CapTCR-seq: hybrid capture for T-cell receptor repertoire profiling.

Mature T-cell lymphomas consisting of an expanded clonal population of T cells that possess common rearrangements of the T-cell receptor (TCR) encoding genes can be identified and monitored using molecular methods of T-cell repertoire analysis. We have developed a hybrid-capture method that enriches DNA sequencing libraries for fragments encoding rearranged TCR genes from all 4 loci in a single reaction. We use this method to describe the TCR repertoires of 63 putative lymphoma clinical isolates, 7 peripheral blood mononuclear cell (PBMC) populations, and a collection of tumor infiltrating lymphocytes. Dominant Variable (V) and Joining (J) gene pair rearrangements in cancer cells were confirmed by polymerase chain reaction (PCR) amplification and Sanger sequencing; clonality assessment of clinical isolates using BIOMED-2 methods showed agreement for 73% and 77% of samples at the ß and γ loci, respectively, whereas ß locus V and J allele prevalence in PBMCs were well correlated with results from commercial PCR-based DNA sequencing assays (r 2 = 0.94 with Adaptive ImmunoSEQ, 0.77-0.83 with Invivoscribe LymphoTrack TRB Assay). CapTCR-seq allows for rapid, high-throughput and flexible characterization of dominant clones within TCR repertoire that will facilitate quantitative analysis of patient samples and enhance sensitivity of tumor surveillance over time.

Landscape of genomic alterations in high-grade serous ovarian cancer from exceptional long- and short-term survivors.

BACKGROUND: Patients diagnosed with high-grade serous ovarian cancer (HGSOC) who received initial debulking surgery followed by platinum-based chemotherapy can experience highly variable clinical responses. A small percentage of women experience exceptional long-term survival (long term (LT), 10+ years), while others develop primary resistance to therapy and succumb to disease in less than 2 years (short term (ST)). To improve clinical management of HGSOC, there is a need to better characterize clinical and molecular profiles to identify factors that underpin these disparate survival responses. METHODS: To identify clinical and tumor molecular biomarkers associated with exceptional clinical response or resistance, we conducted an integrated clinical, exome, and transcriptome analysis of 41 primary tumors from LT (n = 20) and ST (n = 21) HGSOC patients. RESULTS: Younger age at diagnosis, no residual disease post debulking surgery and low CA125 levels following surgery and chemotherapy were clinical characteristics of LT. Tumors from LT survivors had increased somatic mutation burden (median 1.62 vs. 1.22 non-synonymous mutations/Mbp), frequent BRCA1/2 biallelic inactivation through mutation and loss of heterozygosity, and enrichment of activated CD4+, CD8+ T cells, and effector memory CD4+ T cells. Characteristics of ST survival included focal copy number gain of CCNE1, lack of BRCA mutation signature, low homologous recombination deficiency scores, and the presence of ESR1-CCDC170 gene fusion. CONCLUSIONS: Our findings suggest that exceptional long- or short-term survival is determined by a concert of clinical, molecular, and microenvironment factors.

Somatic BRCA1/2 Recovery as a Resistance Mechanism After Exceptional Response to Poly (ADP-ribose) Polymerase Inhibition.

Purpose Durable and long-term responses to the poly (ADP-ribose) polymerase inhibitor olaparib are observed in patients without BRCA1/2 mutations. However, beyond BRCA1/2 mutations, there are no approved biomarkers for olaparib in high-grade serous ovarian cancer (HGSOC). To determine mechanisms of durable response and resistance to olaparib therapy, we performed an analysis of HGSOC tumors from three patients without germline BRCA1/2 mutations who experienced exceptional responses to olaparib. Patients and Methods We performed integrated exome, low-pass genome, and RNA sequence analysis of tumors at diagnosis and upon relapse from patients with platinum-sensitive HGSOC recurrence who were treated > 5 years with olaparib therapy as a single agent. Results We observed somatic disruption of BRCA1/2 in all three patients at diagnosis, followed by subsequent BRCA recovery upon progression by copy number gain and/or upregulation of the remaining functional allele in two patients. The third patient with ongoing response (> 7 years) had a tumor at diagnosis with biallelic somatic deletion and loss-of-function mutation, thereby lacking a functional allele for recovery of BRCA1 activity and indicating a potential cure. Conclusion Olaparib has durable benefit for patients with ovarian cancer beyond germline BRCA1/2 carriers. These data suggest that biallelic loss of BRCA1/2 in cancer cells may be a potential marker of long-term response to poly (ADP-ribose) polymerase inhibition and that restoration of homologous repair function may be a mechanism of disease resistance.

Circulating tumour DNA sequence analysis as an alternative to multiple myeloma bone marrow aspirates.

The requirement for bone-marrow aspirates for genomic profiling of multiple myeloma poses an obstacle to enrolment and retention of patients in clinical trials. We evaluated whether circulating cell-free DNA (cfDNA) analysis is comparable to molecular profiling of myeloma using bone-marrow tumour cells. We report here a hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq) method used to sequence all protein-coding exons of KRAS, NRAS, BRAF, EGFR and PIK3CA in 64 cfDNA specimens from 53 myeloma patients to >20,000 × median coverage. This method includes a variant filtering algorithm that enables detection of tumour-derived fragments present in cfDNA at allele frequencies as low as 0.25% (median 3.2%, range 0.25-46%). Using LB-Seq analysis of 48 cfDNA specimens with matched bone-marrow data, we detect 49/51 likely somatic mutations, with subclonal hierarchies reflecting tumour profiling (96% concordance), and four additional mutations likely missed by bone-marrow testing (>98% specificity). Overall, LB-Seq is a high fidelity adjunct to genetic profiling of bone-marrow in multiple myeloma.

Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer.

Sustained expression of the estrogen receptor-α (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers, suggesting that other mechanisms underlie the persistent expression of ESR1. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk-associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.

The genomic landscape of schwannoma.

Schwannomas are common peripheral nerve sheath tumors that can cause debilitating morbidities. We performed an integrative analysis to determine genomic aberrations common to sporadic schwannomas. Exome sequence analysis with validation by targeted DNA sequencing of 125 samples uncovered, in addition to expected NF2 disruption, recurrent mutations in ARID1A, ARID1B and DDR1. RNA sequencing identified a recurrent in-frame SH3PXD2A-HTRA1 fusion in 12/125 (10%) cases, and genomic analysis demonstrated the mechanism as resulting from a balanced 19-Mb chromosomal inversion on chromosome 10q. The fusion was associated with male gender predominance, occurring in one out of every six men with schwannoma. Methylation profiling identified distinct molecular subgroups of schwannomas that were associated with anatomical location. Expression of the SH3PXD2A-HTRA1 fusion resulted in elevated phosphorylated ERK, increased proliferation, increased invasion and in vivo tumorigenesis. Targeting of the MEK-ERK pathway was effective in fusion-positive Schwann cells, suggesting a possible therapeutic approach for this subset of tumors.

Long-range PCR and next-generation sequencing of BRCA1 and BRCA2 in breast cancer.

Individuals and families carrying mutations in BRCA1 and BRCA2 (BRCA1/2) have a markedly elevated risk of developing breast and ovarian cancers. The first-generation of BRCA1/2 mutation analysis targeted only the coding exons and has implicated protein-truncating mutations (indel, nonsense) in BRCA1/2 inactivation. Recently, heritable breast cancers have also been attributed to other exonic mutations (missense, silent) and mutations in introns and untranslated regions. However, analysis of these alterations has been prohibitively laborious and cost intensive, and the proportion of cases carrying mutations in unscreened regions of BRCA1/2 and other predisposition genes is unknown. We have developed and validated a next-generation sequencing (NGS) approach for BRCA1/2 mutation analysis by applying long-range PCR and deep sequencing. Genomic DNA from familial breast cancer patients (N = 12) were screened and NGS successfully identified all 19 distinct (51 total) BRCA1 and 35 distinct (63 total) BRCA2 sequence alterations detectable by the Sanger sequencing, with no false-negative or positive results. In addition, we report the robust detection of variants from introns and untranslated regions. These results illustrate that NGS can provide comprehensive genetic information more quickly, accurately, and at a lower cost than conventional approaches, and we propose NGS to be a more effective method for BRCA1/2 mutational analysis. Advances in NGS will play an important role in enabling molecular diagnostics and personalized treatment of breast and ovarian cancers.

Application of post-surgical stimulated thyroglobulin for radioiodine remnant ablation selection in low-risk papillary thyroid carcinoma.

BACKGROUND: We present our ongoing experience in the use of postsurgical stimulated serum thyroglobulin (Stim-Tg) to assist in radioiodine remnant ablation (RRA) decision-making. METHODS: Patients with low-risk well-differentiated thyroid carcinoma (WDTC) with undetectable anti-Tg antibodies were prospectively followed after total thyroidectomy and therapeutic central compartment neck dissection, when indicated.Stim-Tg was performed 3 months postoperatively and used to base RRA selection. RESULTS: Of 104 patients, 59 patients (56.7%) had an undetectable Stim-Tg after thyroidectomy, 35 (33.7%) had Stim-Tg values of 1-5 microg/L, and 10 (9.6%) had Stim-Tg values >5 microg/L. RRA was administered to 1 patient (1.7%) with undetectable Stim-Tg, 6 patients (17.1%) with Stim-Tg1-5 microg/L, and 9 patients (90%) with Stim-Tg >5 microg/L, for a total of 16 patients (15.4%) receiving RRA. When compared to current RRA selection guidelines, the proposed protocol achieved a significantly lower RRA administration rate. CONCLUSION: Stim-Tg measurement performed several months after total thyroidectomy is a useful objective parameter in assisting RRA decision-making for patients with low-risk WDTC. (

Influence of age and primary tumor size on the risk for residual/recurrent well-differentiated thyroid carcinoma.

BACKGROUND: Though age and primary tumor size predict cancer-specific survival in well-differentiated thyroid carcinoma (WDTC), their influence on residual/recurrent disease has not been elucidated. METHODS: In a retrospective study, residual/recurrent disease was defined by the surrogate outcome of positive (>or=2 microg/L) follow-up stimulated thyroglobulin after surgery and radioactive remnant ablation. Age, primary tumor size, and clinical staging systems were examined in the context of stimulated thyroglobulin outcome. RESULTS: A total of 246 patients were followed up for a mean of 5.8 years. No significant difference in age (t(239) = 0.61, p > .05) or tumor size (t(237) = 0.16, p > .05) was found among patients with positive follow-up stimulated thyroglobulin compared with those with negative results. pTNM staging failed to demonstrate significant, stage-dependent increase in the percentage of patients with positive stimulated thyroglobulin, chi(2)(2, N = 229) = 0.17, p > .05, unlike staging based solely on surgical pathology, chi(2)(2, N = 241) = 34.97, p < .001. CONCLUSION: Age, primary tumor size, and pTNM staging do not predict risk for residual/recurrent WDTC, whereas extrathyroidal extension at initial surgery is predictive.

Prognostic value of postsurgical stimulated thyroglobulin levels after initial radioactive iodine therapy in well-differentiated thyroid carcinoma.

BACKGROUND: In well-differentiated thyroid carcinoma, predictors of future positivity of stimulated thyroglobulin (>2 microg/L) after initial radioactive iodine treatment are not known. METHODS: In a retrospective study, we used logistic regression analysis to determine whether postoperative stimulated thyroglobulin measurements and pathologic stage independently predict future stimulated thyroglobulin positivity. RESULTS: We followed 141 patients with well-differentiated thyroid carcinoma for a median of 35 months; follow-up stimulated thyroglobulin measurements were positive in 20.6% (29/141). The natural logarithm of the postsurgical stimulated thyrogolobulin was independently associated with a positive stimulated thyroglobulin at long-term follow-up (odds ratio [OR], 4.44; 95% confidence interval [CI], 2.33-8.45; p < .001); there was a trend for a positive association of TNM stage with positive follow-up stimulated thyroglobulin (p = .054). Lymph node positivity predicted a positive stimulated thyroglobulin in papillary cancer. CONCLUSIONS: Stimulated thyroglobulin measurements prior to initial radioactive iodine treatment independently predict future stimulated thyroglobulin positivity in well-differentiated thyroid carcinoma.