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Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations.

Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. PCR-based methods to enrich mutations during amplification have limited multiplexing capability, are mostly restricted to known mutations and are prone to polymerase or mis-priming errors. Here, we present Differential Strand Separation at Critical Temperature (DISSECT), a method that enriches unknown mutations of targeted DNA sequences purely based on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions. Target DNA is pre-amplified in a multiplex reaction and hybridized onto complementary probes immobilized on magnetic beads that correspond to wild-type DNA sequences. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature and selectively separated from wild-type DNA. We demonstrate multiplexed enrichment by 100- to 400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using two to four successive cycles of DISSECT. Cancer and plasma-circulating DNA samples containing traces of mutations undergo mutation enrichment allowing detection via Sanger sequencing or high-resolution melting. The simplicity, scalability and reliability of DISSECT make it a powerful method for mutation enrichment that integrates well with existing downstream detection methods.

Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80-150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05-1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: This study characterizes the tumor-immune microenvironment in pretreatment, localized anal squamous cell carcinoma (ASCC), including two markers that have not previously been studied in ASCC: indoleamine 2,3 dioxygenase 1 (IDO1) and human leukocyte antigen (HLA) class I., Materials and Methods: Retrospective review identified 63 patients with ASCC receiving definitive chemoradiation between 2005 and 2016 with pretreatment tissue available. Immunohistochemistry was used to quantify cluster of differentiation 8 (CD8), programmed cell death protein 1, programmed death-ligand 1, HLA class I, and IDO1. Cox proportional hazards models evaluated associations between outcomes and immune markers, controlling for clinical characteristics., Results: With a median follow-up of 35 months, 3-year overall survival was 78%. The only marker found to have a robust association with outcome was tumor IDO1. In general, the percentage of tumor cells expressing IDO1 was low (median 1%, interquartile range 0%-20%); however, patients with >50% of tumor cells expressing IDO1 had significantly worse overall survival (hazard ratio [HR] 4.7, p  = .007) as well as higher local recurrence (HR 8.6, p  = .0005) and distant metastasis (HR 12.7, p  = .0002). Tumors with >50% IDO1 were also more likely to have the lowest quartile of CD8 infiltrate (<40 per high-power field, p  = .024)., Conclusion: ASCC has a diverse immune milieu. Although patients generally do well with standard therapy, IDO1 may serve as a prognostic indicator of poor outcome and could help identify a patient population that might benefit from IDO-targeted therapies., Implications for Practice: After definitive chemoradiation, patients with locally advanced anal cancer may experience significant treatment morbidity and high risk of recurrence. The goal of the current study is to identify novel prognostic factors in the tumor-immune microenvironment that predict for poor outcomes after definitive chemoradiation. This study characterizes the tumor-immune microenvironment in pre-treatment, localized anal squamous cell carcinoma (ASCC), including two markers which have not previously been studied in ASCC: indoleamine 2,3 dioxygenase 1 (IDO1) and HLA class I. With a median follow-up of 3 years, this study demonstrated that high IDO1 expression is correlated with significantly worse 3-year overall survival (88% vs. 25%). Whereas recent studies of IDO1 inhibitors have shown mixed results, this study suggests that patients with anal cancer with high IDO1 expression have dismal prognosis and may represent a patient population primed for response to targeted IDO1 inhibition., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2019.)

Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatellite changes, due to the high false-positive artifacts generated by polymerase slippage (stutter-bands). Here, we describe a nuclease-based approach, NaME-PrO, that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR. By appropriate design of the overlapping oligonucleotides, NaME-PrO eliminates WT alleles in long single-base homopolymers ranging from 10 to 27 nucleotides in length, while sparing targets containing variable-length indels at any position within the homopolymer. We evaluated 5 MSI targets individually or simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from cell-lines, biopsies and circulating-DNA from colorectal cancer patients. NaME-PrO enriched altered microsatellites and detected alterations down to 0.01% allelic-frequency using high-resolution-melting, improving detection sensitivity by 500-1000-fold relative to current HRM approaches. Capillary-electrophoresis also demonstrated enhanced sensitivity and enrichment of indels 1-16 bases long. We anticipate application of this highly-multiplex-able method either with standard 5-plex reactions in conjunction with HRM/capillary electrophoresis or massively-parallel-sequencing-based detection of MSI on numerous targets for sensitive MSI-detection.

Background: We evaluated the efficacy and safety of risk-adapted, proton-based stereotactic body radiation therapy (SBRT) for liver metastases from solid tumors., Methods: This single-arm phase II single institutional study (NCT01239381) included patients with limited extrahepatic disease, 800 mL or greater of uninvolved liver, and no cirrhosis or Child-Pugh A, who had received proton-based SBRT to one to four liver metastases from solid tumors. Treatment comprised 30 to 50 Gray equivalent (GyE) in five fractions based on the effective volume of liver irradiated. Sample size was calculated to determine if local control (LC) at one year was greater than 70%. The cumulative incidence of local failure was used to estimate LC. The association of tumor characteristics, including genetic alterations in common cancer genes such as BRAF, EGFR, HER2, KRAS, NRAS, PIK3CA, and TP53 with local tumor control, was assessed. All statistical tests were two-sided., Results: Eighty-nine patients were evaluable (colorectal, n = 34; pancreatic, n = 13; esophagogastric, n = 12; other, n = 30). Median tumor size was 2.5 cm (range = 0.5-11.9 cm). Median dose was 40 GyE (range = 30-50 GyE), and median follow-up was 30.1 months (range = 14.7-53.8 months). There was no grade 3 to 5 toxicity. Median survival time was 18.1 months. The one- and three-year LC rates were 71.9% (95% confidence limit [CL] = 62.3% to 80.9%) and 61.2% (95% CL = 50.8% to 71.8%), respectively. For large tumors (≥6 cm), one-year LC remained high at 73.9% (95% CL = 54.6% to 89.8%). Mutation in the KRAS oncogene was the strongest predictor of poor LC (P = .02). Tumor with both mutant KRAS and TP53 were particularly radioresistant, with a one-year LC rate of only 20.0%, compared with 69.2% for all others (P = .001)., Conclusions: We report the largest prospective evaluation to date of liver SBRT for hepatic metastases, and the first with protons. Protons were remarkably well tolerated and effective even for metastases that were 6 cm or larger. KRAS mutation is a strong predictor of poor LC, stressing the need for tumor genotyping prior to SBRT and treatment intensification in this patient subset., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Esophageal symptoms are common during radiation and chemotherapy. It is unclear how often these symptoms persist after therapy. We retrospectively reviewed medical records of 320 adults treated for nonmetastatic breast cancer (84), lung cancer (109), or Hodgkin and non-Hodgkin lymphoma (127) who were disease-free at 10-14 months after therapy. Treatment included chemotherapy with or without nonmediastinal radiation therapy (150 patients), chemotherapy plus sequential mediastinal radiation therapy (MRT) (48 patients), chemotherapy plus concurrent MRT (61 patients), or non-MRT only (61 patients). Proton pump inhibitor use was documented. All treatment groups had similar prevalence of the esophageal symptom of heartburn before therapy. Rates were higher during treatment in those who received MRT with or without chemotherapy, but declined by 10-14 months after treatment. However, low baseline rates of dysphagia (4%) and odynophagia (2%) increased significantly after combined chemotherapy and MRT to 72% for dysphagia and 62% for odynophagia (P < 0.01) during treatment and stayed significantly elevated over baseline with 27% of the patients having dysphagia and 11% having odynophagia at 10-14 months after treatment. The use of proton pump inhibitors by patients who had MRT with chemotherapy was significantly increased during and after treatment (P = 0.002). Dysphagia, odynophagia and the use of proton pump inhibitors were significantly more common both during and after treatment than before treatment in patients who received both chemotherapy and mediastinal radiation. Our data highlight the important challenge for clinicians of managing patients with lung cancer and lymphoma who have persistent esophageal problems, particularly dysphagia and odynophagia, at approximately 1 year after treatment., (© 2016 International Society for Diseases of the Esophagus.)

Background: Autophagy is a catabolic pathway that permits cells to recycle intracellular macromolecules, and its inhibition reduces pancreatic cancer growth in model systems. We evaluated hydoxychloroquine (HCQ), an inhibitor of autophagy, in patients with pancreatic cancer and analyzed pharmacodynamic markers in treated patients and mice., Methods: Patients with previously treated metastatic pancreatic cancer were administered HCQ at 400 mg (n = 10) or 600 mg (n = 10) twice daily. The primary endpoint was 2-month progression-free survival (PFS). We analyzed peripheral lymphocytes from treated mice to identify pharmacodynamic markers of autophagy inhibition that were then assessed in peripheral lymphocytes from patients., Results: Among 20 patients enrolled, 2 (10%) were without progressive disease at 2 months. Median PFS and overall survival were 46.5 and 69.0 days, respectively. Treatment-related grade 3/4 adverse events were lymphopenia (n = 1) and elevated alanine aminotransferase (n = 1). Tolerability and efficacy were similar at the two dose levels. Analysis of treated murine lymphocytes suggested that LC3-II expression by Western blot is a reliable marker for autophagy inhibition. Analysis of LC3-II in patient lymphocytes demonstrated inconsistent autophagy inhibition., Conclusion: Mouse studies identified LC3-II levels in peripheral lymphocytes as a potential pharmacodynamic marker of autophagy inhibition. In patients with previously treated metastatic pancreatic cancer, HCQ monotherapy achieved inconsistent autophagy inhibition and demonstrated negligible therapeutic efficacy., (©AlphaMed Press; the data published online to support this summary is the property of the authors.)

Purpose: To determine the efficacy and toxicity of weekly neoadjuvant cetuximab combined with irinotecan, cisplatin, and radiation therapy in patients with locally advanced esophageal or gastroesophageal junction cancer., Methods and Materials: Patients with stage IIA-IVA esophageal or gastroesophageal junction cancer were enrolled in a Simon's two-stage phase II study. Patients received weekly cetuximab on weeks 0-8 and irinotecan and cisplatin on weeks 1, 2, 4, and 5, with concurrent radiotherapy (50.4 Gy on weeks 1-6), followed by surgical resection., Results: In the first stage, 17 patients were enrolled, 16 of whom had adenocarcinoma. Because of a low pathologic complete response (pCR) rate in this cohort, the trial was discontinued for patients with adenocarcinoma but squamous cell carcinoma patients continued to be enrolled; two additional patients were enrolled before the study was closed as a result of poor accrual. Of the 19 patients enrolled, 18 patients proceeded to surgery, and 16 patients underwent an R0 resection. Three patients (16%) had a pCR. The median progression-free survival interval was 10 months, and the median overall survival duration was 31 months. Severe neutropenia occurred in 47% of patients, and severe diarrhea occurred in 47% of patients. One patient died preoperatively from sepsis, and one patient died prior to hospital discharge following surgical resection., Conclusions: This schedule of cetuximab in combination with irinotecan, cisplatin, and radiation therapy was toxic and did not achieve a sufficient pCR rate in patients with localized esophageal adenocarcinoma to undergo further evaluation.

Background: Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3 °C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle., Methods: We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA., Results: A single thermocycling program with a denaturation-temperature window of 2.5-3.0 °C enriches mutations in all DNA amplicons simultaneously, despite their different T(m)s. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR., Conclusions: Low-level mutations in diverse amplicons with different T(m)s can be mutation enriched via TT-COLD-PCR provided that their T(m)s fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.

Background: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality., Methods: We modified one of the 2 PCR primers by use of an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3' quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer., Results: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r(2) >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens., Conclusion: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.