NTRK gene aberrations in triple-negative breast cancer: detection challenges using IHC, FISH, RT-PCR, and NGS.

Triple-negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type-agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan-TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), and RNA-based next-generation sequencing (NGS). Of 83 cases, 16 showed pan-TRK positivity although no cases had NTRK-fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT-PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan-TRK IHC. Our data demonstrate that IHC has a high false-positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.

Impact of Breast Tumor Onset on Blood Count, Carcinoembryonic Antigen, Cancer Antigen 15-3 and Lymphoid Subpopulations Supported by Automatic Classification Approach: A Pilot Study.

BACKGROUND: Recent observations showed that systemic immune changes are detectable in case of breast cancer (BC). In this preliminary study, we investigated routinely measured peripheral blood (PB) parameters for malignant BC cases in comparison to benign breast conditions. Complete blood count, circulating lymphoid subpopulation, and serological carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) levels were considered. METHODS: A total of 127 female patients affected by malignant (n = 77, mean age = 63 years, min = 36, max = 90) BC at diagnosis (naïve patients) or benign breast conditions (n = 50, mean age = 33 years, min = 18, max = 60) were included in this study. For each patient, complete blood count and lymphoid subpopulations (T-helper, T-cytotoxic, B-, NK-, and NKT-cells) analysis on PB samples were performed. Hormonal receptor status, Ki-67 expression, and serological CEA and CA15-3 levels were assessed in the case of patients with malignant BC via statistical analysis. RESULTS: Women with malignant BC disclosed increased circulating T-helper lymphocytes and CD4/CD8 ratio in PB when compared to those affected by benign breast conditions (2.345 vs 1.894, P < .05 Wilcoxon rank-sum test). In the case of malignant BC patients, additive logistic regression method was able to identify malignant BC cases with increased CA15-3 levels (CA15-3 >25 UI/mL) via the hematocrit and neutrophils/lymphocytes ratio values. Moreover, in the case of women with aggressive malignant BC featured by high levels of Ki-67 proliferation marker, an increasing number of correlations were found among blood count parameters and lymphocytes subpopulations by performing a Spearman's correlation analysis. CONCLUSIONS: This preliminary study confirms the ability of malignant BC to determine systemic modifications. The stratification of malignant BC cases according to the Ki-67 proliferation marker highlighted increasing detectable alterations in the periphery of women with aggressive BC. The advent of novel and more sensitive biomarkers, as well as deep immunophenotyping technologies, will provide additional insights for describing the relationship between tumor onset and peripheral alterations.