Molecular pathology selected abstracts

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Editors: Donna E. Hansel, MD, PhD, chief, Division of Anatomic Pathology, and professor, Department of Pathology, University of California, San Diego; James Solomon, MD, PhD, resident, Department of Pathology, UCSD; Richard Wong, MD, PhD, molecular pathology fellow, Department of Pathology, UCSD; and Sounak Gupta, MBBS, PhD, molecular pathology fellow, Memorial Sloan Kettering Cancer Center, New York.

Cell-free DNA tumor mutational burden predicts efficacy of immune checkpoint inhibitors

October 2018—Immune checkpoint inhibitors have emerged as a potent class of therapy for a variety of malignancies. The biologic rationale for these drugs is that somatic mutations, not necessarily in cancer driver genes, may accumulate in tumor cells, resulting in amino acid changes that create neoantigens (epitopes not present in normal cells during maturation of the immune system). Tumors expressing neoantigens can evade immune clearance by turning on immune inhibitory mechanisms, or immune checkpoints. PD-1 binding to PD-L1 is an immune checkpoint-signaling pathway that has been shown to be exploited by tumor cells. Antibodies targeting PD-1 and PD-L1 have been used successfully to treat non-small cell lung cancer (NSCLC). PD-L1 can be directly expressed by tumor cells, induced to be expressed on tumor cells, or expressed in the local microenvironment, while PD-1 is expressed on effector and regulatory T cells. This supports the rationale for assessing PD-L1 and PD-1 when considering patients for checkpoint inhibitor therapy. A standardized scoring system exists in which tumor cells or immune cells are graded for PD-L1 or PD-1 expression by immunohistochemistry. In an effort to identify other biomarkers for predicting the effectiveness of checkpoint inhibitors, studies have been conducted, which have shown that tumor mutation burden (TMB) may be a surrogate for overall neoantigen load. TMB, as measured by next-generation sequencing, is calculated as the number of somatic coding mutations (single nucleotide variants, insertions, deletions) per million bases surveyed. Initial studies measured TMB over whole exomes, but TMB assessed over smaller sets of genes also correlated with better outcomes in checkpoint inhibitor-treated patients. After the standard pathology evaluation of NSCLCs, there is often limited tissue available for TMB or other molecular evaluations. The authors of this study described a blood-based assay to measure TMB in the blood plasma (bTMB) of cell-free tumor DNA (ctDNA). Using a retrospective analysis of specimens from two large randomized trials, they showed that bTMB reproducibly identified patients who derived clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1 antibody) in second-line and higher NSCLC. They assessed tissue TMB (tTMB) and bTMB in a subset of patients. Tissue mutations were defined as somatic coding mutations, including single nucleotide variants, insertions, and deletions detected at an allele frequency of five percent or more. Blood mutations were calculated from only somatic coding single nucleotide changes at an allele frequency of 0.5 percent or more. In patients with high TMB count (more than 30 mutations/MB) in blood and tissue, one-third of the variants, on average, were unique to the blood and one-fourth were unique to the tissue sample. The remaining variants were identified in both. Concordance was found for patients with lower tissue or blood TMB, or both. Using a bTMB cut-point of 16/MB or more, clinical trial patients on atezolizumab showed improved progression-free survival. The prevalence of a bTMB of 16/MB or more in the evaluated study patients was approximately 30 percent. Interestingly, bTMB was independent of a high tumor cell/immune cell score. Patients with a bTMB of 16/MB or more and a high tumor cell/immune cell score seemed to derive the most clinical benefit from atezolizumab. Notably, patients with no PD-L1 expression or low expression and a bTMB of 16 or more showed a trend toward progression-free survival. Identifying biomarkers that can stratify patients who may derive benefit from immune checkpoint inhibition is an important goal in this emerging class of therapy. Combining bTMB with the now near-standard PD-1/PD-L1 assessment of NSCLCs may one day provide greater granularity in the evaluation of these patients.

Gandara DR, Paul SM, Kowanetz M, et al. Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab. Nat Med. 2018. doi:10.1038/s41591-018-0134-3.

Correspondence: Dr. David R. Gandara at drgandara@ucdavis.edu, or Dr. Tony Mok at tony@clo.cuhk.edu.hk, or Dr. David S. Shames at shames.david@gene.com

Epigenetic immune cell counting analogous to flow cytometry on dried blood spots

Quantitative enumeration of the various immune cell types in blood provides valuable diagnostic information for hematologic and immunologic diseases. Morphologic counting of cells on blood smears and use of electrical impedance hematology analyzers and flow cytometry are standard methods employed in most clinical laboratories. A common limitation of these methods is the necessity for fresh blood specimens with relatively narrow windows of sample viability for testing. The authors conducted a study in which they described a method for quantitatively assessing immune cells using methylation-based epigenetic quantitative PCR (qPCR). The novel aspect of this technique is its ability to provide relative and absolute immune cell counts on not only fresh blood specimens but also fresh-frozen and paper-spotted dried blood. DNA methylation is an epigenetic modification whereby a methyl group is added to a cytosine nucleotide in DNA. As various cell types reach maturity, different sets of genes are stably downregulated by methylation. Maintenance of methylation results in mitotically heritable lineage-specific methylation profiles. Bisulfite sequencing is a method used to identify the methylation status of specific DNA sites. Bisulfite treatment of DNA converts unmethylated cytosine bases to uracil, and the change is determined by various PCR or array hybridization techniques. The authors identified lineage-specific methylation profiles for CD3+, CD4+, and CD8+ T cells, as well as regulatory T cells, neutrophils, B cells, and natural killer cells from genome-wide discovery and profiling of candidate genes. Although some lineage-specific sites had clear biologic relevance for the cell type, others had undefined roles. To calculate relative methylation and, thereby, the relative number of a cell type, and to correct for variable bisulfite conversion efficiencies, the authors used various synthetic DNA constructs to act as internal standards and bisulfite conversion calibrators. In validating their assay, they compared cell counts from the liquid blood samples of 25 healthy donors assessed by flow cytometry and epigenetic qPCR. After quantifying various leukocyte lineages by both methods, the joint comparison of all markers showed high correlation (Spearman rank correlation coefficients, 0.96 and 0.97 [P < .001], respectively). To test more clinically relevant samples, 97 HIV-positive subjects were assessed for CD4+ and CD8+ T cell counts by standard flow cytometry and epigenetic qPCR using liquid blood or dried blood spots as samples. Again, samples from the different methods showed high correlation in quantifying cell types and total leukocyte numbers. It was noted that epigenetic qPCR of dried blood spots for counting CD4+ T cells showed greater variation when compared to flow cytometric and epigenetic measurements from liquid blood samples. Recognizing the prominent use of dried blood spot collection in newborn screening, the authors showed how their assay could be used to identify various primary immunodeficiencies (PIDs). Severe combined immunodeficiency (SCID), delayed-onset SCID, X-linked agammaglobulinemia, and severe congenital neutropenia are clinically characterized by a decrease or absence of one or more immune cell lineages. In a case-control study consisting of original dried blood spot neonatal screening cards from 24 PID patients and 250 randomly selected newborns, epigenetic qPCR identified almost all PID patients through quantitative deficiencies of specific leukocyte subpopulations. Gene methylation status of disease and treatment-relevant targets is a well-established clinical test for a variety of neoplastic and congenital diseases. This study showed how specific leukocyte methylation profiles can be used to enumerate blood leukocytes as part of an effort to identify and follow a spectrum of immunologic diseases from fresh blood samples and paper-spotted dried blood.