for ITB diagnosis and discrimination from CD with LTBI. The study was conducted prospectively from September 2020 until December 2023. We recruited patients with undetermined intestinal ulcers and positive interferon-gamma release assay. We compared tNGS (using fresh biopsy tissue samples from ulcer bases) to pathological detection of caseous necrotising granuloma, acid-fast bacillus (AFB) staining, tuberculosis polymerase chain reaction (TB-PCR) for diagnostic efficiency. ITB was diagnosed based on cure by anti-tuberculosis therapy and comprehensive clinical evaluation. Of the 100 patients included, 66 had ITB and 34 had CD with LTBI. The sensitivity, specificity, positive predictive value and negative predictive value of tNGS for ITB were 83% (72%-91%), 100% (87%-100%), 100% (92%-100%) and 76% (60%-87%), respectively. TNGS had significantly higher diagnostic sensitivity than AFB staining [15% (4%-39%), p < 0.05], TB-PCR [22% (12%-36%), p < 0.05] and detection of caseous necrotising granulomas [17% (9%-28%), p < 0.05]. The models combining multiclinical factors increased sensitivity (97% vs. 83%) than tNGS alone. No patients with CD and LTBI had positive tNGS. TNGS using fresh biopsy tissue from ulcer bases is highly sensitive and specific, with superiority over other traditional diagnostic methods for ITB detection. TNGS could facilitate rapid diagnosis of ITB and discrimination from CD with LTBI, particularly in high TB-endemic countries.

Identification of Somatic Genetic Variants in Superficial Vascular Malformations by Liquid Biopsy in a Cohort of 88 Patients from a French Hospital.

Superficial vascular anomalies are complex disorders characterized by abnormal vascular growth. Next-generation sequencing has recently identified somatic genetic alterations associated with these malformations, offering new insights for targeted treatments. However, tissue biopsies for genetic testing can be invasive and difficult to obtain, especially in arteriovenous malformations (AVM) with hemorrhagic risks. A liquid biopsy, a non-invasive approach, offers a promising solution by detecting genetic mutations in cell-free DNA. This pilot study aimed to evaluate the feasibility of using a liquid biopsy for the genetic analysis of patients with superficial vascular anomalies through cell-free DNA sampling. Additionally, it explored whether specific sampling sites, such as the afferent artery, nidus, and efferent vein, could enhance the sensitivity of detecting pathogenic variants in patients with AVM. A total of 88 patients were enrolled, including 55 with AVM and 33 with lymphatic malformations. For patients with AVM, cell-free DNA samples were collected from peripheral blood, efferent veins, afferent arteries, and the AVM nidus. In patients with lymphatic malformations, cystic lymphatic fluid was collected by a direct puncture during diagnostic procedures. A molecular analysis was performed using a targeted gene panel relevant to somatic alterations in solid tumors. Pathogenic variants were validated by digital polymerase chain reaction for patients with lymphatic malformations. Pathogenic variants were identified in 23.6% of patients with AVM, predominantly in MAP2K1 and KRAS genes, with higher sensitivity near the AVM nidus. In addition, pathogenic variants were identified in 27.3% of patients with lymphatic malformations, all involving the PIK3CA gene. Despite the lower sensitivity of a cell-free DNA analysis compared with a tissue biopsy, especially in patients with AVM, the detection rate suggests the utility for a cell-free DNA analysis, particularly when a tissue biopsy is not feasible. This study confirms the feasibility of using a cell-free DNA liquid biopsy for genotyping patients with superficial vascular anomalies, although a tissue biopsy remains the gold standard for comprehensive genetic profiling because of its higher sensitivity. A liquid biopsy offers a non-invasive option for molecular analysis that is useful as a preliminary or alternative approach when direct tissue sampling is not possible. Importantly, the sensitivity of cell-free DNA sampling in AVM appeared highest when obtained close to the nidus, indicating an optimal sampling location for future studies. Further research is needed to improve detection sensitivity, especially for samples taken near the nidus, to validate and strengthen these findings.Although our study focused on superficial/extra-cranial AVM, further research should assess the applicability of this approach to cerebral AVM, where a tissue biopsy is particularly risky.

Short-Term Metformin Protects Against Glucocorticoid-Induced Toxicity in Healthy Subjects: A Randomized, Double-Blind, Placebo-Controlled Trial.

Glucocorticoids (GCs) are potent anti-inflammatory drugs, but strategies to prevent side effects are lacking. We investigated whether metformin could prevent GC-related toxicity and explored the underlying mechanisms. This single-center, randomized, placebo-controlled, double-blind, crossover trial compared metformin with placebo during high-dose GC treatment in 18 lean, healthy, male study participants. The trial was conducted at the University Hospital Basel, Switzerland. Participants received prednisone 30 mg/d in combination with metformin or placebo for two 7-day periods (1:1 randomization). The primary outcome, change in insulin sensitivity, was assessed using a two-sided paired t test. Before and after each study period, we conducted a mixed-meal tolerance test, blood metabolomics, and RNA sequencing of subcutaneous adipose tissue biopsy specimens. Metformin improved insulin sensitivity as assessed by the Matsuda index (n = 17; mean change: -2.73 ± 3.55 SD for placebo, 2.21 ± 3.95 for metformin; mean difference of change -4.94 [95% CI, -7.24, -2.65)]; P < 0.001). Metabolomic and transcriptomic analyses revealed that metformin altered fatty acid flux in the blood and downregulated genes involved in fatty acid synthesis in adipose tissue. Metformin reduced markers of protein breakdown and bone resorption. Furthermore, metformin downregulated genes responsible for AMPK inhibition and affected GLP1 and bile acid metabolism. Metformin prevents GC-induced insulin resistance and reduces markers of dyslipidemia, myopathy, and, possibly, bone resorption through AMPK-dependent and -independent pathways.

Immunotherapy engagement in pancreatic adenocarcinoma: Provisional results of iLSTA study— Durvalumab, LSTA1 (certepetide), gemcitabine, and nab-paclitaxel for locally advanced pancreatic ductal adenocarcinoma.

746 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterised by a dense, extracellular matrix-rich stroma, which creates a physical barrier against drug penetration. Evidence suggests that the iRGD peptide, LSTA-1, can increase the penetration of anti-cancer drugs to tumours through selective targeting of tumour endothelial cell receptors. Additionally, LSTA-1 administration promotes CD8+ immune cell tumour infiltration, potentially enabling immunotherapy as a treatment modality. iLSTA examined safety, tolerability and effect of the combination. Methods: Participants diagnosed with locally advanced PDAC were divided into 3 cohorts in a 1:1:4 ratio. Cohort 1 (n=5) received gemcitabine (1000mg/m 2 ), nab-paclitaxel (125mg/m 2 ), placebo LSTA-1 (3.2mg/kg) and placebo durvalumab (750mg). Cohort 2 (n=5) received gemcitabine, nab-paclitaxel, active and placebo LSTA-1, and placebo durvalumab. Cohort 3 (n=20) received gemcitabine, nab-paclitaxel, LSTA-1 and durvalumab. Tissue biopsies via endoscopic ultrasound were obtained pre-treatment and between weeks 12 and 16 for analysis of tumour infiltrating lymphocytes (TILs). Patient tumour sizes were assessed every 8 weeks using radiological imaging according to RECIST v1.1, and serum CA19-9 levels were analysed monthly. Results: 6/17 patients showed significant RECIST partial response after 2 cycles of treatment (5 patients in cohort 3), with the remaining 11 patients exhibiting stable disease. After 4 cycles of treatment, 10/17 patients demonstrated partial response (9 patients in cohort 3). Of the remaining 7 patients, 6 demonstrated stable disease, and 1 patient (cohort 2) exhibited a RECIST complete response. 13/17 patients who completed 4 treatment cycles showed a decrease in CA19-9 levels. 6 patients demonstrated &gt;90% reduction in CA19-9 (5 in cohort 3), with the remaining 7 patients showing a &gt;50% reduction in CA19-9 levels (5 in cohort 3). 12 patients underwent repeat biopsies 12-16 weeks post-treatment to assess TILs. 10 patients showed immune cell infiltration (5% to 50% stroma infiltration), 2 had no tumour found (Cohort 3). The combination was safe with no unexpected toxicities. Conclusions: The preliminary results of this study suggest that the combination of gemcitabine and nab-paclitaxel with LSTA-1 and durvalumab is safe and potentially induces tumour infiltrating lymphocytes and RECIST response in locally advanced pancreatic adenocarcinomas. Clinical trial information: ACTRN12623000223639 .

Managing Calciphylaxis: Insights from Real-World Cases at a Tertiary Academic Center.

Calciphylaxis is a rare and potentially fatal condition involving chronic, nonhealing wounds caused by microvascular calcification. There is currently no approved treatment for calciphylaxis, contributing to its devastating impacts on quality of life. In this case series, the authors highlight instances of potentially misdiagnosed calciphylaxis in a cohort of patients and emphasize the importance of accurate diagnosis through multidisciplinary management approaches.The authors report on three patients treated at a large tertiary academic center between March 2021 and March 2023. Consent was obtained from all patients, including for the use of patient images. The first and second patients, a 75-year-old man and 62-year-old woman, respectively, both underwent an extensive course of local wound care as well as sodium thiosulfate therapy. The third patient, a 61-year-old woman, was treated with a combination of medication adjustments, lymphedema therapy, pulse lavage treatment, and cadaveric skin substitute. Tissue biopsy was also obtained in two of the three subjects to aid in the diagnosis of calciphylaxis. Unfortunately, these procedures lead to diagnostic challenges and misadventures, highlighting the risks associated with biopsy of calciphylaxis.This case series demonstrates how correctly diagnosing calciphylaxis relies heavily on providers maintaining a high index of clinical suspicion based on patient history, presentation, and risk factors. The primary goals of treatment for calciphylaxis include appropriate wound care, management of infection and pain, and close monitoring.

Safety and efficacy of combining PD-L1 and CTLA-4 blockade with transarterial chemoembolization in intermediate-stage hepatocellular carcinoma: A phase II study.

609 Background: Transarterial chemoembolization (TACE) is the standard treatment for intermediate-stage hepatocellular carcinoma (HCC) defined as Barcelona Clinic Liver Cancer (BCLC) B, but the benefit of adding systemic therapy remains unclear. While durvalumab and tremelimumab are approved for advanced HCC, the safety and efficacy in combination with TACE for intermediate-stage disease is undefined. Combining these PDL1 and CTLA4 inhibitors with TACE may enhance anti-tumor activity by leveraging TACE’s proinflammatory effects while blocking immune evasion and reducing immune suppression. This phase IIb study evaluates the safety and efficacy of this combination in BCLC-B HCC. Methods: We enrolled patients with intermediate-stage HCC that are also unresectable and ineligible for transplant. All patients received drug-eluting bead TACE (DEB-TACE) followed by durvalumab (1500 mg Q4W) and tremelimumab (300 mg once). Durvalumab monotherapy was continued until disease progression or unacceptable toxicity, for a maximum of 13 cycles. Primary endpoints include objective response rate (ORR) based on mRECIST. Secondary endpoints were overall survival (OS), and safety/adverse events (AEs). The trial is designed to rule out ORR of 30%. We obtained 3 serial biopsies and plasma for participating patients prior and one week after TACE and after the administration of immunotherapy for correlative studies. Results: 21 patients were enrolled. Of these, 20 patients were considered evaluable after receiving at least one dose of TACE and immunotherapy. ORR was 55% (95% CI: 32%-77%, CR 10% (n=2), PR 45% (n=9), meeting the primary endpoint. Five patients had PD (25%) per mRECIST, of which two were unconfirmed on repeat imaging and continued treatment beyond progression. Three patients received curative intent treatment as the result of treatment: surgical resection (n=1) and transplant (n=2). At a median follow-up of 18.4 months, median OS was 28.8 mo (95% CI: 15.1, NA) and mPFS was 6.1mo (95% CI: 3.3, NA). Grade 3 or higher immune related adverse events were rash (n=2), diarrhea, myalgia, nausea, elevated AST/ALT. One patient developed multiple irAEs after first dose (myocarditis, myositis, pericarditis) and was taken off study. Conclusions: The combination of DEB-TACE with PD-L1/CTLA-4 blockade is safe and shows promising activity in patients with BCLC-B HCC. The ongoing immune analysis and biomarker studies on serial blood and tissue biopsies will provide further insights into the mechanistic effects of this combination approach and inform future therapeutic strategies. Clinical trial information: NCT03638141 .

Clinical relevance of tumor fraction assessment from circulating tumor DNA in metastatic colorectal cancer.

237 Background: Both tissue (TBx) and liquid (LBx) biopsies are indicated for detecting actionable alterations in metastatic colorectal cancer (mCRC). LBx is easier to obtain and often has faster turnaround time, but its informative results depend on the ctDNA tumor fraction (TF) content. We aimed to (1) evaluate the concordance between LBx and TBx in CRC; and (2) determine baseline lab and clinical factors associated with TF. Methods: CRC patients (pts) with both tissue and liquid Foundation Medicine comprehensive genomic profiling within an interval of up to 90 days between samples collection were analyzed. Positive percent agreement (PPA) and negative predictive value (NPV) for RAS (KRAS/NRAS) and BRAFV600E detection were calculated with tissue as reference. Clinical data was obtained by the US-wide de-identified Flatiron Health-Foundation Medicine real-world clinicogenomic CRC database, from ~280 cancer clinics (~800 sites of care) between 1/2011-12/2023. mCRC pts with LBx collected up to 60 days prior to therapy (Tx) start were analyzed for association between TF and baseline factors using logistic regression with TF≥1% as the binary outcome. Baseline factors include age, ECOG, race/ethnicity, line of therapy, practice type, metachronous vs. synchronous disease, tumor side, albumin, alkaline phosphatase (AP), serum creatinine, hemoglobin, lactate dehydrogenase (LDH), neutrophil-to-lymphocyte ratio, opioid and steroid pre-Tx. Foundation Medicine’s ctDNA TF is a composite algorithm prioritizing aneuploidy at higher levels to avoid germline signal and prioritizing variant allele frequency of canonical alterations at lower levels to maximize dynamic range. Results: A total of 402 pts had TBx and LBx (268 [67%] TF≥1% and 134 [33%] TF&lt;1%). Without accounting for TF, the overall PPA for RAS and BRAFV600E was 77% and 83%, and the NPV was 65% and 99%. In LBx with TF≥1%, the PPA for RAS and BRAFV600E was 99% and 100%, and NPV was 98% and 100%. In contrast, for TF&lt;1% samples, the PPA for RAS and BRAFV600E was 37% and 50%, and NPV was 57% and 97%. Of the 633 pts with LBx collected prior to Tx, 474 (75%) had TF≥1%. TF≥1% was independently associated with ECOG 1+ (odds ratio [OR]: 1.58, p=0.038), community oncology care site (OR: 2.58, p=0.002), elevated levels of AP (OR: 3.00, p&lt;0.001) and LDH (OR: 3.97,p=0.002). TF≥1% was less likely to occur in right-side tumors (OR: 0.53, p=0.012) and metachronous disease (OR: 0.48, p&lt;0.001). Conclusions: Approximately 70% of LBx samples from CRC pts have TF≥1%, with near-perfect concordance for RAS/BRAFV600E. For LBx with TF&lt;1%, interpretation of negative results can be challenging due to low tumor shedding, especially for RAS mutations, where 43% may be false negatives and a subsequent TBx will provide more confidence in the negative results. Clinical factors such as right-sided tumors and metachronous disease may help anticipate low TF and guide decisions to pursue TBx.

Genomic profiling of matched tumor samples in patients with rectal cancer undergoing total neoadjuvant therapy.

213 Background: Total neoadjuvant therapy (TNT) is increasingly used for locally advanced rectal cancer. However, which patients benefit most from TNT remains to be elucidated. In this study we sequenced a TNT cohort with long-term follow-up to identify genomic correlates of TNT efficacy. Methods: Patients with locally advanced rectal cancer who underwent TNT followed by surgery (Oct 2016-June 2020) were identified at our institution. MSS patients with paired tissues (normal, biopsy, and surgery) with successful DNA extraction and sequencing were analyzed. Retrospective chart review collected patient demographics and clinical outcomes. Genomic analyses were performed with the R maftools package. Descriptive statistics were calculated with Fisher’s exact, Wilcoxon rank sum, and Wilcoxon rank sum exact tests. Univariate survival analyses (UVA) were calculated with Cox proportional hazards regressions. All statistical analyses were performed using R (v4.3.2). Results: 53 patients met inclusion criteria and 32 patients with paired tissues (64 samples) were obtained and successfully sequenced. All patients received chemotherapy, most commonly FOLFOX (81.3%) as part of TNT and a 5-FU based chemoradiation therapy (RT) regimen. Almost all patients received 50.4 Gy (93.8%). Median followup was 57 months (IQR: 47-68). There were 24 pathological responders (R) [complete/partial] and 8 nonresponders (NR) [stable disease/progression]. R vs NR were not statistically different across demographic or treatment characteristics. Local recurrences were infrequent (9.4% 3/32). No covariates were prognostic on UVA survival analyses. However, NR was associated with worse distant metastases free survival (DMFS) (HR = 4.11, CI 1.01-16.8, p = 0.048) on UVA. 5-year DMFS was 83% for R and 50% for NR, and the median DMFS was not reached for R, 41 mo for NR. Tumor mutation burden (TMB) did not change with treatment (pre = 4.5 vs post = 6.2 mutations/Mb; r, 1.7-10.3, p=0.59) and was not different on biopsy between R and NR (4.1 vs 5.9, p=0.09). Pre-treatment, the most mutated gene was APC (81%), [majority nonsense/frameshift (88%)], followed by TP53 (69%), [missense (59%)]. When comparing frequent mutations (n ≥ 5), PIEZO1 was more mutated in NR (63%, 5/8) than R (4% 1/24 patients) (p = 0.023 [multiple testing corrected]). Putative radioresistant KRAS-TP53 co-mutations were uncommon with similar representation in both groups (R 12.5%, 3/24, NR 12.5%, 1/8). Conclusions: Pathologic response positively correlated with DMFS in this cohort with long-term followup. PIEZO1 mutations were negatively prognostic for pathologic NR to TNT. This uncovers a potential new role in chemoRT resistance in addition to a previously reported association with metastatic spread. Further evaluation of PIEZO1 as a prognostic biomarker may help personalize appropriate treatment for patients with locally advanced rectal cancer.

Pembrolizumab and stereotactic radiotherapy combined in subjects with advanced HCC: A phase II study (PEMRAD).

602 Background: Combination approaches of immune-checkpoint inhibitors and locoregional therapies have an increasing role in advanced HCC. Methods: The PEMRAD phase II trial investigated the efficacy of combination pembrolizumab and stereotactic body radiation (SBRT) following progression on sorafenib. Patients with advanced HCC, ECOG 0-1 and Childs-Pugh A were eligible. Patients received pembrolizumab on day 1 followed by SBRT starting Day 2 and delivered in five fractions. Pembrolizumab was continued every 21 days until progression, unacceptable toxicity or patient withdrawal. The primary endpoint was objective response rate (ORR) as measured by RECISTv 1.1. hypothesizing an increase to 40% with the combination. Tissue and liquid biopsies were collected for correlative analyses including multiplex IHC, longitudinal CyTOF and dynamic cytokine changes. Results: The trial was stopped early due to a shift in the treatment landscape. Between March 2018 and March 2023 18 patients of a planned 22 patients were enrolled and treated with combination therapy. Of these 10 (55%) had evidence of macrovascular invasion (MVI) and 15 (83%) had extrahepatic disease; viral hepatitis was the underlying etiology in 50%. The ORR was 41% meeting the proposed endpoint. Median number of cycles of pembrolizumab was 7.5 (2-35) .The median number of lesions treated by SBRT was 3 (1-5) and the median dominant gross tumour volume was 184 cc (IQR 69-285). The median PFS was 5.4 (2.8-9.9) months and median OS 12.6 (5.7-25.8) months. Of the 10 patients with macrovascular invasion 5 (50%) had evidence of a vascular thrombus response by RECIST criteria including 1 complete response. Treatment related adverse events ≥ grade 3 were reported in 5 patients (28%). One death due to myocarditis was attributed to treatment. Conclusions: In a poor prognostic group of patients in the second line setting, the combination of SBRT and pembrolizumab demonstrated high ORR and may have a specific role in patients with MVI. Correlative analyses will be presented at the meeting. Clinical trial information: NCT03316872 .

Knockdown of GSDMD inhibits pyroptosis in psoriasis by blocking the NOD-like receptor signaling pathway.

Psoriasis is a chronic inflammatory skin disease regulated by autoimmunity, and pyroptosis plays an important role in this condition. This research sought to examine the function and potential molecular pathway of Gasdermin D (GSDMD) in psoriasis. GSDMD expression was examined by immunohistochemistry in biopsied skin tissues from patients with psoriasis. Pyroptosis-related genes and inflammatory factors were quantified using qRT-PCR and ELISA, respectively. HaCaT cells were treated with M5 cytokines to develop an in vitro psoriasis model, while imiquimod (IMQ) was administered to construct an in vivo psoriasis model. To counteract the inhibition of the NOD-like receptor (NLR) pathway caused by GSDMD knockdown, the pathway activator M-TriDAP was employed. In the lesional skin tissues of patients with psoriasis, GSDMD expression was highly expressed. The levels of pro-pyroptosis mediators were increased, whereas the level of anti-inflammatory factor was lowered. GSDMD knockdown and disulfiram treatment inhibited pyroptosis and promoted apoptosis in M5-induced HaCaT cells. In the IMQ-induced psoriasis-like mouse model, GSDMD knockdown suppressed pyroptosis and improved skin lesion severity, alleviating erythema, epidermal thickness, and inflammatory cell infiltration. Mechanistically, GSDMD knockdown inhibited the NLR pathway, accompanied by reduced protein levels of NLRP3, NOD1, NOD2, and PYCARD. NLR pathway activator, M-TriDAP treatment significantly reversed the effects of GSDMD knockdown on psoriasis progression. Knockdown of GSDMD inhibits pyroptosis in psoriasis by blocking the NLR signaling pathway, presenting a novel potential strategy for psoriasis treatment.