Giovanna Rossi

Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer.

The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial–mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.

Cell-free DNA and circulating tumor cells: Comprehensive liquid biopsy analysis in advanced breast cancer

Purpose: Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). We evaluated the utility of combining circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) to predict prognosis in MBC. Experimental Design: We conducted a retrospective study of 91 patients with locally advanced breast cancer and MBC. CTCs were enumerated by CellSearch; the plasma-based assay was performed utilizing Guardant360 and the survival analysis using Kaplan–Meier curves. Results: Eighty-four patients had stage IV cancer, and 7 patients had no metastases. Eighty patients had CTC analysis: median number 2 (0–5,612). Blood samples [232 of 277 (84%)] had mutations. The average ctDNA fraction was 4.5% (0–88.2%) and number of alterations 3 (0–27); the most commonly mutated genes were TP53 (52%), PIK3CA (40%), and ERBB2 (20%). At the time of analysis, 36 patients (39.6%) were dead. The median follow-up for CTCs was 9 months; for ctDNA, it was 9.9 months. For CTCs and ctDNA, respectively, progression-free survival (PFS) was 4.2 and 5.2 months and overall survival (OS) was 18.7 and 21.5 months. There was a statistically significant difference in PFS and OS for baseline CTCs < 5 versus CTCs ≥ 5 (P = 0.021 and P = 0.0004, respectively); %ctDNA < 0.5 versus ≥ 0.5 (P = 0.003 and P = 0.012); number of alterations < 2 versus ≥ 2 (P = 0.059 borderline and P = 0.0015). A significant association by Fisher exact test was found between the number of alterations and the %ctDNA in the baseline sample (P < 0.0001). Conclusions: The study demonstrated that liquid biopsy is an effective prognostic tool. Clin Cancer Res; 24(3); 560–8. ©2017 AACR.

Developmental therapeutics for inflammatory breast cancer: Biology and translational directions.

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, which accounts for approximately 3% of cases of breast malignancies. Diagnosis relies largely on its clinical presentation, and despite a characteristic phenotype, underlying molecular mechanisms are poorly understood. Unique clinical presentation indicates that IBC is a distinct clinical and biological entity when compared to non-IBC. Biological understanding of non-IBC has been extrapolated into IBC and targeted therapies for HER2 positive (HER2+) and hormonal receptor positive non-IBC led to improved patient outcomes in the recent years. This manuscript reviews recent discoveries related to the underlying biology of IBC, clinical progress to date and suggests rational approaches for investigational therapies.

Detection of Activating Estrogen Receptor Gene (ESR1) Mutations in Single Circulating Tumor Cells

Purpose: Early detection is essential for treatment plans before onset of metastatic disease. Our purpose was to demonstrate feasibility to detect and monitor estrogen receptor 1 (ESR1) gene mutations at the single circulating tumor cell (CTC) level in metastatic breast cancer (MBC). Experimental Design: We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot mutations in ESR1 and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies allowed recovery of 71 single CTCs and 12 WBC from 3 ER-positive MBC patients. Forty CTCs and 12 WBC were subjected to whole genome amplification by MALBAC and Sanger sequencing. Results: Among 3 selected patients, 2 had an ESR1 mutation (Y537). One showed two different ESR1 variants in a single CTC and another showed loss of heterozygosity. All mutations were detected in matched cell-free DNA (cfDNA). Furthermore, one had 2 serial blood samples analyzed and showed changes in both cfDNA and CTCs with emergence of mutations in ESR1 (Y537S and T570I), which has not been reported previously. Conclusions: CTCs are easily accessible biomarkers to monitor and better personalize management of patients with previously demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important information on clonal heterogeneity and can be a source of discovery of novel and potential driver mutations. Finally, we also validate a workflow for liquid biopsy that will facilitate early detection of ESR1 mutations, the emergence of endocrine resistance and the choice of further target therapy. Clin Cancer Res; 23(20); 6086–93. ©2017 AACR.

Abstract P1-01-05: Prognostic values of circulating tumor cells (CTC) and cancer associated macrophage-like cells (CAML) enumerations in metastatic breast cancer: The role for innate immunity in the metastatic process

Background: The enumeration of circulating tumor cells (CTCs) using the CellSearch assay is a well-established prognostic and predictive marker for metastatic breast cancer (MBC). However, additional prognostic markers are lacking in patients with ≥ 5 CTCs in 7.5 ml of blood. Tumor-associated macrophages (TAMs) are derived from circulating monocytes or tissue-resident macrophages. TAMs have a controversial role in metastasis and anti-tumor processes. Recent studies showed that circulating cancer associated macrophage-like cells (CAMLs) are specialized phagocytic myeloid cells and found in the peripheral blood of patients with solid tumors including breast cancer, but not in healthy individuals. The presence of CAMLs may indicate the activation of innate immunity in cancer patients. The function and prognostic value of CAMLs in MBC is unknown. In the current study, we measured CTCs and CAMLs on the CellSearch™ platform and investigated their prognostic values in MBC. Methods: Peripheral blood samples from 127 stages IV breast cancer patients were collected at baseline before starting first-line therapy. The detection and enumeration of CTCs and CAMLs in 7.5 ml blood sample were performed on the CellSearch™ system. CTCs were identified by cytokeratins (CK-8, 18, and 19) positive and CD45 negative staining. CAMLs were defined by positive staining for cytokeratins and CD45 (Adams et al, PNAS,111(9):3514-9, 2014). CTCs and CAMLs enumeration in associations with the progression-free survival (PFS) and overall survival (OS) of patients were evaluated using Kaplan Meier curves and Cox proportional hazards modeling. Results: The image review of CAMLs by using CellSearch analysis showed heterogeneous morphological phenotypes. CAMLs are large cells presenting enlarged nuclei or multiple individual nuclei, and both cytokeratin and CD45 positive with diffused cytoplasmic staining. Among the 127 MBC patients, 38 (29.9%) had elevated CTCs (≥5 CTCs), and 21 (16.5%) had at least one CAML detected. Patients with CAMLs had a significantly increased PFS (p=0.0374) and OS (p=0.0042), compared to patients without CAMLs at baseline. Patients with elevated baseline CTCs and CAMLs had worse PFS with a hazard ratio (HR) of 4.04 (95% CI 2.16 -7.56, P Conclusion: Baseline enumerations of both individual CTCs and CAMLs are feasible and increase our ability to accurately predict outcome in MBC patients. Evaluation of CAMLs in peripheral blood may be a marker of innate immunity and provide additional prognostic values for MBC. Citation Format: Mu Z, Wang C, Ye Z, Rossi G, Austin L, Yang H, Cristofanilli M. Prognostic values of circulating tumor cells (CTC) and cancer associated macrophage-like cells (CAML) enumerations in metastatic breast cancer: The role for innate immunity in the metastatic process [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-05.