Melanoma clonal heterogeneity leads to secondary resistance after adoptive cell therapy with tumor-infiltrating lymphocytes.

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) is effective in melanoma patients, although long-term responses seem restricted to patients who have complete remissions. Many patients develop secondary resistance to TIL-ACT but the involved mechanisms are unclear. Here, we describe a case of secondary resistance to TIL-ACT likely due to intratumoral heterogeneity and selection of a resistant tumor cell clone by the transferred T cells. To our knowledge, this is the first case of clonal selection of a pre-existing non-dominant tumor cell clone and it demonstrates a mechanism involved in secondary resistance to TIL-ACT that could potentially change current clinical practice, because it advocates for T-cell collection from multiple tumor sites and analysis of tumor heterogeneity before the treatment with TIL-ACT.

Response to tumor-infiltrating lymphocyte adoptive therapy is associated with preexisting CD8+ T-myeloid cell networks in melanoma.

Adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell-intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8+ TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor-reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

PD-1-expressing macrophages and CD8 T cells are independent predictors of clinical benefit from PD-1 inhibition in advanced mesothelioma.

BACKGROUND: Few tissue biomarkers exist to date that could enrich patient with cancer populations to benefit from immune checkpoint blockade by programmed cell death protein 1/ligand-1 (PD-/L-1) inhibitors. PD-L1 expression has value in this context in some tumor types but is an imperfect predictor of clinical benefit. In malignant pleural mesothelioma, PD-L1 expression is not predictive of the benefit from PD-1 blockade. We aimed to identify novel markers in malignant pleural mesothelioma to select patients better. METHODS: We performed a multiplex-immune histochemistry analysis of tumor samples from the phase III PROMISE-meso study, which randomized 144 pretreated patients to receive either pembrolizumab or standard second-line chemotherapy. Our panel focused on CD8+T cell, CD68+macrophages, and the expression of PD-1 and PD-L1 on these and cancer cells. We analyzed single and double positive cells within cancer tissues (infiltrating immune cells) and in the stroma. In addition, we performed cell neighborhood analysis. The cell counts were compared with clinical outcomes, including responses, progression-free and overall survivals. RESULTS: We confirmed the absence of predictive value for PD-L1 in this cohort of patients. Furthermore, total CD8 T cells, CD68+macrophages, or inflammatory subtypes (desert, excluded, inflamed) did not predict outcomes. In contrast, PD-1-expressing CD8+T cells (exhausted T cells) and PD-1-expressing CD68+macrophages were both independent predictors of progression-free survival benefit from pembrolizumab. Patients with tumors simultaneously harboring PD1+T cells and PD-1+macrophages benefited the most from immune therapy. CONCLUSION: We analyzed a large cohort of patients within a phase III study and found that not only PD-1+CD8 T cells but also PD-1+CD68+ macrophages are predictive. This data provides evidence for the first time for the existence of PD-1+macrophages in mesothelioma and their clinical relevance for immune checkpoint blockade.

Response and Resistance to Trametinib in MAP2K1-Mutant Triple-Negative Melanoma.

The development of targeted therapies for non-BRAF p.Val600-mutant melanomas remains a challenge. Triple wildtype (TWT) melanomas that lack mutations in BRAF, NRAS, or NF1 form 10% of human melanomas and are heterogeneous in their genomic drivers. MAP2K1 mutations are enriched in BRAF-mutant melanoma and function as an innate or adaptive resistance mechanism to BRAF inhibition. Here we report the case of a patient with TWT melanoma with a bona fide MAP2K1 mutation without any BRAF mutations. We performed a structural analysis to validate that the MEK inhibitor trametinib could block this mutation. Although the patient initially responded to trametinib, he eventually progressed. The presence of a CDKN2A deletion prompted us to combine a CDK4/6 inhibitor, palbociclib, with trametinib but without clinical benefit. Genomic analysis at progression showed multiple novel copy number alterations. Our case illustrates the challenges of combining MEK1 and CDK4/6 inhibitors in case of resistance to MEK inhibitor monotherapy.

Deep and lasting response and acquired resistance to BRAFV600E targeting in a low-grade ovarian cancer patient.

The treatment of BRAFV600E mutant melanoma has been revolutionized by BRAF inhibitors. Furthermore, the BRAF/MEK combination has shown further improvement in clinical outcomes in advanced and in adjuvant melanoma patients. In low-grade ovarian tumors, BRAF inhibitor use has been also proposed. Here we present a patient with an excellent, lasting response to BRAF therapy alone. At first progression, after more than two years on BRAF monotherapy, we could not identify any molecular mechanisms explaining resistance. After a switch to dual BRAF/MEK therapy, the patient responded. However, despite the initial response clinical the patient again progressed, this time with the appearance of a KRAS G12C mutation, which could not be overcome by BRAF/MEK therapy. We provide evidence that BRAF inhibitor alone can be highly beneficial in BRAF mutant low-grade ovarian tumors and the resistance mechanisms are similar to that of other BRAF mutant tumors, including in melanoma.

A neutrophil response linked to tumor control in immunotherapy.

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sellhi state rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12, and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy.

Bispecific PD1-IL2v and anti-PD-L1 break tumor immunity resistance by enhancing stem-like tumor-reactive CD8+ T cells and reprogramming macrophages.

Immunotherapies have shown remarkable, albeit tumor-selective, therapeutic benefits in the clinic. Most patients respond transiently at best, highlighting the importance of understanding mechanisms underlying resistance. Herein, we evaluated the effects of the engineered immunocytokine PD1-IL2v in a mouse model of de novo pancreatic neuroendocrine cancer that is resistant to checkpoint and other immunotherapies. PD1-IL2v utilizes anti-PD-1 as a targeting moiety fused to an immuno-stimulatory IL-2 cytokine variant (IL2v) to precisely deliver IL2v to PD-1+ T cells in the tumor microenvironment. PD1-IL2v elicited substantial infiltration by stem-like CD8+ T cells, resulting in tumor regression and enhanced survival in mice. Combining anti-PD-L1 with PD1-IL2v sustained the response phase, improving therapeutic efficacy both by reprogramming immunosuppressive tumor-associated macrophages and enhancing T cell receptor (TCR) immune repertoire diversity. These data provide a rationale for clinical trials to evaluate the combination therapy of PD1-IL2v and anti-PD-L1, particularly in immunotherapy-resistant tumors infiltrated with PD-1+ stem-like T cells.

Aberrant hyperexpression of the RNA binding protein FMRP in tumors mediates immune evasion.

Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP's immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators-interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP's cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.

First communication on the efficacy of combined 177Lutetium-PSMA with immunotherapy outside prostate cancer.

Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy is a validated treatment option for patients with advanced prostate cancer. Although PSMA expression is not limited to prostate tissue, little is known about its relevance to other types of cancer. Here, we present a case report of a patient with uterine leiomyosarcoma that is progressing while on immunotherapy and treated with 177Lu-PSMA radionuclide therapy. We report for the first time that 177Lu-PSMA radionuclide therapy combined with immunotherapy outside of prostate cancer. We did observe post-treatment reduction of tumor growth rate, although we did not notice disease response based on RECIST criteria. We suggest that 177Lu-PSMA treatment especially combined with immunotherapy may be an option for patients with cancer without other therapeutic options. Insights: 177Lu-PSMA radionuclide therapy should be considered for any tumor stained positive for PSMA.

Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity.

Glioblastoma (GBM) is poorly responsive to therapy and invariably lethal. One conceivable strategy to circumvent this intractability is to co-target distinctive mechanistic components of the disease, aiming to concomitantly disrupt multiple capabilities required for tumor progression and therapeutic resistance. We assessed this concept by combining vascular endothelial growth factor (VEGF) pathway inhibitors that remodel the tumor vasculature with the tricyclic antidepressant imipramine, which enhances autophagy in GBM cancer cells and unexpectedly reprograms immunosuppressive tumor-associated macrophages via inhibition of histamine receptor signaling to become immunostimulatory. While neither drug is efficacious as monotherapy, the combination of imipramine with VEGF pathway inhibitors orchestrates the infiltration and activation of CD8 and CD4 T cells, producing significant therapeutic benefit in several GBM mouse models. Inclusion up front of immune-checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) in eventually relapsing tumors markedly extends survival benefit. The results illustrate the potential of mechanism-guided therapeutic co-targeting of disparate biological vulnerabilities in the tumor microenvironment.

Proton Pump Inhibitor Use and Efficacy of Nivolumab and Ipilimumab in Advanced Melanoma.

The impact of proton pump inhibitors (PPIs) on clinical outcomes with first-line immune checkpoint inhibitors (ICIs) in patients with metastatic melanoma was previously analyzed in the phase II study, CheckMate 069. This retrospective analysis utilized data from three phase II/III studies of first-line ICI therapy in untreated advanced melanoma: CheckMate 066, 067, and 069. All randomized patients with PPI use ≤ 30 days before initiating study treatment were included in the PPI-use subgroup. Possible associations between baseline PPI use and efficacy were evaluated within each treatment arm of each study using multivariable modeling. Approximately 20% of 1505 randomized patients across the studies reported baseline PPI use. The median follow-up was 52.6-58.5 months. Objective response rate (ORR), progression-free survival (PFS), and overall survival analyses provided insufficient evidence of a meaningful association between PPI use and efficacy outcomes with nivolumab-plus-ipilimumab, nivolumab, or ipilimumab therapy. In five of the six ICI treatment arms, 95% confidence intervals for odds ratios or hazard ratios traversed 1. Significant associations were observed in the CheckMate 069 combination arm between PPI use and poorer ORR and PFS. This multivariable analysis found insufficient evidence to support meaningful associations between PPI use and ICI efficacy in patients with advanced melanoma.

Sphingolipids control dermal fibroblast heterogeneity.

Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.

Safety and Efficacy of Ipilimumab plus Nivolumab and Sequential Selective Internal Radiation Therapy in Hepatic and Extrahepatic Metastatic Uveal Melanoma.

To assess the safety and efficacy of ipilimumab plus nivolumab around selective internal radiation therapy (SIRT) in patients with metastatic uveal melanoma (mUM). We present a retrospective, single center study of 32 patients with mUM divided into two groups based on the treatment received between April 2013 and April 2021. The SIRT_IpiNivo cohort was treated with Yttrium-90 microspheres and ipilimumab plus nivolumab before or after the SIRT (n = 18). The SIRT cohort underwent SIRT but did not receive combined immunotherapy with ipilimumab plus nivolumab (n = 14). Twelve patients (66.7%) of the SIRT_IpiNivo arm received SIRT as first-line treatment and six patients (33.3%) received ipilimumab plus nivolumab prior to SIRT. In the SIRT group, seven patients (50.0%) received single-agent immunotherapy. One patient treated with combined immunotherapy 68 months after the SIRT was included in this group. At the start of ipilimumab plus nivolumab, 94.4% (n = 17) presented hepatic metastases and 72.2% (n = 13) had extra liver disease. Eight patients (44.4%) of the SIRT_IpiNivo group experienced grade 3 or 4 immune related adverse events, mainly colitis and hepatitis. Median overall survival from the diagnosis of metastases was 49.6 months (95% confidence interval (CI); 24.1-not available (NA)) in the SIRT_IpiNivo group compared with 13.6 months (95% CI; 11.5-NA) in the SIRT group (log-rank p-value 0.027). The presence of extra liver metastases at the time of SIRT, largest liver lesion more than 8 cm (M1c) and liver tumor volume negatively impacted the survival. This real-world cohort suggests that a sequential treatment of ipilimumab plus nivolumab and SIRT is a well-tolerated therapeutic approach with promising survival rates.

Trabectedin and Durvalumab Combination Is Feasible and Active in Relapsing Ovarian Cancer.

The combination of chemotherapy and immune therapies still promises to synergize for prolonged tumor control. However, the quest for optimal combinations tailored for tumor histology remains ongoing. A recent study provides evidence on the feasibility of the trabectedin/durvalumab combination and reports on interesting preliminary efficacy. See related article by Toulmonde et al., p. 1765.

Estimated Costs of the Ipilimumab-Nivolumab Therapy and Related Adverse Events in Metastatic Melanoma.

Combined ipilimumab and nivolumab significantly improve outcomes in metastatic melanoma patients but bear an important financial impact on the healthcare system. Here, we analyze the treatment costs, focusing on irAE. We conducted a retrospective analysis of 62 melanoma patients treated with ipilimumab-nivolumab at the Lausanne University Hospital between 1 June 2016 and 31 August 2019. The frequency of irAEs and outcomes were evaluated. All melanoma-specific costs were analyzed from the first ipilimumab-nivolumab dose until the therapy given subsequently or death. A total of 54/62 (87%) patients presented at least one irAE, and 31/62 (50%) presented a grade 3-4 irAE. The majority of patients who had a complete response 12/14 (86%) and 21/28 (75%) of overall responders presented a grade 3-4 toxicity, and there were no responses in patients without toxicity. Toxicity costs represented only 3% of the total expenses per patient. The most significant contributions were medication costs (44%) and disease costs (39%), mainly disease-related hospitalization costs, not toxicity-related. Patients with a complete response had the lowest global median cost per week of follow up (EUR 2425) and patients who had progressive disease (PD), the highest one (EUR 8325). Except for one patient who had a Grade 5 toxicity (EUR 6043/week), we observe that less severe toxicity grades (EUR 9383/week for Grade 1), or even the absence of toxicity (EUR 9922/week), are associated with higher median costs per week (vs. EUR 3266/week for Grade 4 and EUR 2850/week for Grade 3). The cost of toxicities was unexpectedly low compared to the total costs, especially medication costs. Patients with higher toxicity grades had better outcomes and lower total costs due to treatment discontinuation.

Ovarian cancer with high-level focal ERBB2 amplification responds to trastuzumab and pertuzumab.

Epithelial ovarian cancer (EOC) is usually diagnosed at an advanced stage and significantly contributes to cancer mortality in women. Despite multimodal treatment associating chemotherapy and surgery, most patients ultimately progress and require palliative systemic therapy. In EOC, the efficacy of anti-HER2 agents is minimal even after selecting patients for HER2 expression. ERBB2 gene amplification is observed in 3-10% of patients, depending on the specific method of detection and cutoffs. We report the case of a young woman with a FIGO stage IV high-grade serous ovarian cancer with an amplification of ERBB2. She was treated with the association of trastuzumab - pertuzumab after two lines of standard treatment and presented an excellent long-lasting partial response after 36 months of treatment. The association of trastuzumab and pertuzumab, without chemotherapy, has not been previously tested in this context and could be more efficacious than monotherapy with either agent. In addition, the significant benefit observed in this case could be attributed to the presence of a high-level focal amplification that is relatively rare and probably more specific than an increase in HER2 expression. In conclusion, prospective trials of the trastuzumab and pertuzumab combination should be considered in an appropriately selected EOC patient population.

Cancer Cells Retrace a Stepwise Differentiation Program during Malignant Progression.

Pancreatic neuroendocrine tumors (PanNET) comprise two molecular subtypes, relatively benign islet tumors (IT) and invasive, metastasis-like primary (MLP) tumors. Until now, the origin of aggressive MLP tumors has been obscure. Herein, using multi-omics approaches, we revealed that MLP tumors arise from IT via dedifferentiation following a reverse trajectory along the developmental pathway of islet ß cells, which results in the acquisition of a progenitor-like molecular phenotype. Functionally, the miR-181cd cluster induces the IT-to-MLP transition by suppressing expression of the Meis2 transcription factor, leading to upregulation of a developmental transcription factor, Hmgb3. Notably, the IT-to-MLP transition constitutes a distinct step of tumorigenesis and is separable from the classic proliferation-associated hallmark, temporally preceding accelerated proliferation of cancer cells. Furthermore, patients with PanNET with elevated HMGB3 expression and an MLP transcriptional signature are associated with higher-grade tumors and worse survival. Overall, our results unveil a new mechanism that modulates cancer cell plasticity to enable malignant progression. SIGNIFICANCE: Dedifferentiation has long been observed as a histopathologic characteristic of many cancers, albeit inseparable from concurrent increases in cell proliferation. Herein, we demonstrate that dedifferentiation is a mechanistically and temporally separable step in the multistage tumorigenesis of pancreatic islet cells, retracing the developmental lineage of islet ß cells.This article is highlighted in the In This Issue feature, p. 2355.

Trametinib Induces the Stabilization of a Dual GNAQ p.Gly48Leu- and FGFR4 p.Cys172Gly-Mutated Uveal Melanoma. The Role of Molecular Modelling in Personalized Oncology.

We report a case of an uveal melanoma patient with GNAQ p.Gly48Leu who responded to MEK inhibition. At the time of the molecular analysis, the pathogenicity of the mutation was unknown. A tridimensional structural analysis showed that Gαq can adopt active and inactive conformations that lead to substantial changes, involving three important switch regions. Our molecular modelling study predicted that GNAQ p.Gly48Leu introduces new favorable interactions in its active conformation, whereas little or no impact is expected in its inactive form. This strongly suggests that GNAQ p.Gly48Leu is a possible tumor-activating driver mutation, consequently triggering the MEK pathway. In addition, we also found an FGFR4 p.Cys172Gly mutation, which was predicted by molecular modelling analysis to lead to a gain of function by impacting the Ig-like domain 2 folding, which is involved in FGF binding and increases the stability of the homodimer. Based on these analyses, the patient received the MEK inhibitor trametinib with a lasting clinical benefit. This work highlights the importance of molecular modelling for personalized oncology.

Organoid technology and applications in cancer immunotherapy and precision medicine.

Tumors are complex ecosystems of multiple cell types in addition to cancer cells. The response to therapies can be shaped by not only cancer cell vulnerabilities but also by the cells of the tumor microenvironment (TME). For cellular immune therapies, cancer cells are the direct target, but for most of the immune checkpoint blockades (ICB), the direct therapeutic targets are the immune cells. Current immune therapy approaches, especially immune checkpoint blockades, only work for select patient populations. A pre-treatment, ex vivo assay could help in selecting between immune therapy options. However, for immune therapy applications, ex vivo assays would require the co-culturing of both cancer cells and cells of the TME. New results show that it is now feasible to co-culture both cell types with organoid technologies. However, many challenges remain to both optimize organoid cultures as well as to validate ex vivo assays as predictors of therapeutic benefits from immune therapies.

High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays.

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.