Concentrations of S100B and neurofilament light chain in blood as biomarkers for checkpoint inhibitor-induced CNS inflammation.

BACKGROUND: Cancer treatment with immune checkpoint inhibition (ICI) can cause immune-related adverse events in the central nervous system (CNS irAE). There are no blood biomarkers to detect CNS irAE. We investigated if concentrations of S100-calcium-binding protein B (S100B) and neurofilament light chain (NfL) in blood can be used as biomarkers for CNS irAE and assessed the incidence of CNS irAE in a cohort of ICI-treated patients. METHODS: In this single-centre, retrospective cohort study, we examined medical records and laboratory data of 197 consecutive patients treated with combined CTLA-4 and PD-1 inhibition (ipilimumab; ipi + nivolumab; nivo) for metastatic melanoma or renal cell carcinoma. CNS irAE was diagnosed using established criteria. Concentrations of S100B and NfL in blood were measured in patients with CNS irAE and in 84 patients without CNS irAE. FINDINGS: Nine of 197 patients (4.6%) fulfilled criteria for CNS irAE. S100B and NfL in blood increased during CNS inflammation and normalized during immunosuppression. CNS irAE was detected with a sensitivity of 100% (S100B) and 79% (NfL) and a specificity of 89% (S100B) and 74% (NfL). Patients with CNS irAE had simultaneous increased concentration of C-reactive protein (CRP) (9/9) and alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in blood (8/9). INTERPRETATION: Analysis of S100B, NfL and CRP in blood facilitates the diagnosis of CNS irAE. CNS irAE may be more common than previously reported. There may be shared immune mechanisms between CNS and hepatitis irAE. FUNDING: Supported by funding from the Swedish Cancer Foundation, the ALF-agreement, and Jubileumsklinikens Cancerfond.

Immune mapping of human tuberculosis and sarcoidosis lung granulomas.

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas "multifocal" granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40-60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.

Intussusceptive Angiogenesis in Human Metastatic Malignant Melanoma.

Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, in vitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation in vitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.

Early rise in brain damage markers and high ICOS expression in CD4+ and CD8+ T cells during checkpoint inhibitor-induced encephalomyelitis.

We report a case of rapid eradication of melanoma brain metastases and simultaneous near-fatal encephalomyelitis following double immune checkpoint blockade. Brain damage marker S-100B and C reactive protein increased before symptoms or signs of encephalomyelitis and peaked when the patient fell into a coma. At that point, additional brain damage markers and peripheral T cell phenotype was analyzed. The analyses were repeated four times during the patient's recovery. Axonal damage marker neurofilament light polypeptide (NFL) and astrocytic damage marker glial fibrillar acidic protein (GFAP) were very high in blood and cerebrospinal fluid and gradually normalized after immunosuppression and intensive care. The costimulatory receptor inducible T cell costimulatory receptor (ICOS) was expressed on a high proportion of CD4+ and CD8+T cells as encephalomyelitis symptoms peaked and then gradually decreased in parallel with clinical improvement. Both single and double immune checkpoint inhibitor-treated melanoma patients with other serious immune-related adverse events (irAE) (n=9) also expressed ICOS on a significantly higher proportion of CD4+ and CD8+T cells compared with controls without irAE (n=12). In conclusion, our results suggest a potential role for ICOS on CD4+ and CD8+T cells in mediating encephalomyelitis and other serious irAE. In addition, brain damage markers in blood could facilitate early diagnosis of encephalitis.

Response to BRAF/MEK Inhibition in A598_T599insV BRAF Mutated Melanoma.

Approximately 50% of patients with metastatic melanoma harbor an activating BRAF mutation. Tumors with activating mutation BRAF gene proliferate excessively and can be treated with targeted BRAF-inhibitors in combination with MEK inhibitors. The most common BRAF mutation occurs at amino acid position 600. Other BRAF mutations are rare and their predictive value for treatment response to BRAF/MEK inhibition is low. Here, we report on a patient with a BRAF A598_T599insV mutated melanoma, a mutation that has only been described in one previous melanoma patient in which the treatment response to BRAF/MEK inhibition was transient. Our patient had a large ulcerated metastasis that showed a durable complete response implying that BRAF/MEK inhibition should be considered a treatment option for this mutation. We analyzed circulating cell-free tumor DNA (ctDNA) carrying the BRAF A598_T599insV mutation throughout treatment. The allele frequency of BRAF A598_T599insV decreased during regression of the tumors, indicating that this method has potential to monitor treatment response. Our case demonstrates durable response to BRAF/MEK inhibition in a melanoma patient carrying a BRAF A598_T599insV mutation. In addition, we show that allele frequency analysis of A598_T599insV mutation in blood using ultrasensitive sequencing can be used to monitor treatment response.