Tumor Mutational Burden and Other Immunotherapy Markers in Histiocytic Neoplasms Using Next Generation Sequencing
Authors: Goyal Gaurav , Vassallo Robert , Rech Karen L , Ryu Jay H , Davidge-Pitts Caroline J , Tobin W Oliver , Koster Matthew J , Bennani Nabila Nora , Shah Mithun Vinod , Liu Minetta C , Go Ronald S. 
Published: October 2019

Abstract
Introduction
Histiocytic neoplasms are rare hematological malignancies that have protean clinical manifestations and can pose significant management challenges. Recently, vemurafenib was approved by the US-FDA for treatment of BRAF-V600-mutant Erdheim-Chester disease (ECD). However, there is a lack of FDA-approved therapies for other histiocytic neoplasms such as Langerhans cell histiocytosis (LCH) and Rosai-Dorfman disease (RDD). Over the last 5 years, immune checkpoint inhibitors such as programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors have shown significant improvement in outcomes among patients with several hematological and solid organ malignancies. In order to identify appropriate treatment candidates for these therapies, predictive biomarkers have been developed in various cancers. Evidence from solid tumors has suggested a favorable response to checkpoint inhibitor therapy with higher tumor mutational burden (TMB), defined as the number of mutations within a tumor genome. Next generation sequencing (NGS) of various tumors has shown an association between TMB of < 5 mutations/megabase (mut/Mb) to be associated with an absence of benefit from checkpoint inhibitors. In addition, high levels of PD-1/PD-L1 expression and microsatellite instability (MSI) are also correlated with response to therapy. The latter may be a result of somatic or germline alterations in DNA mismatch repair genes. In this study, we report the results for these biomarkers using NGS in patients with histiocytic neoplasms.
Methods
We utilized TempusTM NGS platform to analyze the tissue specimen of patients with histiocytic neoplasm. The Tempus xO Assay (Tempus Labs; Chicago, IL) combines a 1,711 gene targeted somatic and germline DNA sequencing panel with RNA-sequencing to detect both germline and somatic single nucleotide polymorphisms, indels, copy number variants, and gene rearrangements causing chimeric mRNA transcript expression in a wide array of solid tumor types. The assay utilizes formalin-fixed paraffin-embedded tumor samples and matched blood samples. TMB was calculated and reported as somatic mutations in tumor tissue per million base-pairs or mut/Mb. RNA sequencing was utilized to assess for PD-L1 and PD-1 gene expressions as compared to matched tumor and normal reference sets. Both PD-L1 and PD-1 gene expressions were reported as percentiles. DNA mismatch repair status was predicted by analysis of alterations in five common mismatch repair genes in somatic and germline DNA (MSH2, MSH6, MLH1, PMS2, and EPCAM). If there were no alterations identified in these genes, the mismatch repair status was predicted as microsatellite stable (MSS).
Results
A total of 13 patients with histiocytic neoplasms were included in the study. The distribution of individual histiocytic neoplasms was as follows: RDD (n=9), ECD (n=3), and LCH (n=1). The median TMB for RDD and ECD patients was 0.17 mut/Mb. For the one patient with LCH, the TMB level was 0.51 mut/Mb. Individual TMB levels are shown in figure 1. PD-L1 and PD-1 expression levels are depicted in table 1. Compared to normal reference sets, the PD-L1 expression was elevated in one patient each with RDD and ECD, and PD-1 expression was elevated in two patients each with RDD and ECD. For both ECD patients with higher PD-1 expression, NGS also showed presence of BRAF-V600E in the tumor tissue. The LCH patient had a low level of PD-L1 and PD-1 expression. For patients where evaluation of DNA mismatch repair was feasible on the tissue specimen (n=4), none showed related somatic or germline alterations.
Conclusions
In our series, the histiocytic neoplasms RDD, ECD, and LCH demonstrated low levels of TMB. None of the RDD and ECD patients were found to have alterations in DNA mismatch repair genes. Other markers of immunotherapy such as PD-L1/PD-1 expression appeared to be higher in ECD patients with BRAF-V600E, but in only a small subset of RDD patients. The low TMB seen in our study suggests that these histiocytic neoplasms may be less likely to respond to immune checkpoint inhibitors such as anti-PD-1 and anti-PD-L1 agents as compared to the tumors with high TMB.

Disclosures
No relevant conflicts of interest to declare.