ScRNA-seq defines dynamic T-cell subsets in longitudinal colon and peripheral blood samples in immune checkpoint inhibitor-induced colitis.

Immune checkpoint inhibitors (ICIs) are increasingly being used to manage multiple tumor types. Unfortunately, immune-related adverse events affect up to 60% of recipients, often leading to treatment discontinuation in settings where few alternative cancer therapies may be available. Checkpoint inhibitor induced colitis (ICI-colitis) is a common toxicity for which the underlying mechanisms are poorly defined. To better understand the changing colon-specific and peripheral immune environments over the course of progression and treatment of colitis, we collected blood and colon tissue from a patient with Merkel cell carcinoma who developed colitis on treatment with pembrolizumab. We performed single-cell RNA sequencing and T-cell receptor sequencing on samples collected before, during and after pembrolizumab and after various interventions to mitigate toxicity. We report T-cells populations defined by cytotoxicity, memory, and proliferation markers at various stages of colitis. We show preferential depletion of CD8+ T cells with biologic therapy and nominate both circulating and colon-resident T-cell subsets as potential drivers of inflammation and response to immune suppression. Our findings highlight the need for further exploration of the colon immune environment and rationalize future studies evaluating biologics for ICI-colitis, including in the context of ICI re-challenge.

Immune dysfunction revealed by digital spatial profiling of immuno-oncology markers in progressive stages of renal cell carcinoma and in brain metastases.

BACKGROUND: The tumor microenvironment (TME) contributes to cancer progression and treatment response to therapy, including in renal cell carcinoma (RCC). Prior profiling studies, including single-cell transcriptomics, often involve limited sample sizes and lack spatial orientation. The TME of RCC brain metastases, a major cause of morbidity, also remains largely uncharacterized. METHODS: We performed digital spatial profiling on the NanoString GeoMx platform using 52 validated immuno-oncology markers on RCC tissue microarrays representing progressive stages of RCC, including brain metastases. We profiled 76 primary tumors, 27 adjacent histologically normal kidney samples, and 86 metastases, including 24 brain metastases. RESULTS: We observed lower immune checkpoint (TIM-3 and CTLA-4), cytolytic (GZMA and GZMB), and T cell activation (CD25) protein expression in metastases compared with primary tumors in two separate cohorts. We also identified changes in macrophages in metastases, with brain metastases-susceptible patients showing less M1-like, inflammatory macrophage markers (HLA-DR and CD127) in metastatic samples. A comparison of brain metastases to extracranial metastases revealed higher expression of the anti-apoptotic, BCL-2-family protein BCL-XL and lower expression of the innate immune activator STING in brain metastases. Lower TIM-3 and CD40 in the TME of brain metastases appear to be associated with longer survival, a finding that requires further validation. CONCLUSIONS: Compared with primary tumors, RCC metastases, including brain metastases, express lower levels of numerous markers of immune activation and current or investigational therapeutic targets. Our findings may have important implications for designing future biomarker and treatment studies and may aid in development of brain metastases-specific therapies.

Management of patients with brain metastases from NSCLC without a genetic driver alteration: upfront radiotherapy or immunotherapy?

Lung cancer is the second most common cancer and the most common cause of cancer-related death in the United States. Brain metastases (BM) are detected in 21% of patients with lung cancer at the time of diagnosis and are the sole metastatic site in 35% of patients with stage IV disease. The best upfront therapy for non-small-cell lung cancer depends on both tumor programmed death 1 ligand-1 (PD-L1) expression and the presence or absence of a targetable genetic alteration in genes such as epidermal growth factor receptor and anaplastic lymphoma kinase. In the absence of a targetable genetic alteration, options include chemotherapy, immune checkpoint inhibitors (ICIs), and ICI combined with chemotherapy. Upfront local therapy followed by systemic therapy is the current standard of care for the management of BM, and may include whole brain radiotherapy, stereotactic radiosurgery (SRS), or craniotomy for surgical resection followed by consolidative SRS. This paradigm is effective in achieving local control, but it remains unclear if this approach is necessary for every patient. Prospective and retrospective data suggest that ICIs with or without chemotherapy can have activity against BM; however, appropriately selecting patients who are able to safely forgo local therapy and start an ICI-based treatment remains a challenge. To be considered for upfront ICI-based therapy, a patient should be free of neurologic symptoms, lesions should be small and not located in a critical region of the central nervous system, if corticosteroids are indicated the requirement should be low (prednisone 10 mg/d or less), and PD-L1 expression should be high. The decision to proceed with upfront ICI without local therapy to BM should be made in a multidisciplinary fashion and patients should undergo frequent surveillance imaging so that salvage local therapy can be administered when necessary. Prospective clinical trials are needed to validate this approach before it can be widely adopted.

Location matters: LAG3 levels are lower in renal cell carcinoma metastatic sites compared to primary tumors, and expression at metastatic sites only may have prognostic importance.

While great strides have been made in the treatment of advanced renal cell carcinoma (RCC) with the emergence of immune checkpoint inhibitors (ICIs) and VEGFR-targeting drugs, sizable proportions of patients still do not respond to upfront therapy and long-term responses only occur in a minority of patients. There is therefore a great need for the development of better predictors of response and an increased understanding of mechanisms of resistance to these therapies. Alternative immune checkpoints outside the PD-1/PD-L1 axis, such as LAG3, have been implicated as one mechanism of resistance to ICIs. These checkpoints thus represent attractive therapeutic targets, and indeed the LAG3 inhibitor relatlimab was recently approved for the treatment of metastatic melanoma in combination with anti-PD-1 therapy. LAG3 inhibitors are being evaluated for RCC as well. In this context, a better understanding of LAG3 expression patterns in RCC and how they relate to clinicopathologic features of disease and response to immunotherapy may give insight into mechanisms of resistance to PD-1 inhibitors and aid in the identification of subgroups of patients more likely to benefit from certain drug regimens. In this study, we assessed LAG3 protein levels in leukocytes in normal kidney adjacent to RCC, primary RCC tumors, and matched metastatic tumors, including large numbers of brain metastases. We found that LAG3 protein levels are on average lower at metastatic sites compared to matched primary tumors, and that the difference was more pronounced in patients with high-risk clinical characteristics, including those with larger primary tumor size, grade 4 tumors, IMDC poor-risk disease, and initial presentation with brain metastases. We further saw that the prognostic value of LAG3 levels varies depending on the tissue site queried (i.e., primary tumor versus metastases), and that relatively higher LAG3 levels at metastatic sites may predict a better response to immunotherapy and longer overall survival after the development of metastatic disease. These findings may have important implications for the design of future studies involving LAG3 or other immunotherapies in RCC.

Keratin 17 is overexpressed and predicts poor survival in estrogen receptor-negative/human epidermal growth factor receptor-2-negative breast cancer.

Clinicopathological features of breast cancer have limited accuracy to predict survival. By immunohistochemistry (IHC), keratin 17 (K17) expression has been correlated with triple-negative status (estrogen receptor [ER]/progesterone receptor/human epidermal growth factor receptor-2 [HER2] negative) and decreased survival, but K17 messenger RNA (mRNA) expression has not been evaluated in breast cancer. K17 is a potential prognostic cancer biomarker, targeting p27, and driving cell cycle progression. This study compared K17 protein and mRNA expression to ER/progesterone receptor/HER2 receptor status and event-free survival. K17 IHC was performed on 164 invasive breast cancers and K17 mRNA was evaluated in 1097 breast cancers. The mRNA status of other keratins (16/14/9) was evaluated in 113 ER-/HER2- ductal carcinomas. IHC demonstrated intense cytoplasmic and membranous K17 localization in myoepithelial cells of benign ducts and lobules and tumor cells of ductal carcinoma in situ. In ductal carcinomas, K17 protein was detected in most triple-negative tumors (28/34, 82%), some non-triple-negative tumors (52/112, 46%), but never in lobular carcinomas (0/15). In ductal carcinomas, high K17 mRNA was associated with reduced 5-year event-free survival in advanced tumor stage (n = 149, hazard ratio [HR] = 3.68, P = .018), and large (n = 73, HR = 3.95, P = .047), triple-negative (n = 103, HR = 2.73, P = .073), and ER-/HER2- (n = 113, HR = 2.99, P = .049) tumors. There were significant correlations among keratins 17, 16, 14, and 9 mRNA levels suggesting these keratins (all encoded on chromosome 17) could be coordinately expressed in breast cancer. Thus, K17 is expressed in a subset of triple-negative breast cancers, and is a marker of poor prognosis in patients with advanced stage and ER-/HER2- breast cancer.

99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer.

UNLABELLED: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and small-molecule radiopharmaceuticals targeting PSMA rapidly detect the location and extent of disease. Here we evaluated preclinically 4 novel (99m)Tc-labeled small-molecule inhibitors of PSMA with the potential for clinical translation for molecular imaging of prostate cancer in humans. METHODS: Four PSMA inhibitors derived from the glutamate-urea-glutamate or glutamate-urea-lysine pharmacophores conjugated to CIM or TIM chelators were radiolabeled with (99m)Tc and evaluated in vitro and in vivo. RESULTS: High-affinity, saturable binding to PSMA on LNCaP cells was observed with Kd values of 0.64 ± 0.46 nM for (99m)Tc-MIP-1427, 1.07 ± 0.89 nM for (99m)Tc-MIP-1404, 1.75 ± 0.32 nM for (99m)Tc-MIP-1428, and 4.35 ± 0.35 nM for (99m)Tc-MIP-1405. (99m)Tc-labeled PSMA inhibitors did not bind human prostate cancer PC3 cells, which lack PSMA, demonstrating specificity, and binding was abolished with 2-(phosphonomethyl)pentanedioic acid (PMPA), a structurally unrelated PSMA inhibitor. (99m)Tc-labeled PSMA inhibitors were shown to internalize at 37 °C. Uptake in LNCaP xenografts ranged from 9.3% to 12.4% injected dose per gram at 1 h after injection and from 7.2% to 11.0% at 4 h, with tumor-to-blood ratios ranging from 29:1 to 550:1 and tumor-to-skeletal muscle ratios ranging from 31:1 to 157:1 at 4 h. (99m)Tc-MIP-1404 exhibited the best combination of high tumor uptake and rapid clearance from kidney and nontarget tissues. (99m)Tc-MIP-1404 specifically bound to PSMA in vivo as demonstrated by the absence of uptake in PC3 xenografts and by competition with PMPA. SPECT/CT imaging corroborated the tissue distribution results, demonstrating uptake only in PSMA-expressing kidney and tumor tissue and clearance through the urinary bladder. CONCLUSION: These (99m)Tc-labeled radiopharmaceuticals targeting PSMA may provide a SPECT molecular imaging option to assist in the initial diagnosis of prostate cancer and the management of patient care by monitoring disease progression.