The Origin of Highly Elevated Cell-Free DNA in Healthy Individuals and Patients with Pancreatic, Colorectal, Lung, or Ovarian Cancer.

Cell-free DNA (cfDNA) concentrations from patients with cancer are often elevated compared with those of healthy controls, but the sources of this extra cfDNA have never been determined. To address this issue, we assessed cfDNA methylation patterns in 178 patients with cancers of the colon, pancreas, lung, or ovary and 64 patients without cancer. Eighty-three of these individuals had cfDNA concentrations much greater than those generally observed in healthy subjects. The major contributor of cfDNA in all samples was leukocytes, accounting for ∼76% of cfDNA, with neutrophils predominating. This was true regardless of whether the samples were derived from patients with cancer or the total plasma cfDNA concentration. High levels of cfDNA observed in patients with cancer did not come from either neoplastic cells or surrounding normal epithelial cells from the tumor's tissue of origin. These data suggest that cancers may have a systemic effect on cell turnover or DNA clearance. SIGNIFICANCE: The origin of excess cfDNA in patients with cancer is unknown. Using cfDNA methylation patterns, we determined that neither the tumor nor the surrounding normal tissue contributes this excess cfDNA-rather it comes from leukocytes. This finding suggests that cancers have a systemic impact on cell turnover or DNA clearance. See related commentary by Thierry and Pisareva, p. 2122. This article is featured in Selected Articles from This Issue, p. 2109.

Risk Factors for Complications in Ulnar Shortening Osteotomies: A Multicenter Retrospective Review.

PURPOSE: Ulnar shortening osteotomy (USO) is commonly performed to alleviate pathologies causing ulnar-sided wrist pain. Surgical complications include nonunion and hardware removal, with rates up to 18% and 45%, respectively. The primary objective of the study was to report the overall complication rate of USO. The secondary objective was to identify risk factors for complications. METHODS: A retrospective multicenter cohort review was undertaken, including six Canadian cities over a 6-year period (January 2013-December 2018). Chart review was used to collect demographic data, surgical technique, implant used, and postoperative complications. Descriptive statistics of demographics and operative characteristics, including plate positioning, type of osteotomy, plate type, and ulnar variance (mm), were analyzed. Univariate analyses were used to select predictor variables for nonunion and hardware removal. These predictor variables were then entered into an adjusted multivariable logistic regression model. RESULTS: A total of 361 USOs were performed. Mean age was 46 ± 16 years (60.7% men). The overall complication rate was 37.1%, hardware removal rate was 29.6%, and nonunion rate was 9.4%. There was a workers' compensation claim associated with 21.6% of all complications, and it was a risk factor for both hardware removal (odds ratio [OR] = 3.81) and nonunion (OR = 2.88). Neither smoking nor diabetes was associated with complication rates. Seventy percent of plates were placed volarly, 25.5% dorsally, and 3.9% directly ulnar. Osteotomies were oblique in 83.7% of cases and transverse in 15.5%. Adjusted multivariate regression analysis revealed that younger age (OR = 0.98) was a risk factor for hardware removal and male sex (OR = 2.49) was a risk factor for nonunion. A surgical factor associated with hardware removal was direct ulnar plate placement (OR = 9.93). No surgical factors were associated with nonunions. CONCLUSIONS: There are substantial rates of complications with USOs. Direct ulnar plate placement should be avoided. Patients should be thoroughly counseled on the risks of complications prior to proceeding with USO. LEVEL OF EVIDENCE: Therapeutic IV.

Rasch Analysis of Joint Replacement Version for Hip Disability and Osteoarthritis Outcome in Individuals With Advanced Osteoarthritis of Hip Awaiting Total Hip Arthroplasty Surgery.

OBJECTIVE: The objective of this study was to examine measurement properties of the Joint replacement version for Hip Disability and Osteoarthritis Outcome Score (HOOS-JR) using Rasch analysis in patients with osteoarthritis of hip (HOA). DESIGN: Cross-sectional clinical measurement SETTING: Patient outcomes database at a tertiary care hospital PARTICIPANTS: Convenience sampling of patients with HOA scheduled for total hip arthroplasty (N=327) OUTCOME MEASURES AND ANALYSIS: The data for pre-surgery assessments for patients with HOA were extracted from an existing database. Variables extracted included HOOS-JR scores, demographic information (age, sex), health-related data, and anthropometric variables. The assumptions of Rasch model such as the test of fit, fit residuals, ordering of item thresholds, factor structure, DIF, internal consistency and Pearson separation index were examined for the HOOS-JR scores. RESULTS: The HOOS-JR showed adequate overall fit to the Rasch model, logically ordered response thresholds, no floor or ceiling effects, and high internal consistency (Cronbach's alpha of 0.91). The HOOS-JR did not satisfy the assumption of unidimensionality, albeit the violation of this assumption was marginal (6.12% over 5%). Person-item threshold distribution (difference between person and item means were equal to 0.92 which was less than 1 logit unit) confirmed that the HOOS-JR scores were well targeted. CONCLUSIONS: Given that the violation of unidimensionality for HOOS-JR was marginal, we recommend further studies to validate this finding. Results broadly support the use of HOOS-JR for assessing hip health in patients with HOA.

Whole-Slide Imaging, Mutual Information Registration for Multiplex Immunohistochemistry and Immunofluorescence.

Multiplex immunohistochemistry/immunofluorescence (mIHC/mIF) is a developing technology that facilitates the evaluation of multiple, simultaneous protein expressions at single-cell resolution while preserving tissue architecture. These approaches have shown great potential for biomarker discovery, yet many challenges remain. Importantly, streamlined cross-registration of multiplex immunofluorescence images with additional imaging modalities and immunohistochemistry (IHC) can help increase the plex and/or improve the quality of the data generated by potentiating downstream processes such as cell segmentation. To address this problem, a fully automated process was designed to perform a hierarchical, parallelizable, and deformable registration of multiplexed digital whole-slide images (WSIs). We generalized the calculation of mutual information as a registration criterion to an arbitrary number of dimensions, making it well suited for multiplexed imaging. We also used the self-information of a given IF channel as a criterion to select the optimal channels to use for registration. Additionally, as precise labeling of cellular membranes in situ is essential for robust cell segmentation, a pan-membrane immunohistochemical staining method was developed for incorporation into mIF panels or for use as an IHC followed by cross-registration. In this study, we demonstrate this process by registering whole-slide 6-plex/7-color mIF images with whole-slide brightfield mIHC images, including a CD3 and a pan-membrane stain. Our algorithm, WSI, mutual information registration (WSIMIR), performed highly accurate registration allowing the retrospective generation of an 8-plex/9-color, WSI, and outperformed 2 alternative automated methods for cross-registration by Jaccard index and Dice similarity coefficient (WSIMIR vs automated WARPY, P < .01 and P < .01, respectively, vs HALO + transformix, P = .083 and P = .049, respectively). Furthermore, the addition of a pan-membrane IHC stain cross-registered to an mIF panel facilitated improved automated cell segmentation across mIF WSIs, as measured by significantly increased correct detections, Jaccard index (0.78 vs 0.65), and Dice similarity coefficient (0.88 vs 0.79).

Rasch Analysis for the Knee Injury and Osteoarthritis Outcome Score Joint Replacement Version in Individuals Awaiting Total Knee Replacement Surgery.

The aim of this study was to verify the single-factor structure of the joint replacement version of the Knee Injury and Osteoarthritis Outcome Score (KOOS-JR) and examine its measurement properties in the context of Rasch analysis in patients with end-stage osteoarthritis of the knee (KOA) awaiting total knee replacement (TKR). The study design was retrieval of prospectively collected clinical data. The data were extracted from the presurgery visit for individuals with KOA who were scheduled for primary TKR at a tertiary care hospital. Those who were scheduled for revision of TKR had any other lower extremity injury or surgery during 6 months prior to the presurgery visit, or those who had reported pre-existing neurological impairments affecting the lower extremity functions were excluded during data extraction. The assumptions of Rasch analysis that were examined included the test of fit, fit of residuals, ordering of item thresholds, Pearson separation index, differential item functioning (DIF), dependency, and unidimensionality. The main outcome measure was KOOS-JR. Data were extracted for 283 patients, including 112 men and 160 women, from clinical charts. The KOOS-JR demonstrated good overall fit to the Rasch model. However, it failed to meet the assumption of unidimensionality. None of the items demonstrated DIF or concerns with response thresholds. Person-item threshold distribution indicated that the score for KOOS-JR overestimated person traits with floor and ceiling effects. Reliability statistics were equal to 0.9, suggesting that seven items within the KOOS-JR were internally consistent and reliable. The hypothetical unidimensional KOOS-JR could not be reproduced in our sample in that KOOS-JR had a latent construct. Future research should perform exploratory factor analysis to examine this latent construct.

CD4 T Cell-Dependent Rejection of Beta-2 Microglobulin Null Mismatch Repair-Deficient Tumors.

Inactivation of beta-2 microglobulin (B2M) is considered a determinant of resistance to immune checkpoint inhibitors (ICPi) in melanoma and lung cancers. In contrast, B2M loss does not appear to affect response to ICPis in mismatch repair-deficient (MMRd) colorectal tumors where biallelic inactivation of B2M is frequently observed. We inactivated B2m in multiple murine MMRd cancer models. Although MMRd cells would not readily grow in immunocompetent mice, MMRd B2m null cells were tumorigenic and regressed when treated with anti-PD-1 and anti-CTLA4. The efficacy of ICPis against MMRd B2m null tumors did not require CD8+ T cells but relied on the presence of CD4+ T cells. Human tumors expressing low levels of B2M display increased intratumoral CD4+ T cells. We conclude that B2M inactivation does not blunt the efficacy of ICPi in MMRd tumors, and we identify a unique role for CD4+ T cells in tumor rejection. SIGNIFICANCE: B2M alterations, which impair antigen presentation, occur frequently in microsatellite-unstable colorectal cancers. Although in melanoma and lung cancers B2M loss is a mechanism of resistance to immune checkpoint blockade, we show that MMRd tumors respond to ICPis through CD4+ T-cell activation.This article is highlighted in the In This Issue feature, p. 1601.

DNA Sensing in Mismatch Repair-Deficient Tumor Cells Is Essential for Anti-tumor Immunity.

Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-ß in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.

Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

PURPOSE: Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients. EXPERIMENTAL DESIGN: We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA). RESULTS: Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% (n = 163) and sensitivities of 78% (n = 23) and 67% (n = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, P = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade. CONCLUSIONS: These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade.See related commentary by Wang and Ajani, p. 6887.

Comparison of Biomarker Modalities for Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Review and Meta-analysis.

IMPORTANCE: PD-L1 (programmed cell death ligand 1) immunohistochemistry (IHC), tumor mutational burden (TMB), gene expression profiling (GEP), and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) assays have been used to assess pretreatment tumor tissue to predict response to anti-PD-1/PD-L1 therapies. However, the relative diagnostic performance of these modalities has yet to be established. OBJECTIVE: To compare studies that assessed the diagnostic accuracy of PD-L1 IHC, TMB, GEP, and mIHC/IF in predicting response to anti-PD-1/PD-L1 therapy. EVIDENCE REVIEW: A search of PubMed (from inception to June 2018) and 2013 to 2018 annual meeting abstracts from the American Association for Cancer Research, American Society of Clinical Oncology, European Society for Medical Oncology, and Society for Immunotherapy of Cancer was conducted to identify studies that examined the use of PD-L1 IHC, TMB, GEP, and mIHC/IF assays to determine objective response to anti-PD-1/PD-L1 therapy. For PD-L1 IHC, only clinical trials that resulted in US Food and Drug Administration approval of indications for anti-PD-1/PD-L1 were included. Studies combining more than 1 modality were also included. Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines were followed. Two reviewers independently extracted the clinical outcomes and test results for each individual study. MAIN OUTCOMES AND MEASURES: Summary receiver operating characteristic (sROC) curves; their associated area under the curve (AUC); and pooled sensitivity, specificity, positive and negative predictive values (PPV, NPV), and positive and negative likelihood ratios (LR+ and LR-) for each assay modality. RESULTS: Tumor specimens representing over 10 different solid tumor types in 8135 patients were assayed, and the results were correlated with anti-PD-1/PD-L1 response. When each modality was evaluated with sROC curves, mIHC/IF had a significantly higher AUC (0.79) compared with PD-L1 IHC (AUC, 0.65, P < .001), GEP (AUC, 0.65, P = .003), and TMB (AUC, 0.69, P = .049). When multiple different modalities were combined such as PD-L1 IHC and/or GEP + TMB, the AUC drew nearer to that of mIHC/IF (0.74). All modalities demonstrated comparable NPV and LR-, whereas mIHC/IF demonstrated higher PPV (0.63) and LR+ (2.86) than the other approaches. CONCLUSIONS AND RELEVANCE: In this meta-analysis, tumor mutational burden, PD-L1 IHC, and GEP demonstrated comparable AUCs in predicting response to anti-PD-1/PD-L1 treatment. Multiplex immunohistochemistry/IF and multimodality biomarker strategies appear to be associated with improved performance over PD-L1 IHC, TMB, or GEP alone. Further studies with mIHC/IF and composite approaches with a larger number of patients will be required to confirm these findings. Additional study is also required to determine the most predictive analyte combinations and to determine whether biomarker modality performance varies by tumor type.

Tumor genomic alterations in severe-combined immunodeficiency bare-lymphocyte syndrome genes are associated with high mutational burden and disproportional neo-antigen rates.

The progression of cancer requires mutational adaptation to permit unrestrained proliferation. A fraction of cancer mutations are oncogenic drivers, while others are putative 'passengers' that do not contribute to oncogenesis. However, altered peptides arising from passenger mutations may bind MHCs and activate non-self immunologic signals (i.e. neoantigens), thus requiring immunoediting for cancer persistence. Disruption of antigen processing machinery in tumor cells may diminish this requirement. Here, we show that rare mutations in antigen processing machinery are associated with high mutational burden and increased predicted neoantigen load, providing insights into the mechanisms of high mutation burden in some patients.

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.

The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor-1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair-deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair-deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers' tissue of origin.

Short Inverted Repeats Are Hotspots for Genetic Instability: Relevance to Cancer Genomes.

Analyses of chromosomal aberrations in human genetic disorders have revealed that inverted repeat sequences (IRs) often co-localize with endogenous chromosomal instability and breakage hotspots. Approximately 80% of all IRs in the human genome are short (<100 bp), yet the mutagenic potential of such short cruciform-forming sequences has not been characterized. Here, we find that short IRs are enriched at translocation breakpoints in human cancer and stimulate the formation of DNA double-strand breaks (DSBs) and deletions in mammalian and yeast cells. We provide evidence for replication-related mechanisms of IR-induced genetic instability and a novel XPF cleavage-based mechanism independent of DNA replication. These discoveries implicate short IRs as endogenous sources of DNA breakage involved in disease etiology and suggest that these repeats represent a feature of genome plasticity that may contribute to the evolution of the human genome by providing a means for diversity within the population.