Clinical Chemistry

Sequence Now, Later, or Never?

Predictive Relevance of Circulating miR-622 in Patients with Newly Diagnosed and Recurrent High-Grade Serous Ovarian Carcinoma

Abstract Background Identifying patients with high-grade serous ovarian cancer (HGSOC) who will respond to treatment remains a clinical challenge. We focused on miR-622, a miRNA involved in the homologous recombination repair (HRR) pathway, and we assessed its predictive value in serum prior to first-line chemotherapy and at relapse. Methods Serum miR-622 expression was assessed in serum prior to first-line platinum-based chemotherapy in a prospective multicenter study (miRNA Serum Analysis, miRSA, NCT01391351) and a retrospective cohort (Biological Resource Center, BRC), and was also studied at relapse. Progression-free survival (PFS) and overall survival (OS) were used as primary and secondary endpoints prior to first-line chemotherapy and OS as a primary endpoint at relapse. Results The group with high serum miR-622 expression was associated with a significantly lower PFS (15.4 versus 24.4 months; adjusted HR 2.11, 95% CI 1.2 3.8, P = 0.015) and OS (29.7 versus 40.6 months; adjusted HR 7.68, 95% CI 2.2–26.2, P = 0.0011) in the miRSA cohort. In the BRC cohort, a high expression of miR-622 was also associated with a significantly lower OS (22.8 versus 35.9 months; adjusted HR 1.98, 95% CI 1.1–3.6, P = 0.026). At relapse, high serum miR-622 was associated with a significantly lower OS (7.9 versus 20.6 months; adjusted HR 3.15, 95% CI 1.4–7.2, P = 0.0062). Serum miR-622 expression is a predictive independent biomarker of response to platinum-based chemotherapy for newly diagnosed and recurrent HGSOC. Conclusions These results may open new perspectives for HGSOC patient stratification and monitoring of resistance to platinum-based and poly(ADP-ribose)-polymerase-inhibitor-maintenance therapies, facilitating better and personalized treatment decisions.

Methylation-Associated Nucleosomal Patterns of Cell-Free DNA in Cancer Patients and Pregnant Women

Abstract Background Cell-free DNA (cfDNA) analysis offers an attractive noninvasive means of detecting and monitoring diseases. cfDNA cleavage patterns within a short range (e.g., 11 nucleotides) have been reported to correlate with cytosine-phosphate-guanine (CpG) methylation, allowing fragmentomics-based methylation analysis (FRAGMA). Here, we adopted FRAGMA to the extended region harboring multiple nucleosomes, termed FRAGMAXR. Methods We profiled cfDNA nucleosomal patterns over the genomic regions from −800 to 800 bp surrounding differentially methylated CpG sites, harboring approximately 8 nucleosomes, referred to as CpG-associated cfDNA nucleosomal patterns. Such nucleosomal patterns were analyzed by FRAGMAXR in cancer patients and pregnant women. Results We identified distinct cfDNA nucleosomal patterns around differentially methylated CpG sites. Compared with subjects without cancer, patients with hepatocellular carcinoma (HCC) showed reduced amplitude of nucleosomal patterns, with a gradual decrease over tumor stages. Nucleosomal patterns associated with differentially methylated CpG sites could be used to train a machine learning model, resulting in the detection of HCC patients with an area under the receiver operating characteristic curve of 0.93. We further demonstrated the feasibility of multicancer detection using a dataset comprising lung, breast, and ovarian cancers. The tissue-of-origin analysis of plasma cfDNA from pregnant women and cancer patients revealed that the placental DNA and tumoral DNA contributions deduced by FRAGMAXR correlated well with values measured using genetic variants (Pearson r: 0.85 and 0.94, respectively). Conclusions CpG-associated cfDNA nucleosomal patterns of cfDNA molecules are influenced by DNA methylation and might be useful for biomarker developments for cancer liquid biopsy and noninvasive prenatal testing.

High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies

AbstractBackgroundMicrosatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool.MethodsWe developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy.ResultsThe 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response.ConclusionsThis innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

Detection of Multiple HPV Types in Liquid Biopsies of Cervical Neoplasia

Abstract Background More than 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Cell-free (cf) HPV DNA detection in blood samples may serve as a monitoring tool for cervical cancer. Methods In our methodological study, an HPV panel for simultaneous detection of 24 types using mass spectrometry-based analysis was developed for liquid biopsy approaches and tested on HPV positive cell lines, plasmid controls, and cervical high-grade squamous intraepithelial lesions (HSIL) in positive smear samples (n = 52). It was validated in cfDNA blood samples (n = 40) of cervical cancer patients. Results The HPV panel showed proficient results in cell lines and viral plasmids with a limit of detection of 1 IU (international units)/µL for HPV16/18 and 10GE/µL for HPV11/31/33/39/45/51/52/58/59 and a specificity of 100% for the tested HPV types. In cervical smear samples, HPV DNA was detected with a sensitivity of 98.14%. The overall agreement between the new HPV panel and clinical records was 97.2% (κ = 0.84). In cervical cancer cfDNA, 26/40 (65.0%) tested positive for any HPV type, with most infections due to hrHPV (24/26). HPV positive samples were found in all FIGO stages, with the highest positivity ratio in FIGO III and IV. Even the lowest stage, FIGO I, had 12/23 (52.2%) patients with a positive HPV plasma status. Conclusions This proof-of-concept paper shows that the described assay produces reliable results for detecting HPV types in a multiplex mass spectrometry-based assay in cervical smear and cfDNA with high specificity and sensitivity in both cohorts. The assay shows potential for liquid biopsy-based applications in monitoring cervical cancer progression.

Liquid Biopsy to Catch the Epigenetic Changes in Endometrial Cancer

Highly Specific Droplet-Digital PCR Detection of Universally Methylated Circulating Tumor DNA in Endometrial Carcinoma

Abstract Background No circulating biomarker is available for endometrial carcinoma (EC). We aimed to identify DNA positions universally hypermethylated in EC, and to develop a digital droplet PCR (ddPCR) assay for detection of hypermethylated circulating tumor DNA (meth-ctDNA) in plasma from patients with EC. Methods DNA positions hypermethylated in EC, and without unspecific hypermethylation in tissue/cell types releasing circulating cell-free DNA in plasma, were identified in silico from TCGA/Gene Expression Omnibus (GEO) data. A methylation-specific ddPCR (meth-ddPCR) assay following bisulfite conversion of DNA extracted from plasma was optimized for detection of meth-ctDNA according to dMIQE guidelines. Performances were validated on a retrospective cohort (n = 78 tumors, n = 30 tumor-adjacent tissues), a prospective pilot cohort (n = 33 stage I–IV patients), and 55 patients/donors without cancer. Results Hypermethylation of zinc finger and SCAN domain containing 12 (ZSCAN12) and/or oxytocin (OXT) classified EC samples from multiple noncancer samples with high diagnostic specificity/sensitivity [>97%; area under the curve (AUC) = 0.99; TCGA/GEO tissues/blood samples]. These results were confirmed in the independent retrospective cohort (AUC = 0.99). Meth-ddPCR showed a high analytical specificity (limit of blank = 2) and sensitivity (absolute lower threshold of detection = 50 pgmethDNA/mLplasma). In the pilot cohort, meth-ctDNA was detected in pretreatment plasma samples from 9/11 and 5/20 patients with advanced and non-advanced EC, respectively. 2 of 9 patients had ctDNA detected after macroscopic complete surgery and experienced progression within 6 months. No healthy donors had any copy of hypermethylated DNA detected in plasma. Conclusions Meth-ddPCR of ZSCAN12/OXT allows a highly specific and sensitive detection of ctDNA in plasma from patients with EC and appears promising for personalized approaches for these patients.

Splicing Predictions, Splicing Assays, and Variant Classification Using ACMG/AMP Guidelines: Challenges Observed with BRCA1 and BRCA2 Variants

Abstract Background Germline loss-of-function variants in BRCA1 and BRCA2 are established drivers of hereditary breast and ovarian cancer, often acting through aberrant splicing. However, not all spliceogenic changes are pathogenic, and many variants remain classified as uncertain due to insufficient experimental evidence and challenges in applying the ACMG/AMP variant interpretation framework to splicing alterations. Methods In this study, we examined the splicing outcomes of 17 variants—10 in BRCA1 [c.135-2A>G; c.135-5T>C; c.5074+1G>C; c.5332+2_5332+4del; c.5333-8C>T; c.5335C>G p.(Gln1779Glu); c.302-24_302-22del; c.302-23A>G; c.547+57T>C; c.4096+34C>G] and 7 in BRCA2 [c.-39-5delT; c.67+3A>G; c.425G>A p.(Ser142Asn); c.425G>T p.(Ser142Ile); c.517-13_517-9del; c.681+5G>C; c.67+84_67+85del]—identified in families with suspected hereditary breast and/or ovarian cancer. Depending on sample availability, we assessed splicing either on carrier-derived mRNA or via splicing-reporter minigene assay. Results Eight variants triggered aberrant splicing, while 9 showed no spliceogenic effect. Our findings, combined in some cases with previously published data, allowed us to apply the PVS1_(RNA) criterion at full strength to some variants. For others, residual full-length transcripts or in-frame mis-spliced isoforms precluded full application of PVS1_(RNA). Conclusions Following ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel specifications based on ACMG/AMP guidelines, we classified 4 variants as pathogenic or likely pathogenic, 10 as benign or likely benign, and 3 as uncertain significance. This comprehensive analysis of splicing defects refines the clinical classification of BRCA1 and BRCA2 variants and highlights the value of combining experimental and computational evidence to enhance genetic risk assessment in hereditary cancer.