Purpose: The role of plasma-based tumor mutation burden (pTMB) in predicting response to pembrolizumab-based first-line standard of care therapy for metastatic non-small cell lung cancer (mNSCLC) has not been explored. Experimental Design: A 500-gene next-generation sequencing (NGS) panel was used to assess pTMB. Sixty-six patients with newly diagnosed mNSCLC starting first-line pembrolizumab-based therapy, either alone or in combination with chemotherapy, were enrolled (Clinicaltrial.gov identifier: NCT03047616). Response was assessed using RECIST 1.1. Associations were made for patient characteristics, 6-month durable clinical benefit (DCB), progression free survival (PFS), and overall survival (OS).Results: Of 66 patients, 52 (78.8%) were pTMB-evaluable. Median pTMB was 16.8 mutations per megabase (mut/Mb; range 1.9-52.5) and was significantly higher for patients achieving DCB compared to no durable benefit: 21.3 mut/Mb vs. 12.4 mut/Mb, P=0.003. For patients with pTMB >16 mut/Mb, median PFS was 14.1 vs. 4.7 months for patients with pTMB<16 mut/Mb (HR 0.30 [0.16-0.60]) P<0.001. Median OS for patients with pTMB>16 was not reached vs. 8.8 months for patients with pTMB<16 mut/Mb (HR 0.48 [0.22-1.03]) P=0.061. Mutations in ERBB2 exon 20, STK11, KEAP1, or PTEN were more common in patients with no DCB. A combination of pTMB>16 and absence of negative predictor mutations was associated with PFS (HR 0.24 [0.11-0.49]) P<0.001 and OS (HR 0.31 [0.13-0.74]) P=0.009. Conclusions: pTMB>16 mut/Mb is associated with improved PFS after first-line standard of care pembrolizumab-based therapy in mNSCLC. STK11/KEAP1/PTEN and ERBB2 mutations may help identify pTMB-high patients unlikely to respond. These results should be validated in larger prospective studies.

PURPOSE Although the majority of patients with metastatic non–small-cell lung cancer (mNSCLC) lacking a detectable targetable mutation will receive pembrolizumab-based therapy in the frontline setting, predicting which patients will experience a durable clinical benefit (DCB) remains challenging. MATERIALS AND METHODS Patients with mNSCLC receiving pembrolizumab monotherapy or in combination with chemotherapy underwent a 74-gene next-generation sequencing panel on blood samples obtained at baseline and at 9 weeks. The change in circulating tumor DNA levels on-therapy (molecular response) was quantified using a ratio calculation with response defined by a > 50% decrease in mean variant allele fraction. Patient response was assessed using RECIST 1.1; DCB was defined as complete or partial response or stable disease that lasted > 6 months. Progression-free survival and overall survival were recorded. RESULTS Among 67 patients, 51 (76.1%) had > 1 variant detected at a variant allele fraction > 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% v 27.7%, P < .001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% v 49.4%, P < .001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders. CONCLUSION Molecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in independent prospective studies.

The most prevalent microdeletion in humans occurs at 22q11.2, a region rich in chromosome-specific low copy repeats (LCR22s). The structure of this region has defied elucidation due to its size, regional complexity, and haplotype diversity, and is not well represented in the human genome reference. Most individuals with 22q11.2 deletion syndrome (22q11.2DS) carry a de novo hemizygous deletion of ~ 3 Mbp occurring by non-allelic homologous recombination (NAHR) mediated by LCR22s. In this study, optical mapping has been used to elucidate LCR22 structure and variation in 88 individuals in thirty 22q11.2DS families to uncover potential risk factors for germline rearrangements leading to 22q11.2DS offspring. Families were optically mapped to characterize LCR22 structures, NAHR locations, and genomic signatures associated with the deletion. Bioinformatics analyses revealed clear delineations between LCR22 structures in normal and deletion-containing haplotypes. Despite no explicit whole-haplotype predisposing configurations being identified, all NAHR events contain a segmental duplication encompassing FAM230 gene members suggesting preferred recombination sequences. Analysis of deletion breakpoints indicates that preferred recombinations occur between FAM230 and specific segmental duplication orientations within LCR22A and LCR22D, ultimately leading to NAHR. This work represents the most comprehensive analysis of 22q11.2DS NAHR events demonstrating completely contiguous LCR22 structures surrounding and within deletion breakpoints. The 22q11.2 Deletion Syndrome (22q11.2DS) is a congenital malformation disorder and the most frequent microdeletion syndrome in humans 1. It has a prevalence of about one in every 3,000 live births 1,2 and one in every 1,000 pregnancies 3. Significant medical issues present in affected individuals may include: congenital cardiac defects (~ 75%), immune deficiencies, speech/language defects, intellectual disabilities, and a 25-30% risk for developing schizophrenia in adolescence or adulthood 2. The causative deletion usually occurs as a de novo event in meiosis in one of the parents. Ninety percent of affected individuals have a hemizygous ~ 3 million base pair (Mbp) deletion in chromosome 22q11.2 2,4. The mechanism responsible for the deletion is non-allelic homologous recombination (NAHR) between surrounding chromosome 22-specific low copy repeats (LCR22s) 5,6. There are eight LCR22s, labeled alphabetically as LCRA to LCRH from centromere to telomere on 22q 6. The LCR22s are comprised of sequence modules of varying lengths containing interspersed genes and pseudogenes.

<p>Mutations Analyzed</p>

<p>Development and validation of a plasma-based panel for mutation detection and assessment of TMB.</p>

<p>Overall response to pembrolizumab monotherapy (n=31, left) and combination therapy (n=34*, right).</p>

<p>pTMBand PD-L1 expression levels.</p>

<div>AbstractPurpose:<p>The role of plasma-based tumor mutation burden (pTMB) in predicting response to pembrolizumab-based first-line standard-of-care therapy for metastatic non–small cell lung cancer (mNSCLC) has not been explored.</p>Experimental Design:<p>A 500-gene next-generation sequencing panel was used to assess pTMB. Sixty-six patients with newly diagnosed mNSCLC starting first-line pembrolizumab-based therapy, either alone or in combination with chemotherapy, were enrolled (Clinicaltrial.gov identifier: NCT03047616). Response was assessed using RECIST 1.1. Associations were made for patient characteristics, 6-month durable clinical benefit (DCB), progression-free survival (PFS), and overall survival (OS).</p>Results:<p>Of 66 patients, 52 (78.8%) were pTMB-evaluable. Median pTMB was 16.8 mutations per megabase (mut/Mb; range, 1.9–52.5) and was significantly higher for patients achieving DCB compared with no durable benefit (21.3 mut/Mb vs. 12.4 mut/Mb, <i>P</i> = 0.003). For patients with pTMB ≥ 16 mut/Mb, median PFS was 14.1 versus 4.7 months for patients with pTMB < 16 mut/Mb [HR, 0.30 (0.16–0.60); <i>P</i> < 0.001]. Median OS for patients with pTMB ≥ 16 was not reached versus 8.8 months for patients with pTMB < 16 mut/Mb [HR, 0.48 (0.22–1.03); <i>P</i> = 0.061]. Mutations in <i>ERBB2</i> exon 20, <i>STK11, KEAP1</i>, or <i>PTEN</i> were more common in patients with no DCB. A combination of pTMB ≥ 16 and absence of negative predictor mutations was associated with PFS [HR, 0.24 (0.11–0.49); <i>P</i> < 0.001] and OS [HR, 0.31 (0.13–0.74); <i>P</i> = 0.009].</p>Conclusions:<p>pTMB ≥ 16 mut/Mb is associated with improved PFS after first-line standard-of-care pembrolizumab-based therapy in mNSCLC. <i>STK11/KEAP1/PTEN</i> and <i>ERBB2</i> mutations may help identify pTMB-high patients unlikely to respond. These results should be validated in larger prospective studies.</p></div>