Baseline Biopsies Research Articles

High RIPK3 expression is associated with a higher risk of early kidney transplant failure

Renal ischemia-reperfusion injury (IRI) is associated with reduced allograft survival, and each additional hour of cold ischemia time increases the risk of graft failure and mortality following renal transplantation. Receptor-interacting protein kinase 3 (RIPK3) is a key effector of necroptosis, a regulated form of cell death. Here, we evaluate the first-in-human RIPK3 expression dataset following IRI in kidney transplantation. The primary analysis included 374 baseline biopsy samples obtained from renal allografts 10minutes after onset of reperfusion. RIPK3 was primarily detected in proximal tubular cells and distal tubular cells, both of which are affected by IRI. Time-to-event analysis revealed that high RIPK3 expression is associated with a significantly higher risk of one-year transplant failure and prognostic for one-year (death-censored) transplant failure independent of donor and recipient associated risk factors in multivariable analyses. The RIPK3 score also correlated with deceased donation, cold ischemia time and the extent of tubular injury.

Food-specific IgA levels in esophageal biopsies are not sufficiently high to predict food triggers in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an immune-mediated disease, characterized by Th2-type inflammation linked to specific foods. No currently available allergy tests reliably identify food triggers in EoE, leading to empiric dietary elimination strategies. Recently, milk- and wheat-specific IgA in esophageal brushings were linked to clinical food triggers. In this study, we aimed to determine whether food-specific IgA from esophageal biopsies is associated with known food triggers. A prior cohort of 21 patients (median age 39 years) with confirmed EoE underwent empirical elimination diets and subsequent reintroduction of foods to determine triggers. Archived baseline biopsies were used to quantify levels of peanut-, milk-, soy-, egg-, wheat-specific and total IgA by enzyme-linked immunosorbent assay. Overall, 13 patients (62%) responded to the dietary elimination as determined by histology (<15 eos/hpf), with milk and egg being the most common triggers. Biopsies had varying amounts of total IgA, while food-specific IgA was only detectable in 48 of 105 (46%) samples. Food-specific IgA was normalized to total IgA for each sample and stratified by whether a food was a known trigger. For all foods tested, there were no significant differences in IgA between positive and negative triggers. Food-specific IgA in esophageal biopsies was not associated with previously identified food triggers in this cohort. Future studies comparing food-specific IgA in esophageal brushings, mucous scrapings, and biopsies from patients with known triggers will be critical to determining whether food-specific IgA may serve as a biomarker for identification of EoE triggers.

Treatment Response in First-Line Metastatic Pancreatic Ductal Adenocarcinoma Is Stratified By a Composite Index of Tumor Proliferation and CD8 T-Cell Infiltration.

Determinants of treatment outcomes to chemotherapy-based regimens in metastatic pancreatic ductal adenocarcinoma (PDA) remain ill-defined. Our aim was to examine tissue-based correlates of treatment response and resistance using matched baseline and on-treatment biopsies collected from patients with PDA treated in the first-line metastatic setting. Patients with treatment-naïve metastatic PDA were enrolled in a Phase II trial (NCT02077881) investigating gemcitabine plus nab-paclitaxel in combination with indoximod, an orally administered small-molecule inhibitor of the IDO pathway. Baseline and on-treatment biopsies (week 8) of metastatic lesions (88% liver) were collected from a cohort of responders (N = 8) and non-responders (N = 8) based on RECIST v1.1 and examined by multiplex IHC and mRNA sequencing. Treatment altered the transcriptional profile of metastatic lesions with a decrease in tumor cell proliferation independent of treatment response. The antiproliferative response was seen in both basal and classical PDA subtypes. PDA subtype was not associated with survival outcomes; instead, genes involved in immune activation distinguished responders from non-responders. Tumor response was associated with an increase in CD3+ and CD8+ T-cell infiltrates into metastatic lesions. A composite of decreased tumor proliferation in response to treatment and increased CD8 T-cell infiltration in metastatic lesions identified responders and associated with a favorable survival outcome. Our findings suggest that inhibiting cancer cell proliferation alone in PDA is insufficient to produce tumor responses and support a role for tumor-extrinsic mechanisms, such as CD8+ T cells, which combine with the cancer cell proliferation index to define treatment outcomes.

Metabolite Alterations and Interactions with Microbiota in Helicobacter pylori-Associated Gastric Lesions.

Metabolites and their interactions with microbiota may be involved in Helicobacter pylori-associated gastric lesion development. This study aimed to explore metabolite alterations upon H. pylori eradication and possible roles of microbiota-metabolite interactions in progression of precancerous lesions. Targeted metabolomics assays and 16S rRNA gene sequencing were conducted to investigate metabolic and microbial alterations of paired gastric biopsy specimens in 58 subjects with successful and 57 subjects with failed anti-H. pylori treatment. Integrative analyses were performed by combining the metabolomics and microbiome profiles from the same intervention participants. A total of 81 metabolites were significantly altered after successful eradication compared to failed treatment, including acylcarnitines, ceramides, triacylglycerol, cholesterol esters, fatty acid, sphingolipids, glycerophospholipids, and glycosylceramides, with P values of <0.05 for all. The differential metabolites showed significant correlations with microbiota in baseline biopsy specimens, such as negative correlations between Helicobacter and glycerophospholipids, glycosylceramide, and triacylglycerol (P < 0.05 for all), which were altered by eradication. The characteristic negative correlations between glycosylceramides and Fusobacterium, Streptococcus, and Gemella in H. pylori-positive baseline biopsy specimens were further noticed in active gastritis and intestinal metaplasia (P < 0.05 for all). A panel including differential metabolites, genera, and their interactions may help to discriminate high-risk subjects who progressed from mild to advanced precancerous lesions in short-term and long-term follow-up periods with areas under the curve (AUC) of 0.914 and 0.801, respectively. Therefore, our findings provide new insights into the metabolites and microbiota interactions in H. pylori-associated gastric lesion progression. IMPORTANCE In this study, a panel was established including differential metabolites, genera, and their interactions, which may help to discriminate high-risk subjects for progression from mild lesions to advanced precancerous lesions in short-term and long-term follow-up.

Abstract B049: Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence

Abstract Background: In patients who receive definitive therapy for locally advanced prostate cancer, biochemical recurrence is due to local recurrence and/or the presence of undetected metastatic disease at the time of initial diagnosis and treatment. Approximately 25-40% of patients who receive radiotherapy plus androgen deprivation therapy (ADT) with curative intent are expected to recur within 5-10 years. We hypothesized that applying multiparametric (mp) MRI, gene expression, and genomic analyses to prostate tumors prior to EBRT+ADT would identify biomarkers predictive of recurrence (NCT01834001). Methods: At baseline and 6 months after completion of radiotherapy, mpMRI was performed and lesions were manually contoured. MR/ultrasound-fusion biopsies acquired at baseline targeted to up to three distinct MR lesions per patient. 29 patients with intermediate and high risk localized prostate cancer treated with EBRT+ADT with sufficient baseline biopsy tissue were selected for this analysis (median follow-up: 91 months). Tumor tissues were macrodissected to obtain DNA and RNA from 60 distinct biopsy regions (1-4 per patient). DNA was used for generating whole-exome sequencing libraries (92.5 × median coverage); RNA was converted into cDNA and hybridized to Affymetrix Human Exon 1.0 microarrays. Secondary gene expression cohorts with clinical annotation were obtained from the University of Miami and Queen’s University Belfast. Gene expression signatures were processed using Ingenuity Pathway Analysis (IPA) and the Decipher GRID. Results: Five of the 29 patients experienced biochemical failure by the time of data analysis; four of these patients were identified to have distant metastases with no evidence of local recurrence at the time of biochemical failure. Baseline and posttreatment lesion volumes by MRI and posttreatment ADCmax (apparent diffusion coefficient) were positively associated with recurrence (p&amp;lt;0.05). The Decipher signature positively correlated with these three MR features. Comparison of gene expression using a linear mixed-effect model identified 1,120 differentially expressed transcripts. IPA nominated TGF-beta signaling as the topmost enriched upstream regulator (z=3.32). This result was validated in the Miami (z=2.54) and Belfast (z=3.67) cohorts. By network analysis, genes that independently tracked with the baseline and posttreatment volumes and posttreatment ADCmax MR features similarly converged to the TGF-beta pathway. Co-occurrence of biallelic TP53 alterations with 1- or 2-copy PTEN losses was observed exclusively in patients who recurred. Conclusions: Genomic, transcriptomic and radiomic analyses have nominated predictive biomarkers that identify the subset of patients with locally advanced prostate cancer destined to recur after definitive EBRT+ADT. Identification of molecular features predictive of failure suggests that aggressive pre-existing subclones harboring these alterations have metastatic potential. Emerging systemic therapies including TGF-beta ligand traps targeted to these tumors may improve overall outcomes. Citation Format: Adam G. Sowalsky, Anson T. Ku, Uma Shankavaram, Shana Y. Trostel, Houssein A. Sater, Stephanie A. Harmon, Nicole V. Carrabba, Yang Liu, Radka Stoyanova, Elai Davicioni, Alan Pollack, Baris Turkbey, Deborah E. Citrin. Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B049.

Phase II randomized window of opportunity trial evaluating cytotoxic and immunomodulatory effects of intratumoral INT230-6 in early stage breast cancer: The INVINCIBLE trial.

573 Background: Most breast cancers are considered immunological cold and are therefore minimally responsive to immunotherapies. Local cytotoxic therapies can induce cell death, expose tumor antigens, provide adjuvants for anti-tumor immune priming, and potentially increase responsiveness to immunotherapies. We conducted a randomized, double blind, Phase 2 presurgical Window-Of-Opportunity trial for intratumoral (IT) INT230-6 (comprising VINblastine (VIN) Cisplatin (VIN)) evaluating clinical and BioLogical Effects in patients with early-stage operable Breast Cancer (the INVINCIBLE trialNCT04781725. The trial evaluated the immune response within the tumor and microenvironment in vivo after IT INT230-6 injections. Methods: Women with newly diagnosed and awaiting surgery for early-stage intermediate or high-grade ER+ T1-T2 invasive breast cancers were recruited to the trial. Participating patients were allocated (2:1) to IT injections of INT230-6 vs saline placebo, prior to resection. RNA profiling of the pre- (baseline biopsy) and post- treatment (surgical resection) tumours using the Ion AmpliSeq Transcriptome Human Gene Expression (Thermo Fisher Scientific) and DNA sequencing using a large (500 gene) comprehensive genomic profiling panel (OCAPlus,) was performed. Results: Significant tumor necrosis was observed in the majority of subjects injected with INT230-6. In the 60 pre-treatment biopsies profiled with OCAPlus, the genes most frequently mutated included PIK3CA (38%), TP53 (23%), CDH1 (22%) and TBX3 (17%). Frequent copy number changes were identified in CCND1 (23%), FGFR1 (18%), and CDH1 (17%). Gene expression analysis showed significant differential gene expression between the baseline biopsy and surgical specimens in patients treated with the drug. Pathway analysis identified genes associated with TCR signaling, macrophage markers, IL-18 signaling and B cell receptor signaling were significantly up-regulated in the post treatment samples. Conclusions: This is the first study evaluating the potential biologic effects of intratumoral cytotoxicity with INT230-6 and its role as a potential immune priming therapy in traditional immune quiescent ER+ breast cancers.

CD8 cell PET imaging with 89-Zr-crefmirlimab berdoxam (crefmirlimab) in patients with metastatic renal cell carcinoma (mRCC) receiving checkpoint inhibitors (CPIs): Association with response and tissue CD8 expression.

4551 Background: Novel imaging modalities using frequently expressed RCC antigens, such as CAIX, have shown promise in early-stage disease (Shuch B et al ASCO GU 2023). In advanced RCC, no tissue-based biomarkers have been well established to predict outcome with contemporary regimens, e.g., checkpoint inhibitors (CPIs) or targeted therapy (TT). We hypothesize that functional imaging of CD8 T-cells (CD8s) with crefmirlimab (a ~80 kDA 89Zr-labelled minibody with high affinity for CD8) may predict response given the essential role of CD8 T-cells (CD8s) in mediating CPI response. Methods: Eligible pts had pathologically verified RCC, metastatic disease and an intent to initiate standard of care CPI therapy. Patients received crefmirlimab PET/CT within 1 wk of CPI infusion and 4-6 weeks after initiating therapy. Baseline biopsy was mandated, along with repeat biopsy 0-2 weeks following the second PET/CT scan. PET signal was characterized as SUVmax, SUVpeak and SUVmean of the biopsied lesions, up to 5 index lesions and representative CD8 avid lymph nodes. Mean SUVmax in responders and non-responders were compared using students t-test (1-sided). CD8 expression in tissue was characterized as the number of positive cells per mm2; PET avidity and CD8 expression were compared using the Spearman correlation coefficient. Results: 17 pts (9 M: 8 F) were enrolled; most pts had clear cell histology (12; 71%) followed by unclassified (3; 17%) and papillary (2; 12%). The most commonly rendered CPI-based regimens were nivolumab alone (6 pts; 35%) and cabozantinib/nivolumab (3 pts; 17%). Follow-up data was available in 15 of the patients. By RECIST v1.1, 3 of 15 patients were classified as responders (best overall response [BOR] of complete response or partial response) and 12 patients were classified as non-responders (BOR of stable disease or progressive disease). Average SUVmax, SUVpeak and SUVmean per patient among all quantified index lesions and representative lymph nodes were 10.02, 6.95 and 6.11 for baseline and 8.82, 6.23 and 5.39 during treatment, respectively. Average SUVmax at baseline was 14.68 in responders to CPI and 8.28 in non-responders (P=0.006). On treatment SUVmax was 10.93 in responders to CPI and 8.22 in non-responders (P=0.19). A strong correlation between CD8 expression in baseline tissue and normalized SUVmean was observed (r=0.77; 95%CI 0.53-0.91). Conclusions: To our knowledge, this is the first series in RCC to demonstrate that functional imaging of immune cells (here, CD8s) may segregate response to CPIs, with responders having a higher baseline SUV and a larger decrement in SUV with therapy. Our results are bolstered by a significant correlation between tissue and imaging CD8 expression. Larger studies are underway to validate this noninvasive strategy. Clinical trial information: NCT03802123 .

SUNMO: A phase III trial evaluating the efficacy and safety of mosunetuzumab in combination with polatuzumab vedotin vs rituximab plus gemcitabine and oxaliplatin in patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma.

TPS7586 Background: Aggressive non-Hodgkin lymphomas (aNHL) are a diverse group of neoplasms, of which diffuse large B-cell lymphoma (DLBCL) is the most common subtype (Thandra, 2021). Patients (pts) with relapsed/refractory (R/R) DLBCL after one prior therapy who are unable to receive, or relapse after an autologous stem cell transplant (ASCT) and/or chimeric antigen receptor T-cell therapy have a poor prognosis (Salles, 2019; Di Blasi, 2022). Mosunetuzumab (Mosun) is an off-the-shelf CD20xCD3 T-cell engaging bispecific antibody that redirects T cells to eliminate malignant B cells (Sun, 2015), with promising efficacy and safety as a single agent, as shown in a Phase I trial in pts with B-cell NHL, including aNHL (Budde, 2022). Mosun has also shown promising safety and efficacy in combination with polatuzumab vedotin (Pola), a CD79b targeted antibody-drug conjugate that delivers the microtubule-disrupting agent monomethyl auristatin E directly to B cells (Dornan, 2009), in a Phase Ib/II trial in pts with R/R aNHL (Budde, ASH 2021). Methods: SUNMO (NCT05171647) is a randomized, open-label, global Phase III trial evaluating the efficacy and safety of subcutaneous Mosun + intravenous (IV) Pola (M-Pola) vs IV rituximab + gemcitabine and oxaliplatin (R-GemOx) in pts with R/R aNHL. Eligible pts have CD20-positive R/R aNHL (DLBCL not otherwise specified [NOS], high-grade B-cell lymphoma double/triple hit or NOS, transformed follicular lymphoma [trFL], or Grade 3B FL), ECOG performance status 0–2, and ≥1 prior systemic therapy (if only one prior line of therapy, pts must be ineligible for ASCT). Exclusion criteria include prior treatment with CD20-directed bispecific antibodies, R-GemOx, or GemOx; prior allogeneic stem cell transplant; and any central nervous system involvement of lymphoma. Pts will be randomized 2:1, stratified by the number of prior lines of therapy (1 vs &gt; 1) and response to last therapy (relapsed vs refractory) to receive M-Pola or R-GemOx IV for a fixed duration of up to 8 cycles (6 cycles for Pola; M-Pola, 21-day cycles; R-GemOx, 14-day cycles). The primary endpoint is progression-free survival (PFS) determined by an independent review facility (IRF). Secondary endpoints include overall survival; IRF- and investigator (INV)-assessed complete response (CR) rate, objective response rate, and duration of response/CR; INV-assessed PFS; patient-reported outcomes; and safety. Biomarker analyses include pre-specified prognostic subsets from baseline biopsies and circulating tumor DNA at baseline and during treatment. Enrollment is ongoing, and an estimated 75 sites globally will enroll approximately 222 pts (M-Pola, 148 pts; R-GemOx, 74 pts). Clinical trial information: NCT05171647 .

Gene expression signatures, stromal tumor infiltrating lymphocytes (sTILs), and change in tumor cellularity to predict pathological complete response (pCR) after 12 week de-escalated neoadjuvant endocrine therapy (ET) vs paclitaxel + dual HER2 blockade in the WSG-TP-II trial.

519 Background: There are limited data on predictive biomarkers for de-escalated ET or chemotherapy with dual anti-HER2 blockade in HR+/HER2+ early breast cancer (BC). In this translational pre-planned project of the WSG-TP-II phase II-trial (NCT03272477), we aimed to identify associations of gene expression signatures and sTILs with pCR. Methods: Patients with cT1c-cT4c, cN0-3 centrally confirmed HR+/HER2+ BC were randomized to 12 weeks of standard ET (n = 100) or paclitaxel (Pac; n = 107). All patients received trastuzumab + pertuzumab (T+P) q3w as neoadjuvant and adjuvant treatment. Gene expression signatures were analyzed using NanoString BC360 panel in baseline biopsies (T+P+ET: n = 72; T+P+Pac: n = 78). sTILs were analyzed in 93 (T+P+ET) and 97 patients (T+P+Pac) at baseline and in 65 (T+P+ET) and 57 patients (T+P+Pac) at week 3. Impacts of standardized BC360 gene expression signatures and sTILs on pCR (ypT0/is ypN0; primary endpoint) expressed in odds ratios (OR) were estimated by logistic regression analysis. Results: pCR rate in patients with BC360 analysis was 39.3% (T+P+ET: 23.6%; T+P+Pac: 53.9%). Overall, ERBB2 (OR 2.37; 95%CI 1.55, 3.62) and cytotoxic cells signature (OR 1.42; 95%CI 1.02, 1.99) were favorable for pCR, while apoptosis (OR 0.64; 95%CI 0.44, 0.92), estrogen receptor 1 (OR 0.60; 95%CI 0.42, 0.85), estrogen receptor signaling (OR 0.64; 95%CI 0.45, 0.91), and progesterone receptor (OR 0.67; 95%CI 0.47, 0.94) signatures were unfavorable. Analyzing by treatment arm, a similar pattern was observed in the T+P+Pac arm (ERBB2: OR 2.01; apoptosis: OR 0.59; estrogen- and progesterone-related signatures: OR 0.41-0.58), but not in the T+P+ET arm where only ERBB2 was prognostic for pCR (OR 7.24; 95%CI 2.12, 24.05). Baseline ≥30% sTIL levels (vs &lt; 30% sTILs; n = 16 vs n = 174) were associated with pCR (OR 5.16; 95%CI 1.60, 16.66); significance was not achieved in either trial arm. Compared to &lt; 30% sTILs (n = 90), low tumor cellularity at week 3 precluding sTILs analysis ( &lt; 500 invasive tumor cells, n = 22), but not ≥30% sTILs (n = 10), was prognostic for pCR in all patients (OR 9.47; 95%CI 2.94, 30.50) and in individual trial arms (OR 6.00-11.79). Conclusions: This hypothesis-generating translational results suggest that gene expression signatures (particularly ERBB2), baseline sTILs, and low tumor cellularity at week 3 predict pCR after ET + double HER2 blockade. Future neoadjuvant trials are needed to prospectively test the use of baseline gene expression and sTILs analysis, and early on-treatment cellularity measurement to select patients with HR+/HER2+ tumors for de-escalated endocrine-therapy-based approaches. Clinical trial information: NCT03272477 .

Tumor-infiltrating lymphocytes (TILs) for prediction of response to platinum-based neoadjuvant chemotherapy (NACT) in triple-negative breast cancer (TNBC).

e12620 Background: According to several previous trials the rate of pathological complete response (pCR) for patients with TNBC is increased when carboplatin is added to NACT. However, it is largely unknown which group of patients would truly benefit from platinum-based NACT and for whom chemotherapy could be de-escalated. Several clinical trials have evaluated the stromal TILs as an important prognostic and predictive biomarker in breast cancer. We assessed the pCR rates and the outcomes in patients with TNBC randomized to receive NACT ± Carboplatin regarding TILs status. Methods: The clinical data were obtained from 132 patients diagnosed with TNBC (ER&amp;PgR &lt; 10%) and treated with either anthracycline-taxane NACT (n = 68) or сarboplatin-based chemotherapy (n = 64) from 2017 to 2022 at N.N. Petrov National Medicine Research Center of Oncology. TILs were evaluated in 132 formalin-fixed paraffin-embedded tumor tissue samples stained with H&amp;E at baseline biopsies according to guidelines from the International TILs Working Group. Classification of TILs into low ( &lt; 10%), intermediate (≤10% to &lt; 40%) and high (≥ 40%) levels was performed. Data are presented using the methods of descriptive statistics. The differences between groups were assessed using the Сhi-square test. P-values below 0.05 were considered statistically significant. Results: The proportion of patients who achieved a pCR was higher in the carboplatin group 66% than in patients without carboplatin 35% (OR 3,5; 95% CI 1,71-7,17; p = 0,0006). There were 25 (18.9%), 77 (58.3%) and 30 (22.7%) patients with low ( &lt; 10%), intermediate (≤10% to &lt; 40%) and high (≥ 40%) levels of TILs, respectively. In total, the tumors with high TILs had a pCR rate of 63.3%, compared with 46.1% for low TILs (OR 2,0; 95% CI 0,87-4,67; p = 0,097). No significant statistical difference in pCR rates was shown between carboplatin-based and standard NACT groups with high TILs - 71% and 54%, respectively (OR 2,06; 95% CI 0,45-9,30; p = 0,346). When analyzing a group with low TILs infiltration, the pCR was more frequent in the platinum-based 64% compared to 31% in the standard NACT group (OR 3,9; 95% CI 1,72-9,00; p = 0,0011). Conclusions: These data may support the de-escalation of carboplatin-based NACT in patients diagnosed with TNBC with high TILs infiltration. Further investigations are warranted to explore the predictive value of TILs in platinum-based NACT. [Table: see text]

Phase 1 clinical trial results for NG-350A, a novel transgene-armed and tumor-selective vector: Differential effects of intravenous (IV) versus intratumoral (IT) dosing on immune pharmacodynamics (PD).

2572 Background: Tumor-selective therapeutic viral vectors are often limited to IT delivery. NG-350A, a blood stable adenoviral vector, selectively replicates in tumors and expresses an agonist anti-CD40 payload. We assessed the impact of IV vs IT delivery during the phase 1 FORTITUDE study (NCT03852511). Methods: NG-350A was dose-escalated in separate IT or IV cohorts of patients (pts) with metastatic/advanced epithelial tumors. Pts received either 1 (Day [D] 1) or 4 IT injections (D1, 8, 15, 22) of up to 1x1012 viral particles (vp) or 3 IV infusions completed in &lt;9 days (doses of 1x1011 to 6x1012 vp). Serum samples were taken immediately after dosing (pharmacokinetics [PK]) and during follow-up (PD). Baseline and on-treatment (D15+) tumor biopsies were taken. Results: In total, 25 pts received NG-350A monotherapy, 9 by IT and 16 by IV dosing. Both IV and IT dosing elicited similar anti-drug (virus) antibody responses and limited viral shedding. Peak vp Cmax was variable with IT dosing but dose-related with IV dosing. Vector DNA was detected by qPCR in tumor biopsies after higher IV dose levels (including up to ~7 weeks post dosing) and IT injection. Dose-dependent, sustained and specific increases in inflammatory cytokines related to the expected effects and timing of the anti-CD40 transgene production (IL-12, IL-17A, IL-2, IFNγ) were detected in serum of pts treated at higher IV doses. By contrast, limited evidence of such PD effects was seen with IT dosing. Transgene was not detectable in core needle biopsies; however, a novel serum biomarker assay detected transgene mRNA at D8 or D15 in five pts who received higher IV dose levels. Importantly, both IV and IT dosed NG–350A was well-tolerated, with no treatment-related SAEs occurring in &gt;1 pt and no DLTs at the highest dose levels tested. No evidence of anti-CD40 transgene-related toxicity was seen with IV or IT dosing, with few hepatic AEs and no Grade ≥3 CRS. Follow-up for imaging was limited; however, stable disease was observed in 3/6 pts at IV Dose Level 3. Conclusions: This analysis provides the first clear evidence that an IV-dosed tumor-selective viral vector can drive expression of immuno-stimulatory payloads with functional PD consequences in advanced cancers. The improved PK and PD profile of IV vs IT dosed NG-350A also highlights the benefits of systemic delivery for such vectors. Dose-escalation of NG-350A + pembrolizumab to identify a dose for efficacy assessments continues in the FORTIFY study (NCT05165433). Clinical trial information: NCT03852511 . [Table: see text]

Molecular predictors and immunomodulatory role of dual checkpoint inhibitor blockade using ipilimumab/nivolumab in patients with extensive stage small cell lung cancer.

8597 Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma with poor prognosis. In extensive stage patients, frontline treatment with chemoimmunotherapy shows modest clinical benefit. However, the biological impact of immunotherapy in SCLC is poorly understood with no clear predictive biomarkers to guide patient selection in this setting. Methods: We collected paired baseline (pre-treatment), on-treatment (week 4), and progression biopsies from patients with relapsed advanced-stage SCLC treated with combination nivolumab (nivo) and ipilimumab (ipi) in a single-arm, phase 2 clinical trial (NCT03670056). Nivo 1 mg/kg and ipi 3 mg/kg were administered every 3 weeks for 4 cycles, followed by nivo maintenance until progressive disease (PD) by RECIST 1.1 or treatment-limiting toxicity. Paired pre/on-treatment samples were available from 16/22 patients, as well as 3 biopsies at progression. The tumor samples were studied using whole exome DNA sequencing (including germline DNA) and RNA-sequencing coupled to Ocean Genomics TxomeAI data analysis pipeline. Results: 6/11 evaluable patients had PD; 5 patients showed clinical activity of treatment (3 with stable disease, 2 with partial response). The frequency of deleterious mutations in TP53 and RB1 was 91% and 64%, respectively. Mutations in HLA-A were more common in baseline samples from patients with PD than those with clinical activity. New TP53 and PLEC mutations were found 4 of 6 patients with PD in week 4 samples vs baseline. The baseline tumor mutational burden was not associated with treatment sensitivity and prominently increased in week 4 biopsies of PD patients. All four molecular SCLC transcriptomic subtypes based on the expression of ASCL1, NEUROD1, and POU2F3 were present in the trial with SCLC-A being the most common (9/16 cases). All patients with clinical activity to ipi/nivo were of SCLC-A subtype. 2/16 of cases showed a different molecular subtype after 4 weeks of treatment: one case with SCLC-N switched to SCLC-A and another case converted from SCLC-A to SCLC-N. Comparison of baseline and on-treatment samples showed upregulation of transcripts associated with T-cell activation and PD-1 signaling. In the 3 biopsies at progression, transcriptomic changes included reduction of neutrophil degranulation, type 1/2 interferon and signatures, as well as down-regulation of β-2 microglobulin, while cell cycle and mitotic prophase pathways were overexpressed. Conclusions: Dual checkpoint blockade using nivo/ipi has a prominent immunomodulatory role in extensive stage SCLC characterized by increased local adaptive immune responses, reduced HLA class-I antigen presentation and change in the molecular subtype in a subset of cases. We identified genomic features associated with treatment sensitivity/resistance. Clinical trial information: NCT03670056 .

Phase I study of MP0317, a FAP-dependent DARPin, for tumor-localized CD40 activation in patients with advanced solid tumors.

2584 Background: CD40 is a master switch for both innate and adaptive immune systems. The clinical development of CD40 agonists has been hampered by dose-limiting toxicity (DLT) due to systemic CD40 activation and peripheral target-mediated drug disposition (TMDD). MP0317 is a CD40-agonistic DARPin (designed ankyrin repeat protein), developed to reduce systemic toxicity. MP0317 is exclusively active in the presence of fibroblast activating protein (FAP) expressed by cancer associated fibroblasts in the tumor microenvironment. This allows reaching serum levels that overcome the TMDD and have the potential to deliver sustained and tumor-localized CD40 activation. Ongoing clinical testing aims at establishing its safety/tolerability profile, PK/PD characteristics, and a recommended dose for combination therapy. Methods: NCT05098405 is a Phase 1, multicenter, open label, dose escalation study followed by a safety expansion part in adult patients with advanced solid tumors. The dose escalation scheme uses an adaptive Bayesian logistic regression model with overdose control. Peripheral blood biomarkers are analyzed by immuno-assays and flow cytometry, and baseline and on-treatment tumor biopsies are characterized by RNA sequencing and immunofluorescence. Eligible patients are enrolled into 9 sequentially escalating dose cohorts of MP0317 (0.03-10 mg/kg), administered IV 3-weekly (Q3W) or 1-weekly (Q1W) until disease progression or unacceptable toxicity. Results: At submission, 23 patients across 6 cohorts completed the study, with no DLT observed. Patient enrolling in higher dose cohorts is ongoing. The most frequent AE was grade 2 infusion related reaction in 5 patients. The 23 patients received ≥2 (range 2-8) doses (range 0.03 – 3 mg/kg) of MP0317 across five Q3W and one Q1W cohorts and completed the 28-day DLT period. PK data confirmed all patients were exposed to MP0317 with broadly dose-dependent Cmax and no sign of accumulation. PD data was derived from patients dosed with 0.03 – 3 mg/kg. Soluble biomarkers (sFAP and sCD40) showed target engagement in periphery with signs of FAP saturation at ≥0.5 mg/kg. Colocalization of MP0317 with FAP and CD40 in the tumor was confirmed in 4 out of 8 evaluable paired tumor biopsies. Whole transcriptome and gene set enrichment analyses showed an upregulation of genes related to B-cell trafficking (CXCL3, CXCR5, CCR6, CCL20) upon treatment. Transient increase of IFNg-induced chemokines (CXCL9, CXCL10) was observed, whereas the pro-inflammatory cytokines (TNFa, IL-2, IL-6, IL-8) were not upregulated. Conclusions: Clinical data and the lack of pro-inflammatory circulating cytokines confirm MP0317 is safe and well-tolerated, while analysis of paired pre- and on-treatment tumor biopsies suggests early evidence of tumor-localized CD40 activation. The current data enables further evaluation in a combination setting. Clinical trial information: NCT05098405 .

Phase 1b/2 trial of pepinemab plus avelumab as second line combination immunotherapy for patients with metastatic pancreatic adenocarcinoma.

TPS4195 Background: Monotherapy with immune checkpoint inhibitors (ICIs) is not effective in metastatic pancreatic adenocarcinoma (PDAC), likely due to the unique immunosuppressive tumor microenvironment (TME) of PDAC, thus safe and novel strategies to overcome resistance and facilitate adaptive immune responses are a major unmet need. Preclinical and clinical studies demonstrated that antibody blockade of semaphorin 4D (SEMA4D) promotes tumoral infiltration and activation of DCs and CD8+ T cells and reverses immunosuppression, including attenuation of MDSC recruitment and function, leading to enhanced efficacy of ICIs without increasing toxicity. Pepinemab, a SEMA4D blocking antibody, in combination (combo) with avelumab provided clinical benefit in some patients with ICI-resistant and PD-L1-low NSCLC (NCT03268057). Pepinemab is also being evaluated in combo with pembrolizumab in patients with metastatic squamous cell carcinoma of the head and neck (NCT04815720). The principal goals of this proof-of-concept study are to investigate the safety, efficacy, and TME biomarker changes of treatment with the combo of pepinemab plus avelumab in patients with metastatic PDAC. Methods: This single-arm, open-label study (NCT05102721) is evaluating the safety, tolerability, and efficacy of pepinemab in combo with avelumab in patients diagnosed with metastatic PDAC who have either progressed on or are otherwise intolerant to first line chemotherapy. Patients must have received 5FU or gemcitabine-based first line therapy with evidence of intolerance or treatment failure, including progression both during or after completing first-line treatment. Accrual for the Phase 1b portion follows a Bayesian Optimal Interval Design (BOIN), beginning at the highest dose combo; 20 mg/kg pepinemab and 800 mg avelumab Q2W. Phase 2 begins after 16 subjects are enrolled at MTD, permitting a Simon’s two stage assessment of futility prior to closing the Phase 1b period. The primary objective for Phase 1b is tolerability, and the primary objective for Phase 2 is therapeutic efficacy compared to historical second-line systemic chemotherapy. The trial anticipates a total evaluable cohort of 40 subjects and is powered to detect a response rate of 23% or greater, with alpha set at 0.1 and 80% power. Exploratory objectives include robust correlative analysis of immune, stromal, and genomic profiling of baseline and on-treatment tumor biopsies to ascertain mechanisms of treatment response and failure. Patient reported outcomes are incorporated throughout the trial to assess disease specific symptoms associated with pancreatic cancer and cancer cachexia. One patient has been enrolled as of Feb 14, 2023. Clinical trial information: NCT05102721 .

Racial disparities in aging-related comorbidity among breast cancer survivors.

10555 Background: Black breast cancer (BC) patients have a higher mortality rate than their white counterparts, potentially due to age acceleration caused by cancer and its treatments. However, epidemiological evidence is limited. Methods: Our study includes 1885 BC patients and 1224 cancer free women (controls) recruited at Vanderbilt University Medical Center from 2004 to 2021. Patient demographics and aging-related outcomes were extracted from the tumor registry or electronic health records. Aging-related outcomes included 28 comorbidities and the Charlson Comorbidity Index (CCI) at baseline (biopsy) and follow-up (latest clinical visit). The associations of BC status with CCI and specific comorbidities were evaluated by linear and logistic regression models, respectively. The associations of race with aging-related outcomes were evaluated among cases. All analyses were adjusted for age, and follow-up outcomes were adjusted for baseline information. Results: The median follow-up time is 10 years. BC patients had a higher mean CCI at baseline (1.32 vs 0.59, p&lt;0.01) and follow-up (beta=1.04, 95% CI=0.94-1.14) than controls. Results from individual comorbidities showed that BC survivors had a higher risk of developing cardiovascular disease, liver disease, hypertension, hyperlipidemia, gastroesophageal reflux disease (GERD), and depression. Among BC patients, Black women were diagnosed at a younger age (52.0 vs 55.8, p&lt;0.01), but had a higher baseline and follow-up CCI than Whites. Black BC survivors were more likely to develop peptic ulcer disease, diabetes, second malignancy, hypertension, and GERD than White BC survivors, even after adjustment for baseline comorbidities. [Table: see text]

Phase IIa study of αDC1 vaccines targeting HER2/HER3 combined with pembrolizumab in patients with asymptomatic brain metastasis from triple negative breast cancer or HER2+ breast cancer.

TPS1112 Background: Brain metastases develop in up to 50% patients (pts) with metastatic triple negative breast cancer (TNBC) and HER2+ BC, constituting an increasing source of morbidity and mortality. HER3, overexpressed in triple negative and HER2+ brain metastatic breast cancer (BMBC), is a resistance factor to HER2-targeted therapies and a driver of CNS metastasis. Disease progression is associated with loss of anti-HER2 and anti-HER3 immunity. Previously, we have demonstrated that glioma-specific peptide-loaded αDC1 which produce CXCL9, CXCL10, CXCL11, CCL5, the chemokines which attract CXCR3- and CCR5- expressing cytotoxic T-lymphocytes (CTLs) and T-helper 1 (Th1) cells, induce clinical responses and long-term disease stabilization in pts with aggressive recurrent primary brain tumors (Okada et al. JCO 2011. PMID: 21149657). We hypothesized that anti-HER2/3-loaded αDC1 in combination with PD1 blockade will result in strong Th1/CTL response against HER2/3 epitopes (Basu A et al. Cancer Immunol Res. 2022 PMID: 34785506), being effective against brain lesions and systemic disease. Methods: This is a phase II single-arm, non-randomized multicenter study (NCT04348747). Eligibility includes pts with triple negative and HER2+ BMBC ≥18 years, ECOG PS ≤1, normal marrow and organ function with asymptomatic untreated brain metastases ≥ 5 mm who will receive αDC1 q3 weeks x 3 along with pembrolizumab every 3 weeks. Thereafter, αDC1 booster doses (if available) would be administered every 3 months until disease progression, intolerable side effects or withdrawal from study, up to 24 months. Baseline and 9-week post-αDC1 peripheral biopsies (non-CNS) are required for six pts. 1 of the planned 21 pts have been enrolled and is undergoing treatment. Primary endpoint is CNS response rate (RR) by RANO-BM criteria. If no CNS response is observed after 12 pts, study will be terminated. If ≥ 1 response observed, then 9 more pts will be enrolled, for a total of 21 pts. If ≥ 3 CR are observed, the proposed therapy will be considered as promising for further evaluation. Secondary endpoints include non-CNS RR per RECIST v1.1, median CNS, non-CNS and overall progression-free survival, overall survival and safety. Exploratory endpoints include changes in intratumoral biomarkers (CTLs, PDL1, chemokines) in pre- and post-treatment peripheral tumor biopsies and immune changes in the blood. Clinical trial information: NCT04348747 .

The role of innate immune system in modulating CHK1 inhibitor (CHK1i) response in BRCA wild-type (BRCAwt), platinum-resistant high-grade serous ovarian cancer (PR-HGSOC): Exploratory analysis from a phase II study of CHK1i prexasertib.

5557 Background: Immune checkpoint blockade, e.g., anti-PD-1/PD-L1, demonstrated limited efficacy in HGSOC despite high levels of tumor-infiltrating lymphocytes, suggesting the innate, rather than adaptive, immune system may play a prominent role in anticancer response. Preclinical studies suggest CHK1i modulates the innate immune milieu. We previously showed clinical activity of prexasertib in BRCAwt, PR-HGSOC patients (pts). Here, we investigated the association of innate immunogenic features with CHK1i response. Methods: Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. Baseline (BL) fresh core biopsies were collected from pts enrolled in the biopsy (Bi) cohort for RNA sequencing. Computational immunogenomic analysis (CIBERSORT) was used for immune cell fractions quantification. Blood samples (BS) were obtained at BL and cycle 1 day 15 (C1D15) from pts enrolled in both Bi and non-Bi (nBi) cohorts for multiparametric flow cytometry (FC) analysis to evaluate dynamic changes in immune cell subsets. Mann-Whitney and Wilcoxon tests were used to compare unpaired and paired data, respectively. Results: 49 BRCAwt, PR-HGSOC pts were enrolled in the Bi cohort (n=25) and nBi cohort (n=24). 39 were evaluable per RECIST v1.1. 18/39 pts (46.2%) had a clinical benefit (CB), defined as progression-free survival≥6 months. Transcriptomic profiles of BL biopsies (n=15) met the quality control and assurance (Gene Similarity Index, Principal Component Analysis) for further in silico analysis. CIBERSORT analysis did not exhibit any differences in the immune cell fractions between CB and non-CB (NCB) groups at BL. FC analysis on paired BS (BL and C1D15) showed an increase of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs, p&lt;0.001) and classical monocytes (CM, p=0.003) in the NCB group (18 paired BS). In the CB group (19 paired BS), CD141+ and CD1c+ dendritic cells (DCs) express higher rates of CD83, a marker of maturity and functionality (p&lt;0.001 and p&lt;0.001, respectively), despite no increase in the absolute number of DCs. At C1D15, a higher rate of M-MDSCs (median 7.8% vs. 5.52%; p=0.03) and CM (median 40.7% vs. 31.7%; p=0.03) were found in the NCB group (n=18) compared to the CB group (n=19); also, CM showed lower expression of HLA-DR in the NCB group (median 19.25% vs. 27.8%; p=0.02). Conclusions: Collectively, our data suggest the possible involvement of innate immunity in modulating CHK1i response. Increasing peripheral immunosuppressive cells and lower immunocompetence of the innate immune system may contribute to CHK1i resistance, whereas the functionality of DCs may be involved in the response. Enhancing innate immunity may represent a strategy to increase CHK1i response in this hard-to-treat population. Clinical trial information: NCT02203513 .

Phase II study of cabozantinib (Cabo) with nivolumab (Nivo) and ipilimumab (Ipi) in advanced renal cell carcinoma with variant histologies (RCCvh).

4520 Background: New therapeutic approaches in RCCvh are needed. Herein, we report on treatment intensification with the combination of Cabo/Nivo/Ipi in patients with metastatic RCCvh in a multi-institutional prospective single arm phase II trial. (NCT04413123). Methods: Eligible patients (pts) had metastatic RCCvh with ECOG performance status of 0-1 and may have received one line of prior therapy excluding immunotherapy or Cabo. Pts underwent a baseline biopsy and received treatment with Nivo 3 mg/kg and Ipi 1 mg/kg intravenously Q3 weeks (W) for 4 cycles followed by Nivo 480 mg IV Q4W. Cabo was given continuously at dose of 40 mg daily; reductions to 20 mg daily and 20 mg every other day were allowed. The primary endpoint was objective response rate (ORR) by RECIST 1.1. Safety was a secondary endpoint. Results: 40 pts have been enrolled. At the time of data cut-off (Dec 9, 2022), 38 pts received at least 1 study drug. 11% (n=4) pts received prior systemic therapy. 45% (n=17) received all 4 doses of Nivo and Ipi; 18% (n=7) received 3 and 37% (n=14) received ≤ 2 doses. 61% (n=23) (15 of whom received 4 cycles Nivo/Ipi) received Nivo maintenance (median number of cycles, 5 (range, 1-21)). 71% (n=27) and 13% (n=5) required Cabo dose reduction to 20 mg and 20 mg every other day, respectively. Median follow-up was 8.4 (range, 2.1-23) months. Objective response was achieved in 8 pts (ORR 21%, two-sided 80% CI, 13%-32%). Median duration of response was not reached with 5 pts maintaining response &gt; 6 months. Median progression-free survival was 8.9 (95% CI, 4.2-12.7) months. 74% (n=28) developed treatment-related grade 3 or higher toxicities; 37% (n=14) developed ≥ grade 3 elevation in AST or ALT. 29% (n=11) required high dose steroids (prednisone ≥ 40 mg daily or equivalent). 13% (n=5) discontinued all study drugs due to toxicity. No grade 5 toxicity has been reported. Conclusions: The study suggests activity for this combination in patients with RCCvh particularly among those without chromophobe histology. An additional cohort of 20 pts is enrolling with Cabo starting dose of 20 mg daily. Clinical trial information: NCT04413123 . [Table: see text]

A phase 1/2 study of combination olaparib and radium-223 in men with metastatic castration-resistant prostate cancer with bone metastases (COMRADE): A trial in progress.

TPS5110 Background: Radium-223, an α-emitting radioisotope inducing DNA double-stranded breaks leading to cell death has demonstrated improvement in overall survival (OS) in men with metastatic castration-resistant prostate cancer (mCRPC) with bone metastases. PARP inhibitors prevent the repair of DNA single-stranded breaks and have demonstrated clinical efficacy as monotherapy in mCRPC patients harboring alterations in the homologous recombination repair (HRR) pathway and in combination with androgen receptor signaling inhibitors irrespective of DNA repair mutation status. In preclinical models, PARP inhibitors have shown efficacy as radio sensitizing agents. We designed a phase 1/2 trial to identify the recommended phase 2 dose (RPD2) of olaparib and radium-223 and test the hypothesis that radium-223 + olaparib will demonstrate anti-tumor activity in mCRPC irrespective of homologous recombination repair deficiency (HRD) status (NCT03317392). Methods: This open-label, multicenter, phase 1/2 study evaluates the dosing, safety and efficacy of olaparib with radium-223 in men with mCRPC with bone metastases. The phase 1 component used a 3+3 dose escalation design to determine the olaparib recommended phase 2 dose (RP2D) + fixed dose radium-223 (55 kBq/kg IV every 4 weeks x 6). The phase 1 portion has completed accrual (n = 12) and demonstrated that the RP2D of olaparib is 200 mg by mouth twice daily when combined with standard dose radium-223. The phase 2 component of the study which was activated in 1/2021 is an open-label, randomized trial (1:1) evaluating olaparib + radium-223 compared to radium-223 alone. The inclusion criteria include patients with mCRPC with any number of prior therapy lines for mCRPC and ≥2 bone metastases by imaging and at least 1 lesion which has not been treated with prior radiation. Patients with visceral metastases or malignant lymphadenopathy ( &gt; 4 cm in short diameter) are excluded. The primary endpoint for phase 2 is radiographic progression-free survival (rPFS) per PCWG3 guidelines for bone metastases and RECIST v1.1 for non-bone disease. The integrated biomarker is Oncopanel assay to determine HRR status. A key secondary endpoint will evaluate rPFS in biomarker positive and negative patients. Other secondary endpoints include time to PSA progression, PSA response, time to subsequent therapy, time to first skeletal event, and OS. Exploratory endpoints include WES/WTS of baseline biopsy tissue, assessment of RAD51 IHC and correlation with outcomes, ctDNA assessment and correlation with responses, evaluation of changes in the tumor immune microenvironment, and quality of life assessment. Target accrual is 133 patients. Clinical trial information: NCT03317392 .

Neoadjuvant immunotherapy with ipilimumab plus nivolumab and radiologically and pathologically quantifiable responses through modulation of the tumour microenvironment in resectable hepatocellular carcinoma.

4129 Background: Liver resection (LR) is a potentially curative option for early-stage hepatocellular carcinoma (HCC), and immune checkpoint inhibitors (ICI) are being investigated in the perioperatory setting to improve long-term relapse rates and survival outcomes. Methods: PRIME‐HCC is a phase Ib study testing nivolumab (3 mg/kg, day 1 and day 22) plus ipilimumab (1mg/kg, day 1 only) (N+I) prior to LR in resectable HCC. Primary endpoint was safety, while secondary endpoints included radiological response (per RECIST v1.1) and pathological response on resection specimens (ReS). Translational endpoints encompassed analysis of tissue samples with multiplex immunohistochemistry (MIHC) and gut microbiota profiling with high-throughput sequencing and their correlation with response. Results: As of 7th of February 2023, 25 patients (pts) were enrolled, all Child-Pugh A, mostly male (n=18, 72%), with cirrhosis (n=13, 52%), and viral hepatitis (44%, 4 HBV and 7 HCV). Any-grade adverse events (AEs) occurred in 88% pts (n=22), of which 24% (n=6) grade (G) 3. No G4-5 AEs were recorded. Any-grade treatment-related (tr)AEs were reported by 72% pts (n=18), including 8% G3 trAEs (n=1 ALT/AST elevation and n=1 dyspnea). After a median follow-up of 14.5 months (95% CI 7.0-22.0), 21 pts were assessed for radiological response and underwent surgery. Median time from treatment start to LR was 2.4 months (interquartile range 2.2-2.7). LR was delayed in 1 pt (4.8%) because of G2 tr-hypothyroidism but occurred uneventfully. Objective response rate was 29% (n=5 partial responses, n=1 complete response [CR]); disease control rate was 95% (n=20), with 1 progressor having a mixed HCC/cholangiocarcinoma. Major pathological response (MPR), defined as ≥70% tumour regression, was observed in 56% (n=9) of the pathologically evaluable ReS (n=16), with 38% pathological CR (n=6). In the MIHC analyses, pretreatment biopsies of MPR pts were significantly enriched in peritumoral CD4+ and CD8+ cells, with no intratumoral difference. When comparing baseline biopsies and ReS, MPR pts had a significant decrease of intratumoral FOXP3+/CD4+ regulatory T cells (Tregs), while peritumoral Tregs increased significantly in non-MPR ReS. In baseline stool samples, α and β diversity did not differ across responders (R) and non-responders (NR), nor it changed after ICI. However, pre-treatment stool samples of NR pts were significantly enriched in Alistipes and Colinsella genera. Conclusions: Neoadjuvant N+I is feasible and tolerable for early-stage HCC, with promising radiological and pathological response rates. Study of tissue and gut microbiota highlights tumor and host-related changes. Immune infiltrate and gut microbiota composition lend themselves as predictors of anti-tumour efficacy of N+I combination. Clinical trial information: NCT03682276 .