Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling.

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

The Origin of Tumor DNA in Urine of Urogenital Cancer Patients: Local Shedding and Transrenal Excretion.

In urogenital cancers, urine as a liquid biopsy for non-invasive cancer detection holds great promise for future clinical application. Their anatomical position allows for the local shedding of tumor DNA, but recent data indicate that tumor DNA in urine might also result from transrenal excretion. This study aims to assess the origin of tumor-associated DNA in the urine of 5 bladder and 25 cervical cancer patients. Besides natural voided urine, paired urine samples were collected in which contact with the local tumor was circumvented to bypass local shedding. The latter concerned nephrostomy urine in bladder cancer patients, and catheter urine in cervical cancer patients. Methylation levels of GHSR, SST, and ZIC1 were determined using paired bladder tumor tissues and cervical scrapes as a reference. Urinary methylation levels were compared to natural voided urine of matched controls. To support methylation results, mutation analysis was performed in urine and tissue samples of bladder cancer patients. Increased methylation levels were not only found in natural voided urine from bladder and cervical cancer patients, but also in the corresponding nephrostomy and catheter urine. DNA mutations detected in bladder tumor tissues were also detectable in all paired natural voided urine as well as in a subset of nephrostomy urine. These results provide the first evidence that the suitability of urine as a liquid biopsy for urogenital cancers relies both on the local shedding of tumor cells and cell fragments, as well as the transrenal excretion of tumor DNA into the urine.

Close Surveillance with Long-Term Follow-up of Subjects with Preinvasive Endobronchial Lesions.

RATIONALE: Autofluorescence bronchoscopy (AFB) and computed tomography (CT) enable lung cancer (LC) detection at the early (pre-)invasive stage. However, LC risk in patients with preinvasive endobronchial lesions is unclear. OBJECTIVES: To assess LC incidence and identify potential risk determinants in patients with preinvasive lesions. METHODS: In our tertiary care referral center, 164 subjects with preinvasive lesions were monitored up to 12.5 years by repeated AFB and CT. Occurrence of LC was monitored. Clinical management depended on histological grade, with cancer patients receiving standard care. Potential risk determinants (smoking status, baseline histology, cancer history, and chronic obstructive pulmonary disease [COPD] status) were evaluated in relation to cancer occurrence, event-free survival (EFS), and overall survival (OS). MEASUREMENTS AND MAIN RESULTS: During surveillance (median of 30 mo, range 4-152) of 164 subjects with preinvasive lesions (80 high grade and 84 low grade at inclusion), 61 LCs were detected in 55 subjects (median time to event 16.5 mo). Twenty-three LCs (38%) were detected by CT, and 38 (62%) were detected by AFB. More cancers (36 of 61; 59%) developed from separate, rather than initial lesional sites. Subjects with high-grade lesions were more likely to be diagnosed with LC at the same or another site in the lungs than those with low-grade lesions (P = 0.03). Independent risk determinants for OS were previous curatively treated cancer and COPD (P ≤ 0.05). CONCLUSIONS: Presence of preinvasive lesions, especially high-grade lesions, may serve as LC risk markers. LCs occur both at preinvasive lesion sites and elsewhere in the bronchial epithelium or lung parenchyma. Prospective validation of biomarkers and randomized intervention studies are needed to determine optimal management strategies.

HPV-associated lung cancers: an international pooled analysis.

Human papillomavirus (HPV) is the etiologic risk factor for cervical cancer. Some studies have suggested an association with a subset of lung tumors, but the etiologic link has not been firmly established. We performed an international pooled analysis of cross-sectional studies (27 datasets, n = 3249 patients) to evaluate HPV DNA prevalence in lung cancer and to investigate viral presence according to clinical and demographic characteristics. HPV16/18 were the most commonly detected, but with substantial variation in viral prevalence between geographic regions. The highest prevalence of HPV16/18 was observed in South and Central America, followed by Asia, North America and Europe (adjusted prevalence rates = 22, 5, 4 and 3%, respectively). Higher HPV16 prevalence was noted in each geographic region compared with HPV18, except in North America. HPV16/18-positive lung cancer was less likely observed among White race (adjusted odds ratio [OR] = 0.33, 95% confidence interval [CI] = 0.12-0.90), whereas no associations were observed with gender, smoking history, age, histology or stage. Comparisons between tumor and normal lung tissue show that HPV was more likely to be present in lung cancer rather than normal lung tissues (OR = 3.86, 95% CI = 2.87-5.19). Among a subset of patients with HPV16-positive tumors, integration was primarily among female patients (93%, 13/14), while the physical status in male cases (N = 14) was inconsistent. Our findings confirm that HPV DNA is present in a small fraction of lung tumors, with large geographic variations. Further comprehensive analysis is needed to assess whether this association reflects a causal relationship.

DNA copy number aberrations in endobronchial lesions: a validated predictor for cancer.

We recently identified a DNA copy number aberration (CNA)-based classifier, including changes at 3p26.3-p11.1, 3q26.2-29, and 6p25.3-24.3, as a risk predictor for cancer in individuals presenting with endobronchial squamous metaplasia. The current study was set out to validate the prediction accuracy of this classifier in an independent series of endobronchial squamous metaplastic and dysplastic lesions. The study included 36 high-risk subjects who had endobronchial lesions of various histological grades that were identified and biopsied by autofluorescence bronchoscopy and were subjected to arrayCGH in a nested case-control design. Of the 36 patients, 12 had a carcinoma in situ or invasive carcinoma at the same site at follow-up (median 11 months, range 4-24), while 24 controls remained cancer free (78 months, range 21-142). The previously defined CNA-based classifier demonstrated 92% (95% CI 77% to 98%) accuracy for cancer (in situ) prediction. All nine subjects with CNA-based classifier-positive endobronchial lesions at baseline experienced cancer outcome, whereas all 24 controls and 3 cases were classified as being low risk. In conclusion, CNAs prove to be a highly accurate biomarker for assessing the progression risk of endobronchial squamous metaplastic and dysplastic lesions. This classifier could assist in selecting subjects with endobronchial lesions who might benefit from more aggressive therapeutic intervention or surveillance.

Can quantifying free-circulating DNA in plasma be used to identify subjects with high-grade pre-invasive endobronchial lesions?

Increased concentrations of free-circulating plasma DNA (cpDNA) are observed in patients with invasive cancer, including lung cancer. Whether cpDNA levels are elevated in subjects with high-grade pre-invasive lesions of lung squamous cell carcinoma (SqCC) and whether its detection may be of value for identifying subjects at the highest risk of developing lung SqCC is currently unknown. The present study assessed cpDNA levels in subjects with high- and low-grade pre-invasive squamous endobronchial lesions relative to patients with clinically overt lung SqCC and healthy controls using real-time quantitative PCR methodology. The median cpDNA levels of the patients with invasive lung SqCC (n=16) were significantly higher compared with those of the healthy controls (n=16; P<0.01), whereas the cpDNA levels in the subjects with pre-invasive lesions (n=20) did not differ from those of the controls (P=0.29). The cpDNA levels in subjects with high-grade pre-invasive lesions were highly similar to those diagnosed with low-grade pre-invasive lesions (P=0.85). Our data suggest that cpDNA levels are not increased during the pre-invasive stages of lung squamous carcinogenesis.

High-risk human papillomavirus-positive lung cancer: molecular evidence for a pattern of pulmonary metastasis.

INTRODUCTION: Infection with high-risk types of human papillomavirus (hrHPV) is associated with cervical, anogenital, and oropharyngeal cancers. Since a causal contribution of hrHPV infection to lung cancer (LC) is still a matter of debate, a comprehensive study was performed to delineate hrHPV involvement in LC, using a Dutch study population. METHODS: Archival tissue specimens from 223 patients (145 men, 78 women, median age 65 years, range 27-87 years), who presented with cancer in the lungs, were subjected to GP5+/6+ polymerase chain reaction and p16 immunohistochemistry. The series included primary lung carcinomas of patients without a history of cancer (n = 175), primary lung carcinomas of patients with an unrelated cancer in the past (n = 36), and carcinomas with primary presentation in the lungs of which the origin (i.e., primary or metastasis) was equivocal at the time of diagnosis (n = 12). GP5+/6+ polymerase chain reaction/p16 double-positive carcinomas were subjected to HPV genotyping, HPVE7 transcript analysis, loss of heterozygosity analysis, and array-comparative genomic hybridization. RESULTS: Whereas all primary lung carcinomas were hrHPV-negative (211 of 211, 100%), three hrHPV-positive equivocal carcinomas (3 of 12, 25%) were identified. These patients (1 male, 2 females) had a history of hrHPV-associated disease; one tonsillar and two cervical carcinomas. A clonal relationship between individual tumor pairs was supported by identical hrHPV genotype, pattern of p16 expression, HPVE7 mRNA expression, and genomic aberrations. CONCLUSIONS: hrHPV presence in a tumor with primary presentation in the lungs signifies pulmonary metastasis from a primary hrHPV-positive cancer elsewhere in the body. No support was found for an attribution of hrHPV infection to the development of primary LC.

DNA copy number alterations in endobronchial squamous metaplastic lesions predict lung cancer.

RATIONALE: Autofluorescence bronchoscopy (AFB) is a valid strategy for detecting premalignant endobronchial lesions. However, no biomarker can reliably predict lung cancer risk of subjects with AFB-visualized premalignant lesions. OBJECTIVES: The present study set out to identify AFB-visualized squamous metaplastic (SqM) lesions with malignant potential by DNA copy number profiling. METHODS: Regular AFB examinations in 474 subjects at risk of lung cancer identified six subjects with SqM lesions at baseline, and carcinoma in situ or carcinoma (carcinoma in situ or greater) at the initial SqM site at follow-up bronchoscopy. These progressive SqM lesions were compared for immunostaining pattern and array comparative genomic hybridization-based chromosomal profiles with 23 SqM lesions of subjects who remained cancer-free. Specific DNA copy number alterations (CNAs) linked to cancer risk were identified and accuracy of CNAs to predict endobronchial cancer in this series was determined. MEASUREMENTS AND MAIN RESULTS: At baseline, p53, p63, and Ki-67 immunostaining were not predictive for a differential clinical outcome of SqM lesions. The mean number of CNAs in baseline SqM of cases was significantly higher compared with control subjects (P < 0.01). Chromosomal regions significantly more frequently altered in SqM of cases were 3p26.3-p11.1, 3q26.2-q29, 9p13.3-p13.2, and 17p13.3-p11.2 (family-wise error rate <0.10). CNAs were specifically detected at the site of future cancer. In cases, baseline-detected CNAs persisted in subsequent biopsies taken from the initial site, and levels increased toward cancer progression. In this series, a model based on CNAs at 3p26.3-p11.1, 3q26.2-29, and 6p25.3-24.3 predicted cancer with 97% accuracy. CONCLUSIONS: The data suggest that the presence of specific CNAs in SqM lesions predict endobronchial cancer.

Methylation-mediated silencing and tumour suppressive function of hsa-miR-124 in cervical cancer.

BACKGROUND: A substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis. RESULTS: Quantitative MSP analysis of all 3 loci encoding the mature hsa-miR-124 (hsa-miR-124-1/-2/-3) showed methylation in cervical cancer cell lines SiHa, CaSki and HeLa as well as in late passages of human papillomavirus (HPV) type 16 or 18 immortalised keratinocytes. Treatment of SiHa cells with a demethylating agent reduced hsa-miR-124 methylation levels and induced hsa-miR-124 expression. In HPV-immortalised keratinocytes increased methylation levels were related to reduced hsa-miR-124 expression and higher mRNA expression of IGFBP7, a potential hsa-miR-124 target gene. Ectopic hsa-miR-124 expression in SiHa and CaSki cells decreased proliferation rates and migratory capacity. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 139 cervical tissue specimens showed an increasing methylation frequency from 0% in normal tissues up to 93% in cervical carcinomas. Increased methylation levels of hsa-miR-124-1 and hsa-miR-124-2 were significantly correlated with reduced hsa-miR-124 expression in cervical tissue specimens. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 43 cervical scrapes of high-risk HPV positive women was predictive of underlying high-grade lesions. CONCLUSIONS: DNA methylation-based silencing of hsa-miR-124 is functionally involved in cervical carcinogenesis and may provide a valuable marker for improved detection of cervical cancer and its high-grade precursor lesions.