Transrenal DNA Research Articles

Detection of Mycobacterium tuberculosis transrenal DNA in urine samples among adults in Peru

Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30–45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62–83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62–100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69–100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78–94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.

Transrenal Mycobacterium tuberculosis DNA in pulmonary tuberculosis patients during the first 14 days of treatment.

We assessed the performance of a novel real-time PCR-based transrenal DNA (trDNA) assay for the specific detection of Mycobacterium tuberculosis as a candidate marker of the early anti-tuberculosis therapy response. The study was performed on 288 urine samples from 72 tuberculosis patients collected at baseline and days 3, 7, and 14 of treatment with amoxicillin-clavulanic acid alone or in combination with meropenem, ertapenem, optimized-dose rifampicin, or standard treatment control in South Africa. trDNA was detected in one-third of the samples. The highest proportion of positive PCR results (cycle threshold < 36) was observed on days 3 and 7, reflecting the point in time when maximum bacterial killing and disintegration are expected. When analyzed by study arms, the trend was observed in groups treated with active antibiotics affecting cell wall integrity (meropenem, control) but not in inactive drugs (ertapenem, amoxicillin/clavulanic acid alone) or active drugs not affecting the cell wall (rifampicin). Overall, however, the trDNA assay did not correlate well with sputum culture-based decline of viable bacteria. This is possibly due to trDNA reflecting the killing of both culturable and non-culturable bacteria and should be explored further.IMPORTANCEThis study presents the results of the evaluation of a novel method for the detection of Mycobacterium tuberculosis, the causative agent of tuberculosis, in urine. Detecting parts of the mycobacteria in urine is of particular interest as it allows us to use a sample that is easy to obtain and that does not require uncomfortable procedures or safety precautions like obtaining sputum for culture, which is the most commonly used sample in the diagnosis of tuberculosis. In certain groups of individuals who cannot produce sputum, for example, children, non-sputum-based methods have particular importance. We found that the method tested was able to detect bacterial killing by active antibiotics that disrupt the cell wall and lead to fragmentation of bacteria. However, the assay can't detect inactive bacteria or bacteria that are active with an intact cell wall.

Impact of Cell-Debris and Room-Temperature Storage on Urine Circulating Tumor DNA from Hepatocellular Carcinoma

This study evaluated the impact of cell debris and 7-day room temperature storage on the quality and yield of transrenal DNA. Archived urine specimens collected from five hepatocellular carcinoma (HCC) patients and two pregnant women carrying male fetuses were used to assess the impact of cell debris on urine cell-free DNA (ucfDNA) isolation as measured by quantitative PCR for Y-chromosome DNA, or HCC-associated mutation and methylation markers, and by capillary electrophoresis. Prospectively collected urine from 21 HCC patients was aliquoted after collection for paired immediate freezing versus 7-day room temperature storage followed by freezing for further analysis. Cell debris contained more Y-chromosome DNA than supernatant in three of the six urine specimens tested from pregnant women, suggesting that cell debris can be associated with 20.6% to 84.9% of transrenal ucfDNA. Ninety-five percent (20 of 21) of frozen and room temperature urine pairs had overlapping DNA size distribution. ucfDNA quantity determined by quantitative PCR for TP53, CTNNB1, TERT, and HCC-associated urine circulating tumor DNA markers were statistically similar between room temperature and frozen samples. This suggests no significant difference in DNA degradation between the groups. The association of transrenal ucfDNA with cell debris and HCC circulating DNA stability at room temperature is significant to further the understanding of transrenal circulating tumor DNA pre-analytical handling for HCC screening.

Роль биомаркеров мочи в диагностике туберкулеза легких (обзор)

Diagnosis of tuberculosis (TB) based on sputum analysis has limitations for certain categories of patients (older adults, children, and people living with HIV). An alternative approach is a non-invasive urine-based method, which provides a large sample volume and a quick result. We searched for English-language publications from 2010 to 2021 in PubMed and Cochrane databases using the terms tuberculosis + urine + biomarkers. Papers on urine lipoarabinomannan testing were excluded as this topic has been sufficiently studied elsewhere. The reviewed publications cover more than 30 urine biomarkers used to diagnose TB and evaluate treatment effectiveness. Urine transrenal DNA extraction continues to be investigated, although its diagnostic sensitivity and specificity depend on the extraction method and patient’s HIV status. IP-10 is likely to be a non-specific inflammatory marker; however, its level correlates with TB/HIV status and may be useful for assessing TB treatment response. Further, the article shows the potential of metabolomic biomarkers and biosignatures for measuring the activity of TB infection and distinguishing it from other respiratory diseases. The number of reliable biomarkers predicting TB treatment outcomes is limited. Numerous untargeted studies have used mass spectrometry to detect metabolomic and proteomic biomarkers in urine. The publications are heterogeneous in design and methods; few studies have analysed the specificity and sensitivity of the diagnostic methods covered. In future, a combination of host and pathogen biomarkers could increase the sensitivity and specificity of TB diagnosis.

Transrenal DNA detection of Mycobacterium tuberculosis in patients with pulmonary tuberculosis.

Multiple attempts have been made to use biological samples other than sputum to diagnose tuberculosis (TB). Sputum acid-fast bacillus (AFB) microscopy is the fastest, most straightforward, and most inexpensive method for diagnosing pulmonary TB. However, urine can be used in place of sputum owing to its various advantages, such as a noninvasive method of collection, convenient handling and storage, and minimal risk of infection in health-care workers involved in sample collection. In this study, we aimed to assess the suitability of urine as a sample to obtain transrenal DNA (trDNA) to diagnose TB. This study involved several patients with TB undergoing inpatient treatment, whose AFB microscopy showed negative inversion. Here, 51 urine samples were collected from 40 patients with TB and examined to confirm the presence of trDNA. First, we compared the efficiency of two trDNA extraction methods. Although molecular diagnosis using GeneXpert yielded negative results, a peculiarity was observed. There was no significant difference between GeneXpert findings and our results nor was there any difference in the sequential trDNA samples obtained. However, even when GeneXpert results were negative, trDNA was detected in seven out of ten samples using the anchor extraction method. Further studies are needed to establish biomarkers for the progression of TB treatment.

Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study

Tuberculous Meningitis (TBM) diagnosis remains a grave challenge. We evaluated the utility of extracellular vesicles (EVs) as a source of cell-free transrenal-mycobacterial DNA (cf-Tr-MTB DNA) for TBM diagnosis from urine samples. We developed a qPCR-assay targeting a highly repetitive 36-bp sequence specific to Mycobacterium tuberculosis complex. EVs were isolated from urine samples of suspected TBM groups (n = 44) [categorized using composite reference standard as ‘Definite’ TBM (n = 8), ‘Probable’ TBM (n = 15), ‘Possible’ TBM (n = 21)] and ‘Non-TBM’ group (n = 26). cf-Tr-MTB DNA-based qPCR assay was applied to DNA isolated from EVs (EV-DNA) and EV-free-fraction (EV-free DNA). ROC-curves were generated using qPCR results of ‘Definite’ TBM and ‘Non-TBM’ category in both EV-DNA and EV-free DNA samples and cut-off values were selected to provide 100% (95%CI:69.1–100) specificity. The cf-Tr-MTB DNA assay gave a sensitivity of 54.5% (95%CI:38.8–69.6) for EV-DNA and 77.3% (95%CI:62.1–88.5) for EV-free DNA in the TBM group (n = 44). The combination of EV-DNA and EV-free DNA results (corresponding to performance cf-Tr MTB DNA assay in urine), gave an overall sensitivity of 81.8% (95%CI:67.2–91.8) in the TBM group. Our results confirmed EVs as one of the sources of cf-Tr-MTB DNA and we believe the cf-Tr-MTB DNA-based qPCR assay has a potential application for TBM diagnosis.

Trans-Renal Cell-Free Tumor DNA for Urine-Based Liquid Biopsy of Cancer.

Cancer biomarkers are a promising tool for cancer detection, personalization of therapy, and monitoring of treatment response or recurrence. “Liquid biopsy” commonly refers to minimally invasive or non-invasive sampling of a bodily fluid (i.e., blood, urine, saliva) for detection of cancer biomarkers such as circulating tumor cells or cell-free tumor DNA (ctDNA). These methods offer a means to collect frequent tumor assessments without needing surgical biopsies. Despite much progress with blood-based liquid biopsy approaches, there are limitations—including the limited amount of blood that can be drawn from a person and challenges with collecting blood samples at frequent intervals to capture ctDNA biomarker kinetics. These limitations are important because ctDNA is present at extremely low levels in plasma and there is evidence that measuring ctDNA biomarker kinetics over time can be useful for clinical prediction. Additionally, blood-based assays require access to trained phlebotomists and often a trip to a healthcare facility. In contrast, urine is a body fluid that can be self-collected from a patient’s home, at frequent intervals, and mailed to a laboratory for analysis. Multiple reports indicate that fragments of ctDNA pass from the bloodstream through the kidney’s glomerular filtration system into the urine, where they are known as trans-renal ctDNA (TR-ctDNA). Accumulating studies indicate that the limitations of blood based ctDNA approaches for cancer can be overcome by measuring TR-ctDNA. Here, we review current knowledge about TR-ctDNA in urine as a cancer biomarker approach, and discuss its clinical potential and open questions in this research field.

Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA.

ABSTRACTTransrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 ml urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0 to 91.5%) and 100% specificity (95% CI: 86.2 to 100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14 to 2,804 copies/ml (median 14.6 copies/ml) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was nonsignificantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine lipoarabinomannan (LAM)-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

Abstract B11: Whole-exome sequencing analysis of urine transrenal tumor DNA in metastatic colorectal cancer patients

Abstract Background: The analysis of circulating free tumor DNA (ctDNA) in blood, commonly referred to as liquid biopsy, is being used to characterize patients with solid cancers. Tumor-specific genetic variants can also be present in DNA isolated from other body fluids such as urine. Unlike blood, urine sampling is noninvasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumor DNA that clears from the glomerular filtration barrier (transrenal tumor DNA, hereafter trtDNA) are largely unexplored. Patients and Methods: Specimens were collected from 24 patients with KRAS or BRAF mutant metastatic colorectal cancer (mCRC). Driver mutations were assessed by droplet digital PCR (ddPCR) in ctDNA from plasma and trtDNA from urine. Whole-exome sequencing (WES) was performed in DNA isolated from tissue, plasma, and urine. Results: Out of the 24 CRC cases, only four had sufficient DNA to allow WES analyses in urine and plasma. We found that tumor alterations reside primarily in low-molecular-weight fragments. In patients where trtDNA was more than 2.69% of the urine-derived DNA, cancer-specific molecular alterations, mutational signatures, and copy number profiles identified in urine DNA are comparable with those detected in plasma ctDNA. Conclusions: With current technologies, WES analysis of trtDNA is feasible in a small fraction of mCRC patients. Tumor-related genetic information is mainly present in shorter DNA fragments. Although the limited amounts of trtDNA pose analytical challenges, enrichment of low-molecular-weight DNA and optimized computational tools improve detection of tumor-specific genetic information in urine. Citation Format: Giovanni Crisafulli, Benedetta Mussolin, Andrea Cassingena, Monica Montone, Alice Bartolini, Ludovic Barault, Antonio Martinetti, Federica Morano, Filippo Pietrantonio, Andrea Sartore-Bianchi, Salvatore Siena, Federica Di Nicolatonio, Silvia Marsoni, Alberto Bardelli, Giulia Siravegna. Whole-exome sequencing analysis of urine transrenal tumor DNA in metastatic colorectal cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B11.

Metagenomic analysis of mycobacterial transrenal DNA in patients with HIV and tuberculosis coinfection

The existence of “transrenal” DNA (tr-DNA), i.e. cell-free DNA that has distributed through the renal barrier to the urine, was first shown from a pathogen in 2000 (Botezatu et al., 2000). However, a targeted search for tr-DNA from Mycobacterium tuberculosis (MBT) started relatively recently (Cannas et al., 2008; Green et al., 2009). While other MBT cellular components found in the urine, e.g. lipoarabinomannan, have been used as an enhanced diagnostic tool, tr-DNA has the potential for strain specific identification or a more persistent biomarker during treatment of active disease. We therefore sought to identify by high-throughput next generation sequencing (NGS) MBT genome fragments in the urine of people with human immunodeficiency virus and tuberculosis (HIV-TB) co-infection living in a co-epidemic setting, and to evaluate whether these DNA targets are suitable for the development a quantitative TaqMan polymerase chain reaction with real-time detection (rt-PCR). Selection and mapping to the reference MBT genome of strain H37Rv (NC_000962) revealed 158 fragments of mycobacterial DNA with length from 19 to 44 base pairs (bp) repeated in different DNA samples. Five targets were chosen for design of rt-PCR primers and probes. Comparative analysis of the newly developed tests that were based on the results of NGS did not reveal a significant increase in sensitivity and specificity relative to the previous empirically designed targets. Howver, highly reproducible NGS reads of mycobacterial tr-DNA were obtained. rt-PCR test development suitable for more practical clinical use was likely limited by the small size of the secreted DNA fragments. It is necessary to develop further molecular approaches for the detection of mycobacterial tr-DNA or rely on NGS techniques with inherent bioinformatics requirements.

Abstract 417: Urine as an alternative to blood for germline genomics and cancer genetics

Abstract Currently, blood is the body fluid of choice to provide comprehensive germline genomics via Peripheral Blood Mononuclear Cells (PBMC) and cancer genetics via the detection of circulating tumor DNA (ctDNA, liquid biopsy) for precision medicine. In this study, we examined if urine can be an alternative to blood for comprehensive germline genomics and for liquid biopsy. We have previously shown that human urine contains DNA derived both from sloughed-off cell debris of the urinary tract (greater than 1 KB/High Molecular weight or HMW) and trans-renal DNA from the circulation (less than 1 KB/Low molecular weight or LMW). We performed whole genome sequencing (WGS) to compare the quality and comprehensiveness of the genomic data between 3 sets of matched PBMC and HMW urine DNA. There was no statistically significant difference between the NGS data obtained from PBMC and urine DNA upon comparing the number of reads, average coverage, percent aligned reads, percent reads passing Phred score Q30 (p value less than 0.05, paired t-test) and coverage of human genome. Next, we compared the detectability of ctDNA in the matching LMW urine and plasma DNA obtained from 44 HCC patients by employing a panel of HCC biomarkers including genetic mutations at TP53 codon 249 [G:C to T:A transversion (TP53 249T)], CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124) and aberrant methylation of RASSF1A (mRASSF1A) promoter. In this study, a systematic analysis of this HCC biomarker panel demonstrated that urine can complement blood for liver cancer screening. A combination of the plasma and urine biomarkers with serum AFP the current most widely used biomarker for HCC can provide higher sensitivity (up to 30% more) for liver cancer detection. In order to characterize and determine if LMW urine DNA is derived from plasma DNA, we performed WGS to compare the size profiles and coverage of these two sources of cell-free DNA in 3 sets of matching HCC urine and plasma samples. Overall shorter read lengths were obtained from urine DNA as compared to the corresponding plasma DNA. A series of peaks occurring at 10 bp periodicity was displayed in both sources of cell-free DNA Further analysis regarding the genome coverage, overlapping fraction and variant detection is ongoing. In conclusion, our data suggests that (i) HMW urine DNA can replace PBMC DNA for providing comprehensive genomic sequencing data and (ii) urine can complement blood for liver cancer liquid biopsy, precision medicine, and potential applications to other cancers. Citation Format: Surbhi Jain, Tai-Jung Lee, Adam Zhang, Jonathan Cheng, Jamin D. Steffen, Chi-Tan Hu, James P. Hamilton, Harry Luu, Hie-Won Hann, Fwu-Shan Shieh, Selena Y. Lin, Wei Song, Ying-Hsiu Su. Urine as an alternative to blood for germline genomics and cancer genetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 417.

Potential use of transrenal DNA for non-invasive monitoring and prognosis of colorectal cancer

Background: Anti-EGFR mAb are recommended treatment for metastatic colorectal cancer (mCRC). Accurate mutation profiling and disease monitoring are challenging. The current study investigates the potential use of transrenal DNA as a biomarker for disease management.Methods: Agreement between archival tissue specimens and transrenal DNA extracted from 200 post-treated mCRC patients was determined. Total DNA concentrations were measured and mutations within the KRAS and EGFR genes were profiled for each specimen. To ascertain therapy resistance; patients were serially monitored monthly.Results: Concordance measurement with matched tissues at baseline was remarkably high (92%) for EGFR and KRAS mutations. Sensitivity and specificity were 98.4% and 89.1% respectively. Newly detectable mutations for a subgroup of patients with initial wildtype characteristics were evident after 4 months of anti-EGFR mAb therapy. Survival analysis using adjusted estimates showed that patients detected by transrenal DNA for key mutations or had higher mutant DNA content had poorer outcome.Conclusion: Transrenal DNA offers new options to follow clinical treatment in mCRC. It demonstrates the ability to capture newly acquired mutations that has strong associative links to therapy resistance. Patients with these mutations fared poorly for survival outcomes and indicated possible prognostic value for transrenal DNA detection.

Whole exome sequencing analysis of urine trans-renal tumour DNA in metastatic colorectal cancer patients

BackgroundThe analysis of circulating free tumour DNA (ctDNA) in blood, commonly referred as liquid biopsy, is being used to characterise patients with solid cancers. Tumour-specific genetic variants can also be present in DNA isolated from other body fluids, such as urine. Unlike blood, urine sampling is non-invasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumour DNA that clears from the glomerular filtration barrier, named trans-renal tumour DNA (trtDNA), are largely unexplored.Patients and methodsSpecimens were collected from 24 patients with KRAS or BRAF mutant metastatic colorectal cancer (mCRC). Driver mutations were assessed by droplet digital PCR (ddPCR) in ctDNA from plasma and trtDNA from urine. Whole exome sequencing (WES) was performed in DNA isolated from tissue, plasma and urine.ResultsOut of the 24 CRC cases, only four had sufficient DNA to allow WES analyses in urine and plasma. We found that tumour alterations primarily reside in low molecular weight fragments (less than 112 bp). In patients whose trtDNA was more than 2.69% of the urine derived DNA, cancer-specific molecular alterations, mutational signatures and copy number profiles identified in urine DNA are comparable with those detected in plasma ctDNA.ConclusionsWith current technologies, WES analysis of trtDNA is feasible in a small fraction of mCRC patients. Tumour-related genetic information is mainly present in low molecular weight DNA fragments. Although the limited amounts of trtDNA poses analytical challenges, enrichment of low molecular weight DNAs and optimised computational tools can improve the detection of tumour-specific genetic information in urine.

Transrenal DNA-based diagnosis of Strongyloides stercoralis (Grassi, 1879) infection: Bayesian latent class modeling of test accuracy.

For epidemiological work with soil transmitted helminths the recommended diagnostic approaches are to examine fecal samples for microscopic evidence of the parasite. In addition to several logistical and processing issues, traditional diagnostic approaches have been shown to lack the sensitivity required to reliably identify patients harboring low-level infections such as those associated with effective mass drug intervention programs. In this context, there is a need to rethink the approaches used for helminth diagnostics. Serological methods are now in use, however these tests are indirect and depend on individual immune responses, exposure patterns and the nature of the antigen. However, it has been demonstrated that cell-free DNA from pathogens and cancers can be readily detected in patient’s urine which can be collected in the field, filtered in situ and processed later for analysis. In the work presented here, we employ three diagnostic procedures—stool examination, serology (NIE-ELISA) and PCR-based amplification of parasite transrenal DNA from urine–to determine their relative utility in the diagnosis of S. stercoralis infections from 359 field samples from an endemic area of Argentina. Bayesian Latent Class analysis was used to assess the relative performance of the three diagnostic procedures. The results underscore the low sensitivity of stool examination and support the idea that the use of serology combined with parasite transrenal DNA detection may be a useful strategy for sensitive and specific detection of low-level strongyloidiasis.

Evaluation of a Urine-Based Rapid Molecular Diagnostic Test with Potential to Be Used at Point-of-Care for Pulmonary Tuberculosis: Cape Town Cohort

Tuberculosis (TB) diagnosis among sputum-scarce patients is time consuming. Thus, a nonsputum diagnostic alternative is urgently needed. The Mycobacterium tuberculosis-specific transrenal (Tr) DNA from urine is a potential target for TB diagnostics. In this study, a new urine-based Tr-DNA molecular assay was evaluated for diagnosis of pulmonary tuberculosis among 428 adults suspected of having pulmonary TB (164 HIV positive, 263 HIV negative) from Cape Town, South Africa. Tr-DNA was isolated from 4 mL of EDTA urine, and a rapid, double-stranded, primer-based PCR method was performed targeting the Mycobacterium tuberculosis-specific direct repeat region. Each Tr-DNA eluate was tested in triplicate using an automated molecular analyzer with controls included in each test. With liquid culture used as the gold standard, the Tr-DNA assay showed sensitivity of 42.9% (n = 75/175; 95% CI, 35.4%-50.5%) and specificity of 88.6% (n = 210/237; 95% CI, 83.9%-92.4%). Among HIV-infected patients with TB, sensitivity and specificity were 45.2% and 89.0%, respectively. The combination of smear microscopy and Tr-DNA increased the sensitivity to 83.8% (smear microscopy alone, 75.1%), with 96.6% specificity. This study indicates that Tr-DNA has a moderate specificity with low sensitivity for diagnosis of pulmonary TB. Despite low sensitivity, this diagnostic test may have potential in combination with smear microscopy to support TB diagnosis in HIV-endemic regions, where sputum-scarce patients are common.

Variations in transrenal DNA and comparison with plasma DNA as a diagnostic marker for colorectal cancer.

Transrenal DNA can potentially be useful in the disease management of colorectal cancer (CRC) and has potential diagnostic utility. Our study aimed to conduct preoperative and postoperative analysis of KRAS-positive patients, using transrenal DNA compared with plasma DNA. This is critically needed for disease diagnostics and treatment response monitoring. CRC patients at different stages of the disease and with different molecular profiles were recruited for serial time-point analysis. Preoperative and postoperative urine specimens were extracted and compared with tumor tissues and plasma DNA. Our analysis demonstrated that transrenal DNA offered comparable sensitivity and specificity to plasma DNA analysis. Collection of transrenal DNA, being noninvasive, is an attractive assay and easily allows serial monitoring of the disease. Results from preoperative detection showed a close correlation to tumor tissue profiling and demonstrated close associations to the disease. We also observed significant decreases in mutant KRAS DNA concentration after surgery, which confirmed transrenal DNA's sensitivity to treatment response. The use of transrenal DNA offers a possible alternative method of disease profiling, detection and tracking. Our study is one of the first to systematically analyze a relatively large number of different CRC patients using transrenal DNA. The positive correlation with disease demonstrates the assay's viability in the clinical setting, and our method further opens up the possibility to use transrenal DNA for clinical intervention investigations.

Abstract 3834: Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade

Abstract A metastatic colorectal cancer (mCRC) patient carrying CAD-ALK translocation achieved partial response to an experimental ALK inhibitor and then progressed after 5 months. We studied whether urine cell-free, trans-renal DNA (tr-DNA) could be used to monitor tumor burden and patient’s response. A NGS panel was developed to interrogate 52 common cancer gene rearrangements and 14 frequently mutated genes in cancer patients. A TP53 p.R248W mutation and the CAD-ALK genomic breakpoint (rearrangement) were identified in the tumor tissue and matched plasma circulating tumor DNA (ctDNA) Urine samples were longitudinally obtained from the patient during ALK inhibitor treatment in parallel with blood. To detect the CAD-ALK translocation in urine tr-DNA we designed ultra-short (51 bp amplicon) primer pairs spanning the genomic breakpoint as a unique tumor marker. This approach allowed the non-invasive monitoring of the gene fusion in urine with amounts paralleling tumor burden. Of note, CAD-ALK gene fusion was apparent in urine tr-DNA before radiological confirmation of disease progression. The same strategy was applied to plasma ctDNA and the results were compared. To detect point mutations in urine tr-DNA, we exploited a peptide nucleic acids (PNA)-CLAMP PCR coupled with droplet digital PCR (ddPCR) analysis to specifically suppress amplification of wild-type DNA fragments. Custom PNA probes were designed for TP53 codon 248, and a ddPCR assay was optimized to detect the TP53 p.R248W mutation, which was then identified in all urine tr-DNA samples, with absolute copies correlating with tumor burden throughout ALK inhibitor treatment. In conclusion, we find that urine tr-DNA can be exploited to non-invasively monitor tumor burden by detecting tumor-specific gene fusions as well as point mutations. Citation Format: Giulia Siravegna, Andrea Sartore-Bianchi, Benedetta Mussolin, Laura Palmieri, Federica Tosi, Andrea Cassingena, Luca Novara, Michela Buscarino, Giorgio Corti, Giovanni Crisafulli, Alice Bartolini, Mark Erlander, Federica Di Nicolantonio, Salvatore Siena, Alberto Bardelli. Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3834. doi:10.1158/1538-7445.AM2017-3834

Tracking aCAD-ALK gene rearrangement in urine and blood of a colorectal cancer patient treated with an ALK inhibitor

Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying a CAD-ALK translocation during treatment with an ALK inhibitor. Using a custom next generation sequencing panel we identified the genomic CAD-ALK rearrangement and a TP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of the CAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient's clinical course. Detection of the CAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identified ALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.

Utility of urinary circulating tumor DNA for EGFR mutation detection in different stages of non-small cell lung cancer patients.

Non-invasive methods of molecular profiling for non-small cell lung cancer (NSCLC) are useful for monitoring disease progression. The aim of the current study was to ascertain if transrenal DNA is sensitive for clinical correlation and EGFR detection in NSCLC patients. 160 patients at various stages of the disease participated and samples were collected prospectively at 2-month intervals. A baseline sample was taken before treatment commencement. To ascertain the sensitivity of transrenal DNA, we compared its results with plasma DNA. ddPCR was used to profile the urine and blood samples for key EGFR mutations. Using tumor tissues as references, our study showed good concordance in EGFR mutations with transrenal DNA before treatment. Results were highly matching in late-stage NSCLC patients, with stage III/IV patients yielding an agreement of more than 90%. The assay was also sensitive to detect early-stage patients after surgical procedures. Profiles were highly concordant with results derived from plasma DNA, demonstrating the specificity of transrenal DNA assays. Serial monitoring of these patients showed stable molecular signatures and correlated to different treatments. Survival analysis showed good prognostic utility for late-stage patients with high transrenal DNA variations and patients that acquired T790M mutation. The study demonstrated the feasibility of using transrenal DNA in mutation profiling for different stages of NSCLC patients. It highlights the importance of continual monitoring and has potential clinical utility in the clinical management of NSCLC.

Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection

Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found an increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types.