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Background: Mature Tertiary Lymphoid Structures (mTLS) are organized lymphoid structures containing B-lymphocytes admixed to CD23+ follicular dendritic cells, which play a key role in antitumor immune response. We and others have demonstrated that presence of mature TLS is higly predictive of benefot of anti-PD1/PDL1 antibodies in patients with solid tumors (Vanhersecke et al Nature Cancer 2021, Italiano et al Nature Medicine 2022). However, the relative prevalence of mature TLS in the different tumor types has not been investigated with an uniform methodological approach. Methods: We screened the presence of mature TLS (mTLS) on tumor samples from patients included in an institutional molecular profiling program (BIP, NCT02534649). mTLS were screened using HES, CD20 (clone L26, Ventana) and CD23 (clone 1B12, Novocastra) stainings, performed on serial sections. TLS were defined as lymphoid aggregates of CD20+ B-lymphocytes of more than 50 immune cells within 1mm of the tumor front. mTLS were defined as TLS containing either a visible germinal center on HES or a meshwork of CD23+ follicular dendritic cells or isolated CD23+ dendritic cell if the cell harbored a morphology fitting with dendritic differentiation. If plasma sample of patient was available, bTMB was assessed by Foundation Medicine, with bTMB-high defined as at least 10 mutations/megabase (mut/Mb). Results: We characterized 1,393 samples. mTLS were present in 467 cases (33.5%) across all tumor histotypes: 36.2% of carcinoma (N = 392/1080 cases), 24.8% of sarcoma (N =69/209 cases) and 25% of neuroendocrine tumor (N= 4/12 cases). Among 569 cases with evaluable bTMB, the proportion of patients with bTMB-high was significantly higher in the mTLS group (16.7%, N=31/185) compared to absence of mTLS group (8.5%, N=30/353) (p=0.036). Conclusion: mTLS are present in variable proportions in almost all tumor types even in those considered as resistant to current immune checkpoint inhibitors. Prostate Bladder Kidney Head and neck Gynecologic (except ovary) Ovary mTLS+% of patients (N) 34% (28) 33% (19) 25% (6) 53% (17) 33% (19) 44% (26) mTLS-% of patients (N) 66% (55) 67% (38) 75% (18) 47% (15) 67% (39) 55% (32) Upper gastro intestinal tract Pancreatic Biliary tract Breast Thyroid Sarcoma mTLS+% of patients (N) 37% (19) 50% (13) 40% (21) 30% (38) 49% (24) 25% (69) mTLS-% of patients (N) 63% (33) 50% (13) 60% (32) 70% (90) 51% (25) 75% (209) Citation Format: Maxime Brunet, Lucile Vanhersecke, Francois Le Loarer, Isabelle Soubeyran, Antoine Italiano. Prevalence of mature tertiary lymphoid structures and association with tumor mutational burden in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2361.

9040 Background: Despite anti-monoclonal antibodies targeting programmed cell death-1 (PD-1) or its ligand PD-L1 revolution, most patients (pts) with advanced NSCLC displayed primary resistance to PD1/PD-L1 blockade. Identifying targetable alterations involved in resistance represent a promising approach to improve immunotherapy efficacy. TROP2 expression is associated with a poor prognosis in NSCLC, and offers a promising target for treatment with the development of new antibody drug conjugate. TROP2 has been suggested to play a role in innate immunity response, but, it's unclear whether TROP2 expression by tumor cells is detrimental specifically in the context of PD1/PD-L1 blockade. The aim of our study was to investigate the the role of TROP2 expression in outcome of NSCLC pts treated with ICI. Methods: We analyzed the largest NSCLC gene expression data set worldwide including the whole-transcriptome profile of 891 pre-treatment tumors from POPLAR and OAK trials, two randomized studies assessing the efficacy of atezolizumab versus docetaxel in NSCLC. We complemented this transcriptional approach with multiplex IF and digital pathology analysis to confirm our findings at the protein level We investigated whether a non-invasive approach focusing on the level of plasma levels of circulating TROP2 may be relevant to predict efficacy of ICI. Results: The PFS of pts with high expression of TROP2 was significantly lower than in pts with low expression 2.5 vs 4.1 months, p < 0.001. Strikingly, high TROP2 expression was also associated with lower clinical benefit rate 15% vs 28%, p = 0.009 and worse overall survival (OS) 12.6 vs 16.3 months, p = 0.007 When adjusting for covariates such as histological subtype, TLS signature, PD-L1 expression, TROP2 remained independently associated with PFS and OS. None of these correlations were observed in the docetaxel arm, suggesting a predictive value of TROP2 gene expression specifically for response to ICI. By using a deconvolution approach, we also observed that TROP2-high tumors were characterized by a significant lowest expression of genes specific to immune populations (T effector memory cells,Th17 cells). Multiplex IF on an independent cohort 53 NSCLC tumors confirmed the correlation between high TROP2, microenvironment features and poor response to PD1 inhibition. Analysis of circulating plasma levels of TROP2 in 97 NSCLC patients revealed a strong correlation with expression in tumor tissue. High plasma levels of circulating TROP2 was also significantly associated with worse ORR, PFS and OS on treatment with PD1/PDL1 antagonist. Conclusions: Our study revealed that TROP2 expression by tumor cells in NSCLC is associated with worse outcome in patients with NSCLC independently of PDL1 expression. Targeting TROP2 in combination with ICI may represent a promising strategy to improve ICI efficacy in pts with TROP2-high NSCLC.

The presence of tertiary lymphoid structures (TLSs) and a high intratumoral density of B lymphocytes have been associated with improved survival in several tumor types. TLSs are heterogenous organized lymphoid aggregates, their mature form containing a germinal center with follicular B cells juxtaposing CD4+ follicular helper T cells and intricated with CD21+CD23+ follicular dendritic cells. We investigated the impact of TLS on responses to immunotherapy. By performing a large-scale retrospective analysis of three independent cohorts of patients with different cancer types (including soft-tissue sarcomas, lung, bladder, colorectal, head and neck, breast cancer and others, n &gt; 500) treated with anti-PD-1 or anti-PD-L1 antibodies, we showed that the presence of mature TLSs was associated with higher objective response rates (ORR), progression-free survival (PFS) and overall survival (OS), independently of PD-L1 expression status and CD8+ T cell density. To decipher the role of B cells in supporting responses to immunotherapy by immune check-point blockers and patients’ survival, we focused on two cancers: clear cell Renal Cell Carcinoma (ccRCC) and Soft Tissue Sarcoma (STS):The randomized phase II BIONIKK trial (NCT02960906) has recently shown that molecular grouping of tumors enabled enhanced response rate to Nivolumab (N), Nivolumab + Ipilimumab (NI) and VEGFR-Tyrosine Kinase Inhibitor (TKI) in patients with frontline metastatic RCC (mRCC). 199 pts were randomized and treated with N (40 pts), NI (101pts) and TKI (58pts). After a median follow-up of 46.5 months, 86 (43%) patients died: 27/58 (46.5%), 39/101 (39%) and 20/40 (50%) in the N, NI, and TKI arm, respectively. Median OS was 35 months with N, not reached with NI and 45 months with TKI. 160/199 mRCC patients had available FFPE samples (N: 42, NI: 84, TKI: 34). In N-treated patients, number of TLS&gt;2 was associated with higher ORR (73% versus 15%, p=0.01), longer PFS (p=0.015) and less upfront progressions (no progression at 6 weeks, p=0.02). In patients treated with NI, TLS&gt;2 correlated with higher ORR (71% versus 40%, p=0.02), fewer events of early progressions (no progression at 6 months) (p=0.04) and a high TLS transcriptomic signature correlated with higher ORR (79%) versus 25% in TKI-treated patients. Analyzing Immunoglobulin (Ig) gene expression and repertoire, we showed that TLS+ tumors depicted higher numbers of IgM, IgG1 and IgA unique clonotypes and a higher number of over-represented clones found more than 30 times in the same tumor. We also performed spatial transcriptomics and examined the nature of B cell responses within TLS structures. B cells were enriched inside TLS, and therein we could identify all B cell maturation stages from naïve B cells to plasma cells (PCs) formation. B cell repertoire analysis revealed clonal diversification, selection, expansion in TLS and the presence of fully mature clonotypes at distance. In TLS+ tumors, IgG producing PCs disseminated into the tumor beds along fibroblastic tracks. TLS+ tumors exhibited high frequencies of IgG-producing PCs and high numbers of IgG-stained and apoptotic malignant cells, suggestive of anti-tumor effector activity. Proximity analyses revealed the presence of activated macrophages located close to IgG-coated cleaved caspase 3 positive tumor cells, reflecting activation of antibody-dependent cell mediated cytotoxicity. Patients with high numbers of IgG-stained tumor cells (above 60%) exhibited higher ORR (62% versus 18%, p=0.03) and PFS (17 versus 6 months, p=0.03) when treated with NI.PEMBROSARC is a multi-cohort phase II study of pembrolizumab combined with low-dose cyclophosphamide in patients with advanced STS. The 6-months non-progression rate (NPR) and the ORR previously reported in cohorts including all comers were 4.9% and 2.4%, respectively. In the cohort enrolling patients selected based on the presence of TLS, the 6-month NPR was 40% and the ORR was 30%. Exploratory analyses revealed that infiltration by mature dendritic cells, activated macrophages and IgG -producing PCs was associated with improved clinical outcome. the Presence of IgG antibodies on tumor cells was documented in some cases. Altogether, these data establish that maturation, selection and amplification of B cells in intratumoral TLSs results in PC generation which may produce anti-tumor antibodies. Macrophage-dependent killing of tumor cells results in the release of immune complexes of IgG with tumor-associated antigens; the latter are endocytosed by dendritic cells that present tumor-specific and self-antigens to T cells in a very efficient way, amplifying “in situ” antitumor immune responses and lowering the threshold for T lymphocytes reinvigoration by anti-checkpoint inhibitors. It represents an amplification loop that may reinforce therapeutic responses to checkpoint inhibitors, particularly in tumors with low mutational burden. Mechanisms underlying B cell maturation, plasma cell generation and antibody production and their regulation in TLS will be discussed. Citation Format: Wolf Herman Fridman, Maxime Meylan, Yann Vano, Guilhem Pupier, Antoine Bougouin, Anne Calvez, Florent Petitprez, Cheng Ming Sun, Margot Mathieu, Lucile Vanhersecke, François Le Loarer, Alban Bessede, Stephane Oudard, Antoine Italiano, Catherine Sautès-Fridman. Generation of B cell immunity in tertiary lymphoid structures supports response to immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY16-02.

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