Circulating Tumor DNA as a Predictive Marker of Recurrence for Patients With Stage II-III Breast Cancer Treated With Neoadjuvant Therapy.

BACKGROUND: Patients with stage II to III breast cancer have a high recurrence rate. The early detection of recurrent breast cancer remains a major unmet need. Circulating tumor DNA (ctDNA) has been proven to be a marker of disease progression in metastatic breast cancer. We aimed to evaluate the prognostic value of ctDNA in the setting of neoadjuvant therapy (NAT). METHODS: Plasma was sampled at the initial diagnosis (defined as before NAT) and after breast surgery and neoadjuvant therapy(defined as after NAT). We extracted ctDNA from the plasma and performed deep sequencing of a target gene panel. ctDNA positivity was marked by the detection of alterations, such as mutations and copy number variations. RESULTS: A total of 95 patients were enrolled in this study; 60 patients exhibited ctDNA positivity before NAT, and 31 patients exhibited ctDNA positivity after NAT. A pathologic complete response (pCR) was observed in 13 patients, including one ER(+)Her2(-) patient, six Her2(+) patients and six triple-negative breast cancer (TNBC) patients. Among the entire cohort, multivariate analysis showed that N3 classification and ctDNA positivity after NAT were independent risk factors that predicted recurrence (N3, hazard ratio (HR) 3.34, 95% confidence interval (CI) 1.26 - 8.87, p = 0.016; ctDNA, HR 4.29, 95% CI 2.06 - 8.92, p < 0.0001). The presence of ctDNA before NAT did not affect the rate of recurrence-free survival. For patients with Her2(+) or TNBC, patients who did not achieve pCR were associated with a trend of higher recurrence (p = 0.105). Advanced nodal status and ctDNA positivity after NAT were significant risk factors for recurrence (N2 - 3, HR 3.753, 95% CI 1.146 - 12.297, p = 0.029; ctDNA, HR 3.123, 95% CI 1.139 - 8.564, p = 0.027). Two patients who achieved pCR had ctDNA positivity after NAT; one TNBC patient had hepatic metastases six months after surgery, and one Her2(+) breast cancer patient had brain metastasis 13 months after surgery. CONCLUSIONS: This study suggested that the presence of ctDNA after NAT is a robust marker for predicting relapse in stage II to III breast cancer patients.

Using next-generation sequencing to redefine BRCAness in triple-negative breast cancer.

BRCAness is considered a predictive biomarker to platinum and poly(ADP-ribose) polymerase (PARP) inhibitors. However, recent trials showed that its predictive value was limited in triple-negative breast cancer (TNBC) treated with platinum. Moreover, tumors with mutations of DNA damage response (DDR) genes, such as homologous recombination (HR) genes, could be sensitive to platinum and PARP inhibitors. Thus, we aim to explore the relationship between mutation status of DDR genes and BRCAness in TNBC. We sequenced 56 DDR genes in 120 TNBC and identified BRCAness by array comparative genomic hybridization. The sequencing results showed that 13, 14, and 14 patients had BRCA, non-BRCA HR, and non-HR DDR gene mutations, respectively. Array comparative genomic hybridization revealed that BRCA-mutated and HR gene-mutated TNBC shared similar BRCAness features, both having higher numbers and longer length of large-scale structural aberration (LSA, >10 Mb) and similar altered chromosomal regions of LSA. These suggested non-BRCA HR gene-mutated TNBC shared similar characteristics with BRCA-mutated TNBC, indicating non-BRCA HR gene-mutated TNBC sensitive to platinum and PARP inhibitors. Among tumors with mutation of non-HR DDR genes, 3 PTEN and 1 MSH6 mutation also contained significant LSAs (BRCAness); however, they had different regions of genomic alteration to BRCA and HR gene-mutated tumors, might explain prior findings that PTEN- and MSH6-mutated cancer cells not sensitive to PARP inhibitors. Therefore, we hypothesize that the heterogeneous genomic background of BRCAness indicates different responsiveness to platinum and PARP inhibitors. Direct sequencing DDR genes in TNBC should be applied to predict their sensitivity toward platinum and PARP inhibitors.

A targeted next-generation sequencing in the molecular risk stratification of adult acute myeloid leukemia: implications for clinical practice.

Conventional cytogenetics can categorize patients with acute myeloid leukemia (AML) into favorable, intermediate, and unfavorable-risk groups; however, patients with intermediate-risk cytogenetics represent the major population with variable outcomes. Because molecular profiling can assist with AML prognosis and next-generation sequencing allows simultaneous sequencing of many target genes, we analyzed 260 genes in 112 patients with de novo AML who received standard treatment. Multivariate analysis showed that karyotypes and mutation status of TET2, PHF6, KIT, and NPM1mutation /FLT3- internal tandem duplication (ITD)negative were independent prognostic factors for the entire cohort. Among patients with intermediate-risk cytogenetics, patients with mutations in CEBPAdouble mutation , IDH2, and NPM1 in the absence of FLT3-ITD were associated with improved Overall survival (OS), similar to those with favorable-risk cytogenetics; patients with mutations in TET2, RUNX1, ASXL1, and DNMT3A were associated with reduced OS, similar to those with unfavorable-risk cytogenetics. We concluded that integration of cytogenetic and molecular profiling improves prognostic stratification of patients into three groups with more distinct prognoses (P < 0.001) and significantly reduces the number of patients classified as intermediate risk. In addition, our study demonstrates that next-generation sequencing (NGS)-based multi-gene sequencing is clinically applicable in establishing an accurate risk stratification system for guiding therapeutic decisions.

Multiple gene sequencing for risk assessment in patients with early-onset or familial breast cancer.

Since BRCA mutations are only responsible for 10-20% of cases of breast cancer in patients with early-onset or a family history and since next-generation sequencing technology allows the simultaneous sequencing of a large number of target genes, testing for multiple cancer-predisposing genes is now being considered, but its significance in clinical practice remains unclear. We then developed a sequencing panel containing 68 genes that had cancer risk association for patients with early-onset or familial breast cancer. A total of 133 patients were enrolled and 30 (22.6%) were found to carry germline deleterious mutations, 9 in BRCA1, 11 in BRCA2, 2 in RAD50, 2 in TP53 and one each in ATM, BRIP1, FANCI, MSH2, MUTYH, and RAD51C. Triple-negative breast cancer (TNBC) was associated with the highest mutation rate (45.5%, p = 0.025). Seven of the 9 BRCA1 mutations and the single FANCI mutation were in the TNBC group; 9 of the 11 BRCA2, 1 of the 2 RAD50 as well as BRIP1, MSH2, MUTYH, and RAD51C mutations were in the hormone receptor (HR)(+)Her2(-) group, and the other RAD50, ATM, and TP53 mutations were in the HR(+)Her2(+) group. Mutation carriers were considered as high-risk to develop malignancy and advised to receive cancer screening. Screening protocols of non-BRCA genes were based on their biologic functions; for example, patients carrying RAD51C mutation received a screening protocol similar to that for BRCA, since BRCA and RAD51C are both involved in homologous recombination. In conclusion, we consider that multiple gene sequencing in cancer risk assessment is clinically valuable.