Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™–HER2-FISH workflow

Background In breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology. Methods 50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany). Results Applying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive. Conclusions The DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

Preliminary Clinical Validation of a Filtration-Based CTC Assay for Tumor Burden and HER2 Status Monitoring in Metastatic Breast Cancer

<b><i>Background:</i></b> Bearing multidimensional tumor-relevant information ranging from genomic alterations to proteomic makeup, circulating tumor cells (CTCs) constitute a promising material for liquid biopsy. The clinical validity of CTCs has been most extensively studied in metastatic breast cancer (MBC). The Cellsearch assay is currently the most widely used, while alternative strategies are pursued. A filtration-based microfluidic device has been described for CTC enrichment, but its clinical relevance remains unknown. <b><i>Methods:</i></b> In this preliminary study, we prospectively enrolled 47 MBC patients and evaluated the performance of the abovementioned CTC assay for tumor burden monitoring and human epidermal growth factor receptor 2 (HER2) status determination. <b><i>Results:</i></b> At baseline, 51.1% patients (24/47) were CTC positive. CTC count and positivity were also significantly higher in samples that accompanied poorer radiographic response evaluations. Serial blood draws suggested that CTC count enabled more accurate monitoring of tumor burden than serum markers carcinoembryonic antigen and cancer antigen 15-3. Also, in contrast to previous reports, CTC-HER2 status was moderately consistent with tumor-HER2 status. CTC-HER2 status assessment was further supported by <i>HER2</i> copy number measurements in select samples. <b><i>Conclusion:</i></b> The preliminary results from this study suggest promise for the interrogated CDC assay in several aspects, including sensitive CTC detection, accurate disease status reflection, and HER2 status determination. More studies are warranted to validate these findings and further characterize the value of CTC assay.

Characteristics of HER2-negative breast cancers with FISH-equivocal status according to 2018 ASCO/CAP guideline

Background According to 2018 ASCO/CAP guideline, HER2 FISH-equivocal breast cancers will be categorized as HER2 negative except those with IHC 3+. However, whether or not HER2 FISH-equivocal breast cancers was a heterogeneous group has not been well illustrated. Methods 195 HER2 FISH-equivocal breast cancer samples were collected from 2014 to 2018. The molecular subtype was identified according to 2013 St Gallen consensus, and HER2 status was also re-determined following 2018 ASCO/CAP guideline. All samples were classified into 4 groups according to the average HER2 copy number (4.0–4.4, 4.5–4.9, 5.0–5.4, 5.5–5.9 signals/cell). The relationship between HER2 copy number and clinicopathological parameters was analyzed. Results 183 (93.8%) of 195 FISH-equivocal cases were classified as luminal-like subtype, while the other 12 (6.2%) were undetermined. Following 2018 ASCO/CAP guideline, all FISH-equivocal cases were recategorized as HER2 negative. Therefore, 31(15.9%) cases were luminal A-like, 152 (77.9%) were luminal B-like (HER2 negative) and 12 (6.2%) were triple negative. The average HER2 copy number showed a positive correlation with chromosome 17 polysomy, but had no significant association with other clinicopathological parameters as well as prognosis. 17 (8.7%) patients were treated with trastuzumab, but showed no difference in prognosis with those who didn’t receive targeted therapy. Conclusions In this study, all HER2 FISH-equivocal breast cancers were recategorized as HER2 negative according to 2018 ASCO/CAP guideline. Most of these patients were luminal B-like (HER2 negative). The average HER2 copy number had no significant association with clinicopathological parameters, as well as prognosis.

Safety and tolerability of andecaliximab as monotherapy and in combination with an anti-PD-1 antibody in Japanese patients with gastric or gastroesophageal junction adenocarcinoma: a phase 1b study

BackgroundMatrix metalloproteinase 9 (MMP9) is implicated in protumorigenic processes. Targeting either stromal or epithelial MMP9 reduces the incidence of metastasis. Andecaliximab is a monoclonal antibody that targets MMP9 with high affinity and selectivity. However, no study has examined whether the inhibition of T-cell programmed death 1 (PD-1) in the presence of andecaliximab increases activated lymphocyte infiltration into the tumor, thereby increasing antitumor activity more than that in anti-PD-1 monotherapy. In this study, we assessed the safety, pharmacokinetics (PK), exploratory biomarkers, and preliminary efficacy of andecaliximab as monotherapy and in combination with nivolumab in Japanese patients with advanced or recurrent gastric or gastroesophageal junction (GEJ) adenocarcinoma.MethodsThis phase 1b study comprised four cohorts enrolling Japanese patients with gastric or GEJ adenocarcinoma. This paper concerns cohorts 1 and 4; cohorts 2 and 3 will be reported subsequently. Cohort 1 enrolled patients with human epidermal growth factor receptor 2 (HER2)-negative tumors (n=8) who received andecaliximab monotherapy (800 mg by intravenous infusion every 2 weeks (Q2W)), and cohort 4 enrolled patients irrespective of their HER2 status (n=10) who received 800 mg of andecaliximab in combination with nivolumab Q2W. Safety, dose-limiting toxicities (DLTs), PK, pharmacodynamics, and biomarkers were assessed in both cohorts.ResultsPK of andecaliximab in Japanese patients with gastric or GEJ adenocarcinoma was similar to that reported in non-Japanese patients with advanced solid tumors. Andecaliximab monotherapy and in combination with nivolumab demonstrated no DLTs in cohort 1 and 4, respectively. Toxicities were manageable and well tolerated in both cohorts. The median progression-free survival was 1.4 months (90% CI, 0.5 to 5.4) and 4.6 months (90% CI, 0.9 to not reached) in cohorts 1 and 4, respectively. The objective response rate was 50% (90% CI, 22% to 78%) in cohort 4, and in some patients, the combination therapy was effective regardless of the biomarker status.ConclusionsThe andecaliximab–nivolumab combination demonstrated a manageable safety profile and promising clinical activity in patients with advanced gastric adenocarcinoma.NCT02862535.

Multivariable Models Based on Baseline Imaging Features and Clinicopathological Characteristics to Predict Breast Pathologic Response after Neoadjuvant Chemotherapy in Patients with Breast Cancer

Introduction Currently, the accurate evaluation and prediction of response to neoadjuvant chemotherapy (NAC) remains a great challenge. We developed several multivariate models based on baseline imaging features and clinicopathological characteristics to predict the breast pathologic complete response (pCR). Methods We retrospectively collected clinicopathological and imaging data of patients who received NAC and subsequent surgery for breast cancer at our hospital from 2014 June till 2020 September. We used mammography, ultrasound and magnetic resonance imaging (MRI) to investigate the breast tumors at baseline. Results A total of 308 patients were included and 111 patients achieved pCR. The HER2 status and Ki-67 index were significant factors for pCR on univariate analysis and in all multivariate models. Among the prediction models in this study, the ultrasound-MRI model performed the best, producing an area under curve of 0.801 (95%CI=0.749-0.852), a sensitivity of 0.797 and a specificity of 0.676. Conclusion Among the multivariable models constructed in this study, the ultrasound plus MRI model performed the best in predicting the probability of pCR after NAC. Further validation is required before it is generalized.

Association Study of BIF-1 Gene Expression with Histopathological Characteristics and Hormone Receptors in Breast Cancer

Background Breast cancer (BC) is a heterogeneous disease that has different clinical outcomes. Bax-interacting Factor-1 (BIF-1) is a member of the endophilin B family that produces the pro-apoptotic BCL2-Associated X (BAX) protein in response to apoptotic signals. Lack of BIF-1 inhibits the intrinsic pathway of apoptosis and increases the risk of tumor genesis. The aim of the present study was to investigate the relationship between hormone receptors (ER, PR, HER2) status and different levels of BIF-1 gene expression in breast cancer patients. Methods BIF-1 gene expression was evaluated in 50 breast cancer tumors and 50 normal breast mammary tissues using SYBR Green Real Time RT-PCR technique. Multivariate and univariate analyses were used to evaluate the relationship between the prognostic significance of the BIF-1 gene using SPSS software. In this study, BIF-1 was selected as a candidate for a molecular biomarker and its expression status in breast cancer patients with hormone receptors (ER, RR, HER2) compared to patients without these hormone receptors. Results The study showed that the relative expression of BIF-1 gene in tissues of patients with hormone receptor in breast cancer compared to those without hormone receptor were not statistically significant. The expression levels of BIF-1 gene in different groups were evaluated for hormone receptor status. No significant relationship was found between BIF-1 gene expression and hormone receptors (ER, PR and HER2) (p> 0.05). Conclusion BIF-1 gene expression may be a useful prognostic marker in breast cancer.

Is It The Time That We Can Depend on Bioscore as a New Staging System in Non-Metastatic Breast Cancer to Predict the Disease-Free Survival?

Purpose: Novel molecular characterization of breast cancer with cellular markers has allowed a new classification that offers prognostic value. This study investigates the prognostic value of the Bioscore among non-metastatic breast cancer patients with respect to disease free survival (DFS).Methods: This study included 317 patients with non-metastatic surgically treated breast cancer; they were identified in the period from January 2015 to December 2018 at Clinical Oncology Department of Assiut University Hospital. Many variables were used; pathologic stage (PS), T stage (T), nodal stage (N), grade (G), estrogen receptor (ER), progesterone receptors (PR), and human epidermal growth factor receptor (HER2) status. Univariate & two multivariate analyses were performed to identify which of these variables are associated with disease-free survival (DFS). Results: The only significant factors in the Univariate analysis were PS3, T2, T3, T4, N3, G2, G3, ER -ve, PR -ve, and HER2 –ve. The factors which were significant in the first multivariate analysis; PS3, G3, ER –ve, and in the second one were; T2, T4, N3, G3, and ER –ve. Two sets of models were built to determine the utility of combining variables. Models incorporating G and E status had the highest C-index (0.72) for T+N + G + ER in comparison with (0.69) for (PS+ G + ER) and the lowest AIC (953.01) for T + N + G + E and (966.9) for PS + G + E. Conclusions: This study confirms the prognostic significance of bioscore in non-metastatic breast cancer in concerning DFS.

Evaluation of Liquid Biopsy in Patients with HER2-Positive Breast Cancer

Breast cancer is one of the common malignant tumors, and liquid biopsy has become a hot spot for clinical testing. To clarify the detection effect of liquid biopsy in breast cancer, we collected peripheral blood of HER2-positive (human epidermal growth factor receptor 2-positive) patients. Circulating tumor cells (CTCs) were isolated and analyzed. HER2 expression on CTCs was detected. The results showed that in the 198 HER2-positive samples, the CTC detection rate was 79.8% (158/198), and the mean number of CTCs was 21, ranging from 1 to 63/7.5 mL peripheral blood. Only 41.1% (65/158) of patients had histology and CTC HER2 status consistent with the remaining 58.9% (93/158) of patients, although their histological HER2 was positive, and CTC HER2 was negative. Our study confirmed the value of CTC HER2 real-time status testing in HER2-positive breast cancer patients. The inconsistency in HER2 status between CTCs and histology may be related to the time interval between CTCs and histological HER2 detection, suggesting that real-time HER2 detection is necessary for histological HER2-positive patients.

Pregnancy-associated breast cancer: the influence of gestational age

Whether PABC tumors arise before or during pregnancy and whether histopathology is affected by gestational age is currently unclear. The present study assesses the influence of gestational age and lactation on the histopathologic profile of PABC. We identified 744 patients with PABC (defined as breast cancer during pregnancy or 6-months following delivery). Histopathologic features were compared between pregnant and postpartum patients. Median age at diagnosis was 34.2 years and majority of cancers were diagnosed during pregnancy (71.3%). Within pregnant patients, tumors were significantly more often ER-negative in second and third trimesters (57.4%), as compared to first trimesters (41.9%) (p=0.036). Similarly, a PR-negative status was reported significantly less often within first trimesters (38.0%) compared to second and third trimesters (57.1%) (p=0.032). For HER2 status no significant differences were observed between gestational trimesters or lactating versus non-lactating patients. In postpartum patients, grade III tumors were found in over 80%, with high percentages of ER-negative tumors reaching 63% in those lactating versus 49% in non-lactating patients. This study demonstrates the varying histopathologic profile of PABC by gestational age and lactation status. Second and third trimester cancers display most typically the common ER/PR-negative phenotype, which is commonly reported in literature. The increased ER-negative status and percentage grade III tumors in lactating versus non-lactating patients also suggest presence of additional factors further diversify histology. This indicates the need for clear definitions of PABC and the role of potential subgroups, which may provide a stepping stone for further in-depth research into PABC-carcinogenesis.