Protein expression in formalin-fixed, paraffin-embedded tissue is routinely measured by IHC or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital spatial profiling ...(DSP) allows spatially informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma.
The NanoString GeoMx DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in non-small cell lung cancer (NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 patients with immunotherapy-treated melanoma on a tissue microarray using a 44-plex immune marker panel measured in three compartments (macrophage, leukocyte, and melanocyte) generating 132 quantitative variables.
The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared with QIF for stromal CD3, CD4, CD8, CD20, and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression-free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68-positive cells (macrophages) and not in tumor cells was a predictive marker for PFS, OS, and response.
DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity, we found a series of expression patterns associated with outcome, including that the expression of PD-L1 in macrophages is associated with response.
The widespread adoption of immune checkpoint inhibitors and small molecule inhibitors of the MAP kinase pathway has transformed the management of locally advanced and metastatic melanoma. Here, we ...provide a broad overview on the use of these agents in the first-line setting, incorporating a review of the clinical literature as well as the practice patterns of our respective melanoma groups. Throughout, we highlight areas of uncertainty that provide opportunities for future clinical investigation and additional improvement in outcomes for patients with melanoma.
Because durable response to programmed cell death 1 (PD-1) inhibition is limited to a subset of melanoma patients, new predictive biomarkers could have clinical utility. We hypothesize that ...pretreatment tumor-infiltrating lymphocyte (TIL) profiles could be associated with response.
Pretreatment whole tissue sections from 94 melanoma patients treated with anti-PD-1 therapy were profiled by multiplex immunofluorescence to perform TIL quantification (CD4, CD8, CD20) and assess TIL activation (CD3, GZMB, Ki67). Two independent image analysis technologies were used: inForm (PerkinElmer) to determine cell counts, and AQUA to measure protein by quantitative immunofluorescence (QIF). TIL parameters by both methodologies were correlated with objective response or disease control rate (ORR/DCR) by RECIST 1.1 and survival outcome.
Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in complete or partial response than in stable or progressive disease, particularly for CD8 (
< 0.0001). Neither TIL activation nor dormancy was associated with outcome. CD8 associations with progression-free survival (HR > 3) were independently significant in multivariable analyses and accounted for similar CD3 associations in anti-PD-1-treated patients. CD8 was not associated with melanoma prognosis in the absence of immunotherapy. Predictive performance of CD8 cell count (and QIF) had an area under the ROC curve above 0.75 (ORR/DCR), which reached 0.83 for ipilimumab plus nivolumab.
Pretreatment lymphocytic infiltration is associated with anti-PD-1 response in metastatic melanoma. Quantitative TIL analysis has potential for application in digital precision immuno-oncology as an "indicative" companion diagnostic.
In a retrospective analysis of patients with unresectable melanoma, higher pretreatment tissue densities of CD16+ macrophages were associated with clinical benefit from combined CTLA-4 and PD-1 ...blockade. With further validation, this biomarker could serve as a tool in selecting between immune checkpoint inhibitor regimens. See related article by Lee et al., p. 2513.
The IMMUNED study is a phase 2, investigator-sponsored, placebo-controlled, double-blind study of adjuvant nivolumab 1 mg/kg plus ipilimumab 3 mg/kg versus nivolumab 3 mg/kg alone versus double ...placebo control in patients with stage IV melanoma who have no evidence of disease following surgical resection or radiotherapy of all stage IV metastases. 167 patients were randomly assigned to receive either nivolumab plus ipilimumab (n=56), nivolumab plus ipilimumab-matching placebo (n=59), or double placebo control (n=52). The authors previously reported the primary endpoint of relapse-free survival; compared with placebo, nivolumab plus ipilimumab and nivolumab alone led to improved relapse-free survival (hazard ratio HR 0·23; 97·5% CI 0·12–0·45; p<0·0001, and HR 0·56; 0·33–0·94; p=0·011, respectively).5 In The Lancet, Elisabeth Livingstone and colleagues7 present updated data on the IMMUNED study's primary recurrence-free survival endpoint at a more mature median follow-up of 49·2 months, as well as the first and final report of the trial's secondary endpoint of overall survival. Among the 42 patients in the placebo group whose disease recurred, 38 (90%) received subsequent systemic therapies known to improve overall survival, including anti-PD-1 (28 67% of 42) within or outside protocol-specified crossover; anti-PD-1 plus anti-CTLA-4 (five 12% of 42); or BRAF plus MEK inhibition (five 12% of 42; post-hoc analysis; figure 4 and appendix p 17 of the Article).7 As the authors suggest, these subsequent treatments probably explain why the recurrence-free survival benefit for adjuvant nivolumab versus placebo (HR 0·60, 97·5% CI 0·36–1·00; p=0·024) did not translate into an overall survival benefit.
Over the past decade, the use of immune checkpoint inhibitors (ICI) has expanded across a wide spectrum of oncology indications. Immune-related adverse events (irAE) from ICIs represent a significant ...source of morbidity, and in rare instances, can lead to treatment-related mortality. There are significant opportunities to better identify patients at increased risk for immune-related toxicity, diagnose irAEs more accurately and earlier in their course, and develop more individualized therapeutic strategies once complications arise. Clinical characteristics, germline and somatic genetic features, microbiome composition, and circulating biomarkers have all been associated with higher risk of developing irAEs in retrospective series. Many of these data suggest that both antitumor and anti-host ICI-associated immune reactions may be driven by common features of either the tumor or the patient's preexisting immune milieu. While irAE diagnosis is currently based on clinical history, exclusion of alternative etiologies, and sometimes pathologic confirmation, novel blood-based and radiographic assays are in development to identify these complications more precisely. Anecdotal reports and small case series have highlighted the potential role of targeted immunomodulatory agents to treat irAEs, though further prospective investigation is needed to evaluate more rigorously their use in these settings. In this review, we highlight the current state of knowledge about predicting, diagnosing, and treating irAEs with a translational focus and discuss emerging strategies which aim to improve each of these domains.
Programmed death 1/programmed death ligand 1 (PD-L1) axis inhibitors have been proven effective, especially in patients with tumors expressing PD-L1. Their clinical efficacy in patients with ...EGFR-activating mutations is still unclear, whereas KRAS mutations seem to be associated with good response.
We used multiplexed quantitative immunofluorescence to investigate PD-L1 expression and to characterize tumor infiltrating lymphocyte (TIL) populations and their activation status in more than 150 NSCLC patients with known mutation status.
PD-L1 expression was significantly lower in EGFR-mutant compared to KRAS-mutant, and EGFR/KRAS wild-type (WT) tumors. KRAS mutant tumors were more inflamed with higher CD4+, CD8+ and CD20+ TILs. Subgroup analysis by TIL activation status revealed that EGFR mutants had a high frequency of inactive TILs even though lymphocytes were present in the tumor microenvironment. In contrast, in KRAS mutants, when TILs were present they were almost always active. Additionally, we found differences between EGFR mutation sites in CD8+ expression and the TIL activation profile. Finally, activated EGFR correlated with increased PD-L1 expression in EGFR mutants but not in EGFR WT, whereas TIL activation was associated with higher PD-L1 only in EGFR/KRAS WT.
Our findings show the unique immune profile of EGFR-mutant tumors. The high frequency of inactive TILs could explain the low immunotherapy response rates in these patients, whereas PD-L1 as a predictive biomarker may reflect the constitutive oncogenic signaling rather than immune signaling, which would be associated with high PD-L1 levels and TILs activation.
Grossmann and colleagues report the results of a large randomized trial demonstrating improved recurrence-free survival with adjuvant pembrolizumab in resected melanoma compared with adjuvant ...ipilimumab or IFNα2b. However, it remains unclear whether adjuvant immunotherapies extend overall survival as outcomes for patients with advanced melanoma continue to improve. See related article by Grossmann et al., p. 644 (1).
With the increasing promise of long-term survival with immune checkpoint blockade (ICB) therapies, particularly for patients with advanced melanoma, clinicians and investigators are driven to ...identify prognostic and predictive factors that may help to identify individuals who are likely to experience durable benefit. Several ICB combinations are being actively developed to expand the armamentarium of treatments for patients who may not achieve long-term responses to ICB single therapies alone. Thus, negative predictive markers are also of great interest. This review seeks to deepen our understanding of the mechanisms underlying the durability of ICB treatments. We will discuss the currently available long-term data from the ICB clinical trials and real-world studies describing the survivorship of ICB-treated melanoma patients. Additionally, we explore the current treatment outcomes in patients rechallenged with ICB and the patterns of ICB resistance based on sites of disease, namely, liver or CNS metastases. Lastly, we discuss the landscape in melanoma in the context of prognostic or predictive factors as markers of long-term response to ICB.
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