CD8+ cell somatic mutations in multiple sclerosis patients and controls—Enrichment of mutations in STAT3 and other genes implicated in hematological malignancies

Somatic mutations have a central role in cancer but their role in other diseases such as common autoimmune disorders is not clear. Previously we and others have demonstrated that especially CD8+ T cells in blood can harbor persistent somatic mutations in some patients with multiple sclerosis (MS) and rheumatoid arthritis. Here we concentrated on CD8+ cells in more detail and tested (i) how commonly somatic mutations are detectable, (ii) does the overall mutation load differ between MS patients and controls, and (iii) do the somatic mutations accumulate non-randomly in certain genes? We separated peripheral blood CD8+ cells from newly diagnosed relapsing MS patients (n = 21) as well as matched controls (n = 21) and performed next-generation sequencing of the CD8+ cells’ DNA, limiting our search to a custom panel of 2524 immunity and cancer related genes, which enabled us to obtain a median sequencing depth of over 2000x. We discovered nonsynonymous somatic mutations in all MS patients’ and controls’ CD8+ cell DNA samples, with no significant difference in number between the groups (p = 0.60), at a median allelic fraction of 0.5% (range 0.2–8.6%). The mutations showed statistically significant clustering especially to the STAT3 gene, and also enrichment to the SMARCA2, DNMT3A, SOCS1 and PPP3CA genes. Known activating STAT3 mutations were found both in MS patients and controls and overall 1/5 of the mutations were previously described cancer mutations. The detected clustering suggests a selection advantage of the mutated CD8+ clones and calls for further research on possible phenotypic effects.

Fine-Needle Aspiration Is Suitable for Breast Cancer BRCA Molecular Assessment: A Case Report

Breast cancer is the most common cause of cancer-related deaths in the female population worldwide. To the best of our knowledge, breast cancer (BRCA)1/2 gene mutations have not been described yet on breast cancer cytological specimens. Here we describe the case of a 38-year old woman with a family and personal history for breast cancer, who underwent a fine needle aspiration (FNA) procedure for a novel 30 mm lesion located in the external quadrants of the contralateral (left) breast. Cytological findings and ancillary immunostaining confirmed the diagnosis of a triple negative NST carcinoma. BRCA1/2 molecular assessment was carried out on DNA extracted from cytological (November 2020), biopsy (December 2014) and surgical resection (July 2015) specimens, as well as on the resection of a benign fibroadenoma, by using a next generation sequencing approach. Molecular analysis showed a pathogenic BRCA1 insertion (c.5266dupC; p.Q1756PfsTer74) in the cytological specimen (allelic fraction 92.0%), biopsy (allelic fraction 84.2%), surgical resection (allelic fraction 87.8%) and fibroadenoma (58.9%), demonstrating a germinal BRCA mutated status.

Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

Whole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

ctDNA pathogenic variants (PVs) in homologous recombination repair (HRR) genes in patients with metastatic CRPC.

138 Background: HRR PVs can serve as predictive biomarkers and two PARP inhibitors are approved for metastasic CRPC (mCRPC) pts. Published data are predominantly focused on tissue-based assays, but obtaining tissue from mCRPC pts is problematic. In a large tissue based series (PROfound), 4047 mCRPC pts had tumor samples submitted for genomic testing but only 69% had interpretable results. No data were published from PROfound enumerating pts without available tissue to submit. Herein we assess frequency of PVs from selected HRR genes using a ctDNA assay. Methods: 292 mCRPC pts at Tulane Cancer Center were assessed for detectable HRR ctDNA changes using the Guardant 360 assay (which assesses the HRR genes BRCA1, BRCA2, and ATM). Results: 20/292 (6.8%) pts had a PV in ATM. However only 4/292 (1.4%) had > 1% mutant allelic fraction. Germline testing occurred in 18/20 of the ctDNA ATM PV pts and 0/18 had a germline PV. The PROfound series had 6.3% somatic PVs in ATM. 18/292 pts (6.2%) had a PV in BRCA2 and 12/292 (4.1%) had a mutant allelic fraction of > 1%. Germline testing was performed in 17/18 with BRCA2 ctDNA PVs and 9/17 had germline PVs. The PROfound series had 9.7% somatic BRCA2 PVs. BRCA1 PVs were detected in 6/292 (2.1%) pts and 3/292 (1%) had a mutant allelic fraction > 1%. 6/6 of the ctDNA PVs has germline testing and 1/6 had a BRCA1 PV. The PROfound series had 1.3% somatic PVs in BRCA1. Conclusions: Using ctDNA essay, it is feasible to measure PVs in only a small subset of HRR genes in mCRPC pts. These assays fail to detect deep deletions, a known and important mechanism of HRR gene loss. The ctDNA mutant allelic fractions are often low. The ability of ctDNA PVs using this assay to predict treatment effects are unknown.

Baseline pathogenic mutations in non-AR/non-TP53 genes and prediction of response to high-dose testosterone.

146 Background: The fact that high dose of testosterone (HDT) elicits positive responses in a subset of prostate cancer patients (pts) is surprising and puzzling. Genomics data differentiating responders (Rs) from non-responders (NRs) is sparse. Pts with mutations in DNA repair pathway genes may be particularly sensitive to HDT (see BA Teply et al. Eur Urol 71:499, 2017). Herein we perform exploratory analyses to better understand the role of pathogenic mutations (muts) in ctDNA as a predictive biomarker for patients treated with HDT. Methods: ctDNA essays were performed with the Guardant360 methodology pre-HDT. Point mutations were classified by cancervar (http://cancervar.wglab.org). Truncating mutations (frameshift and nonsense) were manually curated to assess for pathogenicity. All patients had CRPC and were pre-treated with abiraterone and/or enzalutamide. HDT was typically administered as 400 mg testosterone cypionate q 3-4 weeks. Rs were compared to NRs. All Rs had >3 more cycles of HDT and a >50% PSA decline (N = 17). Non-responders had <3 cycles and no PSA decline (N = 23). Only muts with an allelic fraction of >0.5% were analyzed given dubious importance of mutations with lower allelic fractions. Results: AR muts (4/17 vs 6/23 in Rs and NRs) and TP53 muts (10/17 vs 11/23 in Rs and NRs) were similar ( P= 0.85 and 0.49, respectively) but the number of pts with non-AR/non-TP53 muts was distinct (3/17 for Rs, and 12/23 for NRs; P= 0.026). The average number of non-AR/non-TP53 muts (Rs = 0.23 and NRs = 0.83) was higher in the NRs (P = 0.046). When analyzing DNA repair alterations, no differences were noted in those with BRCA1/BRCA2/ATM mutations in the Rs and NRs (1/17 vs 6/23 respectively; P= 0.09). Conclusions: AR and TP53 pathogenic mutations are common in both Rs and NRs but other pathogenic mutations are more common in non-responders. We hypothesize that genetic pathways outside of the AR/TP53 axis drive resistance to HDT. Additional studies are warranted to assess whether or not these pathways drive resistance to HDT. Limitations are acknowledged with regard to the Guardant assay gene selection for CRPC pts.

Confirming Putative Variants at ≤5% Allele Frequency Using Allele Enrichment and Sanger Sequencing

Whole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~1000x WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5% and 5% VAF. Each variant was then tested using NuProbe NGSureTM, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

Giant Magnetoresistive Nanosensor Analysis of Circulating Tumor DNA Epidermal Growth Factor Receptor Mutations for Diagnosis and Therapy Response Monitoring

Background Liquid biopsy circulating tumor DNA (ctDNA) mutational analysis holds great promises for precision medicine targeted therapy and more effective cancer management. However, its wide adoption is hampered by high cost and long turnaround time of sequencing assays, or by inadequate analytical sensitivity of existing portable nucleic acid tests to mutant allelic fraction in ctDNA. Methods We developed a ctDNA Epidermal Growth Factor Receptor (EGFR) mutational assay using giant magnetoresistive (GMR) nanosensors. This assay was validated in 36 plasma samples of non-small cell lung cancer patients with known EGFR mutations. We assessed therapy response through follow-up blood draws, determined concordance between the GMR assay and radiographic response, and ascertained progression-free survival of patients. Results The GMR assay achieved analytical sensitivities of 0.01% mutant allelic fraction. In clinical samples, the assay had 87.5% sensitivity (95% CI = 64.0—97.8%) for Exon19 deletion and 90% sensitivity (95% CI = 69.9—98.2%) for L858R mutation with 100% specificity; our assay detected T790M resistance with 96.3% specificity (95% CI = 81.7–99.8%) with 100% sensitivity. After 2 weeks of therapy, 10 patients showed disappearance of ctDNA by GMR (predicted responders), whereas 3 patients did not (predicted nonresponders). These predictions were 100% concordant with radiographic response. Kaplan-Meier analysis showed responders had significantly (P &lt; 0.0001) longer PFS compared to nonresponders (N/A vs. 12 weeks, respectively). Conclusions The GMR assay has high diagnostic sensitivity and specificity and is well suited for detecting EGFR mutations at diagnosis and noninvasively monitoring treatment response at the point-of-care.