Human epidermal growth factor receptor-2 (HER2) expression in FIGO3 high-grade endometrial endometrioid carcinoma: Clinicopathologic characteristics and future directions.

OBJECTIVES: To study the expression of HER2 in high-grade FIGO3 endometrial endometroid carcinoma (EEC) and to correlate our findings with the clinicopathologic characteristics of this tumor. METHODS: HER2 expression by immunohistochemistry (IHC) was performed on 10% formalin-fixed paraffin embedded tissue on cases diagnosed as FIGO3 EEC. HER2 expression was interpreted as negative (0), low (1+ and 2+) or positive (3+) using similar criteria as in the breast. HER2 amplification by Fluorescence in situ hybridization (FISH) was performed on cases interpreted as 2+ and 3+ by IHC. RESULTS: One hundred and forty-three FIGO3 EEC were identified. Of these, 70 (49%) cases were HER2 negative (IHC 0), and 73 (51%) cases expressed/amplified HER2 by IHC and/or FISH. Of the 73 cases expressing or amplifying HER2, 59 cases were IHC 1+, 12 cases were IHC 2+, and 2 cases were IHC 3+. FISH testing was performed in 12 cases. Only one of the two HER2 IHC 3+ cases showed HER2 gene amplification by FISH and the other 11 cases were not amplified. The 5-year overall survival (OS) rate for HER2 IHC 1+ cases was 92.20% (95% CI: 83.97-100.00), and the 5-year OS rate for HER2 IHC 2+/3+ cases was 89.50% (95% CI: 56.41-100.00). CONCLUSION: Our findings indicate that about one half of FIGO3 EEC variably expresses HER2 and with the emerging concept of "HER2 low", anti-HER2 agents may be explored as potential therapeutic options in these patients, for possible survival benefits.

Omitting Radiotherapy after Breast-Conserving Surgery in Luminal A Breast Cancer.

BACKGROUND: Adjuvant radiotherapy is prescribed after breast-conserving surgery to reduce the risk of local recurrence. However, radiotherapy is inconvenient, costly, and associated with both short-term and long-term side effects. Clinicopathologic factors alone are of limited use in the identification of women at low risk for local recurrence in whom radiotherapy can be omitted. Molecularly defined intrinsic subtypes of breast cancer can provide additional prognostic information. METHODS: We performed a prospective cohort study involving women who were at least 55 years of age, had undergone breast-conserving surgery for T1N0 (tumor size <2 cm and node negative), grade 1 or 2, luminal A-subtype breast cancer (defined as estrogen receptor positivity of ≥1%, progesterone receptor positivity of >20%, negative human epidermal growth factor receptor 2, and Ki67 index of ≤13.25%), and had received adjuvant endocrine therapy. Patients who met the clinical eligibility criteria were registered, and Ki67 immunohistochemical analysis was performed centrally. Patients with a Ki67 index of 13.25% or less were enrolled and did not receive radiotherapy. The primary outcome was local recurrence in the ipsilateral breast. In consultation with radiation oncologists and patients with breast cancer, we determined that if the upper boundary of the two-sided 90% confidence interval for the cumulative incidence at 5 years was less than 5%, this would represent an acceptable risk of local recurrence at 5 years. RESULTS: Of 740 registered patients, 500 eligible patients were enrolled. At 5 years after enrollment, recurrence was reported in 2.3% of the patients (90% confidence interval [CI], 1.3 to 3.8; 95% CI, 1.2 to 4.1), a result that met the prespecified boundary. Breast cancer occurred in the contralateral breast in 1.9% of the patients (90% CI, 1.1 to 3.2), and recurrence of any type was observed in 2.7% (90% CI, 1.6 to 4.1). CONCLUSIONS: Among women who were at least 55 years of age and had T1N0, grade 1 or 2, luminal A breast cancer that were treated with breast-conserving surgery and endocrine therapy alone, the incidence of local recurrence at 5 years was low with the omission of radiotherapy. (Funded by the Canadian Cancer Society and the Canadian Breast Cancer Foundation; LUMINA ClinicalTrials.gov number, NCT01791829.).

Is HER2 ultra-low breast cancer different from HER2 null or HER2 low breast cancer? A study of 1363 patients.

OBJECTIVE: This study aims to analyze whether there are any differences in clinicopathological features and prognosis between HER2 ultra-low, HER2-null, and HER2-low expression in Chinese breast cancer (BC) patients. METHODS: The clinicopathological data of 1363 HER2-negative BC patients were retrospectively collected (from January 2018 to December 2019). HER2 status was further classified into HER2-null, HER2 ultra-low, and HER2-low. HER2-null expression is defined as infiltrating cancer cells completely free of staining. HER2 ultra-low expression is defined as ≤10% of infiltrating cancer cells showing incomplete and faint/weak membrane staining. HER2-low expression is defined as HER2 immunohistochemistry (IHC) 1+ or 2+ with negative in situ hybridization (ISH) assay. RESULTS: Of 1363 patients, there were 86 (6.3%) HER2-null patients, 395 (29.0%) HER2 ultra-low patients, and 882 (64.7%) HER2-low patients. HER2 ultra-low patients were different from HER2-low patients in terms of N stage, hormone receptor (HR) status, Ki-67 expression, and type of surgery. There were also significant differences in histologic type and postoperative endocrine therapy between HER2 ultra-low and HER2-null patients. HR+ (81.0%) tumors was more common than HR- (19.0%) in HER2 ultra-low patients. In addition, there was a significant difference in HR status between HER2 ultra-low and HER2-low patients (P = 0.001). The survival analysis showed that HER2 status had no effect on disease-free survival (DFS) in HER2-negative patients (all P > 0.05). However, regardless of HER2 status, HR+ patients had better DFS than HR- patients (P = 0.003). Cox multivariate analysis revealed that age (HR [95% CI] = 0.950 [0.928, 0.972], P < 0.001), HR status (HR [95% CI] = 3.342 [1.658, 6.736], P = 0.001), and postoperative endocrine therapy (HR [95% CI] = 0.048 [0.048, 0.023], P < 0.001) were important influencing factors of DFS in HER2-negative BC patients. CONCLUSION: HER2 ultra-low BC patients demonstrated distinct clinicopathological features from HER2-null and HER2-low tumors; while, HER2 status (null, ultra-low, or low) had no prognostic value in these HER2-negative BC population. Consistent with the published literature, HR status was an independent prognostic factor for DFS in HER2-negative BC patients.

Trastuzumab Deruxtecan in Previously Treated HER2-Low Advanced Breast Cancer.

BACKGROUND: Among breast cancers without human epidermal growth factor receptor 2 (HER2) amplification, overexpression, or both, a large proportion express low levels of HER2 that may be targetable. Currently available HER2-directed therapies have been ineffective in patients with these "HER2-low" cancers. METHODS: We conducted a phase 3 trial involving patients with HER2-low metastatic breast cancer who had received one or two previous lines of chemotherapy. (Low expression of HER2 was defined as a score of 1+ on immunohistochemical [IHC] analysis or as an IHC score of 2+ and negative results on in situ hybridization.) Patients were randomly assigned in a 2:1 ratio to receive trastuzumab deruxtecan or the physician's choice of chemotherapy. The primary end point was progression-free survival in the hormone receptor-positive cohort. The key secondary end points were progression-free survival among all patients and overall survival in the hormone receptor-positive cohort and among all patients. RESULTS: Of 557 patients who underwent randomization, 494 (88.7%) had hormone receptor-positive disease and 63 (11.3%) had hormone receptor-negative disease. In the hormone receptor-positive cohort, the median progression-free survival was 10.1 months in the trastuzumab deruxtecan group and 5.4 months in the physician's choice group (hazard ratio for disease progression or death, 0.51; P<0.001), and overall survival was 23.9 months and 17.5 months, respectively (hazard ratio for death, 0.64; P = 0.003). Among all patients, the median progression-free survival was 9.9 months in the trastuzumab deruxtecan group and 5.1 months in the physician's choice group (hazard ratio for disease progression or death, 0.50; P<0.001), and overall survival was 23.4 months and 16.8 months, respectively (hazard ratio for death, 0.64; P = 0.001). Adverse events of grade 3 or higher occurred in 52.6% of the patients who received trastuzumab deruxtecan and 67.4% of those who received the physician's choice of chemotherapy. Adjudicated, drug-related interstitial lung disease or pneumonitis occurred in 12.1% of the patients who received trastuzumab deruxtecan; 0.8% had grade 5 events. CONCLUSIONS: In this trial involving patients with HER2-low metastatic breast cancer, trastuzumab deruxtecan resulted in significantly longer progression-free and overall survival than the physician's choice of chemotherapy. (Funded by Daiichi Sankyo and AstraZeneca; DESTINY-Breast04 ClinicalTrials.gov number, NCT03734029.).

HER2 Overexpression and Amplification in Uterine Carcinosarcomas With Serous Morphology.

Uterine carcinosarcoma (UCS) is an aggressive malignancy with few treatment options. A recent clinical trial has shown an increase in progression-free survival in patients with human epidermal growth factor receptor 2 (HER2)-positive serous endometrial carcinomas treated with anti-HER2-targeted therapies. Few studies have evaluated HER2 expression/amplification in UCS. Similar to serous endometrial carcinoma, the majority of UCS have TP53 mutations and a serous epithelial component, suggesting that UCS may show similar rates of HER2 positivity and therapeutic response. Therefore, we evaluated HER2 expression/amplification in a cohort of UCS over a 5-year period. HER2 immunohistochemistry (IHC) and chromogenic in situ hybridization were performed on tissue microarray and whole tissue sections and scored according to the most recent clinical trial recommendations. Three of 48 UCS (6%) had strong (3+) HER2 IHC expression, and 3 cases (6%) were equivocal (2+). Seven cases (15%) had HER2 amplification by chromogenic in situ hybridization, including all 3 with overexpression and 2 that were equivocal by IHC. Mismatch repair (MMR) protein, p53, and programmed cell death-ligand 1 (PD-L1) expression status was obtained from prior whole section analyses. All HER2-positive cases had a serous morphology and aberrant p53 expression. Only minimal PD-L1 expression was seen in the HER2-positive cases, and none had MMR loss. A subset of UCS with serous morphology have overexpression and/or amplification of HER2, which may predict response to HER2-targeted therapies. HER2-positive UCS may be less susceptible to immune checkpoint inhibition as they uncommonly show MMR deficiency and/or strong PD-L1 expression. Thus, HER2-targeted therapies could be of clinical utility in a subset of UCS without other adjuvant treatment options.

Can We Successfully De-Escalate Axillary Surgery in Women Aged ≥ 70 Years with Ductal Carcinoma in Situ or Early-Stage Breast Cancer Undergoing Mastectomy?

BACKGROUND: Omission of sentinel lymph node biopsy (SLNB) in older women with clinically node-negative, hormone receptor-positive (HR+) early-stage breast cancer undergoing lumpectomy is accepted, given established low rates of regional recurrence. The safety of omitting SLNB in women undergoing mastectomy is unknown and may differ depending on extent of breast disease and variation in radiotherapy use. PATIENTS AND METHODS: From 2006 to 2018, 123 cTis and 328 cT1-2 HR+/HER2- tumors from 410 women aged ≥ 70 years who underwent mastectomy and SLNB were included (41 bilateral cases). The rate of nodal positivity and effect of nodal positivity on adjuvant therapy use were examined. RESULTS: Median age was 74 years; 21% of patients had positive sentinel lymph nodes, 7% had micrometastases, and 14% had macrometastases. Of cases of cTis tumors, 31% were upstaged to invasive carcinoma; 1% had macrometastases. Fewer cases of cT1 than cT2 tumors had macrometastases [13% (26/200) versus 29% (37/128); p < 0.001]. Eight percent of patients with pT1 tumors (18/228) and 27% of patients with pT2 tumors (30/113) received chemotherapy. Most patients with pT1, pN1 disease (78%; 25/32) did not receive chemotherapy. Rates of locoregional recurrence were similar between patients with cT1 or cT2 tumors with and without nodal metastases (median follow-up, 4.5 years). CONCLUSIONS: Women aged ≥ 70 years with cTis and cT1N0 HR+/HER2- tumors who underwent mastectomy had low rates of nodal positivity, similar to rates reported for lumpectomy. Given this and the RxPONDER results, omission of SLNB may be considered, as findings are unlikely to alter adjuvant therapy recommendations.

Evolution of low HER2 expression between early and advanced-stage breast cancer.

BACKGROUND: Low human epidermal growth factor receptor 2 (HER2) expression is emerging as an actionable biomarker for the treatment of breast cancer (BC) with novel anti-HER2 drugs. However, the evolution of this biomarker during the course of disease is still poorly characterised, and controversial data exist on its prognostic implications. METHODS: We reviewed data of patients with HER2-negative BC according to the latest ASCO/CAP guidelines referred between January 2014 and December 2020. We grouped patients based on the immunohistochemistry (IHC) expression of HER2, HER2-zero (IHC 0) and HER2-low subgroup (IHC 1+ or 2+/ISH-negative) and evaluated the evolution of HER2 expression between the primary tumour and the first biopsy collected in the advanced setting. Disease-free survival, overall survival and progression-free survival were compared between patients with HER2-zero and HER2-low expression on the primary tumour. RESULTS: 232 patients were included in the analysis. Among the overall population, there was a relevant discordance in HER2 expression between the primary tumour and the matched biopsy (K = 0.33, 95%CI 0.21-0.44): 44% of the HER2-zero primary tumour showed an increased HER2 score on biopsy, and 22% of the HER2-low primary tumours turned into HER2-IHC 0. The findings in the sub-populations of hormone-receptors positive (K = 0.32, 95%CI 0.19-0.45) and triple-negative tumours (K = 0.18, 95%CI -0.09-0.46) were consistent with the primary analysis. No difference in survival outcomes was observed between HER2-low and HER2-zero primary tumours. CONCLUSIONS: HER2-low expression is dynamic in BC and may be enriched in the advanced-stage setting. No prognostic significance was demonstrated for HER2-low expression.

The efficacy and safety of palbociclib combined with endocrine therapy in patients with hormone receptor-positive HER2-negative advanced breast cancer: a multi-center retrospective analysis.

To explore the efficacy and safety of palbociclib combined with endocrine therapy (ET) in advanced breast cancer (ABC). We conducted a retrospective study involving patients with hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) ABC who received palbociclib combined with ET in the first- to third-line at three centers in China between January 2018 and October 2020. A total of 151 patients were included in this study. The median age of the patients at palbociclib initiation was 56 years (range 30-86 years) with a median follow-up of 10.9 months (range 2.0-41.2 months). Among these patients, 88 patients received palbociclib combined with ET as first-line therapy, and achieved a median progression-free survival (mPFS) of 19.8 months and an objective response rate (ORR) of 40.9%, meanwhile, in the first-line setting, 62 patients received palbociclib at an initial dose of 125 mg, achieving a mPFS of 20.9 months and an ORR of 46.8%. There were 39 and 24 patients who received palbociclib combined with ET as second- and third-line therapy, the mPFS were 10.0 months and 6.1 months, respectively. The most common and serious adverse events (AEs) were leukopenia and neutropenia. A total of 64 patients (42.4%) underwent palbociclib dose reduction due to AEs. Palbociclib combined with ET is an effective therapeutic regimen for HR+/HER2- ABC, particularly in the first-line setting with palbociclib initial dose of 125 mg, and AEs were manageable.

Predictive value of NLR, TILs (CD4+/CD8+) and PD-L1 expression for prognosis and response to preoperative chemotherapy in gastric cancer.

The combination of perioperative chemotherapy plus complete surgical resection is currently accounted as the first-choice strategy in patients with locally advanced Gastric Cancer (LAGC). Nevertheless, the partial response rate makes it necessary to search biological parameters useful to select patients who would benefit most from neoadjuvant chemotherapy (NAD-CT). We performed a retrospective analysis on a cohort of 65 LAGC cases, EBV negative and without MMR defect, submitted to perioperative chemotherapy plus surgical resection. We evaluated the neutrophil-lymphocytes ratio (NLR) in peripheral blood, the TILs density (reported as CD4/CD8 tissue ratio) and PD-L1 expression by immunohistochemistry on bioptic tissues before the treatment. Results were correlated with the biological features, histological response (TRG) and clinical outcome (PFS and OS). We found that NLR, TILs and PD-L1 expression showed a significant correlation with TNM stage, lymphovascular invasion and response to NAD-CT (TRG). Correlating the NLR, TILs and PD-L1 expression with PFS and OS, we found that patients with lower NLR levels (< 2.5 ratio), lower TILs (< 0.2 ratio) and higher PD-L1 level (CPS ≥ 1) had a significantly better PFS and OS than those with higher NLR, higher TILs and lower PD-L1 expression (p < 0.0001). Multivariate and multiple regression analyses confirmed the predictive and prognostic role of all three parameters, especially when all three parameters are combined. Our study demonstrated that pre-treatment NLR, TILs and PD-L1 expression are predictive and prognostic parameters in NAD-CT-treated LAGC suggesting a pivotal role of the systemic and tumor microenvironment immunological profile in the response to chemotherapy.

Successful treatment of a case of hormone receptor-positive metastatic extramammary Paget disease with tamoxifen.

Extramammary Paget disease (EMPD) is a rare cutaneous adenocarcinoma that usually is of epidermal origin and shows glandular differentiation and that is treated by wide local excision depending on the disease extent. For widely metastatic disease, however, a standard treatment remains to be established. Similar to breast cancer, EMPD has been found to overexpress human epidermal growth factor receptor 2 (HER2) or hormone receptors (HRs). Whereas HER2-directed therapy was recently shown to be effective for HER2-positive EMPD, the potential role of endocrine therapy for HR-positive EMPD has remained unknown. We here report a case of metastatic EMPD with HR positivity that was successfully treated with the selective estrogen receptor modulator tamoxifen. This first-line treatment of systemic metastasis resulted in durable tumor regression for > 20 months without any treatment-related toxicities. This is the first report to reveal the promise of tamoxifen as a safe and effective treatment for HR-positive metastatic EMPD.

A review of research progress on mechanisms and overcoming strategies of acquired osimertinib resistance.

Targeted therapy with epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs) is the standard first-line treatment for advanced EGFR-mutated non-small cell lung cancer (NSCLC). Third-generation EGFR-TKIs, represented by osimertinib, have been approved to overcome the EGFR T790M mutation in patients who are resistant to first- or second-generation TKIs, which brings more survival benefits for patients with advanced NSCLC. However, resistance to the third generation of EGFR-TKIs is still inevitable. Acquired drug resistance is the main reason for limiting the long-term effectiveness of targeted therapy in EGFR-mutated NSCLC patients. The mechanism of EGFR-TKI resistance of the third generation has become a focus of research in the field of targeted therapy. In this review, we summarize the research progress in resistance mechanisms of advanced NSCLC to osimertinib and the potential overcoming strategies and hope to provide a clinical basis and ideas for precision treatment of NSCLC.

Case report: ado-trastuzumab as second-line treatment in HER2 positive salivary duct carcinoma.

The objective of this report is to present the use and efficacy of ado-trastuzumab as an advanced line of therapy in salivary duct carcinoma (SDC) patient. An 84-year-old gentleman diagnosed with metastatic salivary duct tumor harboring overexpressed human epidermal growth factor receptor-2 has gone through resection surgery. The first line of treatment included initial doublet chemotherapy combined with trastuzumab, which was continued later as maintenance. PET-CT follow-up for 4 years has demonstrated no evidence of disease. However, upon recurrence, use of Ado-trastuzumab emtansine was prompted as second-line of treatment. This targeted patient-tailored therapy has accomplished a complete response. The effectiveness of ado-trastuzumab emtansine was notable within a short time period of two treatment cycles leading to full recovery. Specific agents aimed at altered oncogenes should be considered as a potential drug of choice in neoadjuvant HER2 positive SDC.

Nuclear Morphological Characteristics in Breast Cancer: Correlation with Hormone Receptor and Human Epidermal Growth Factor Receptor 2.

BACKGROUND: Hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) are the common diagnostic/prognostic markers in breast cancer. Few articles have recently reported the correlation between cytology and molecular subtypes. We combined nuclear morphological characteristics with HR and HER2 status to observe the relationship and provide ideas for machine learning. METHODS: We reanalyzed fine-needle aspiration cytology samples and core-needle puncture histological specimens from 142 patients with invasive breast cancer between March 2019 and December 2019, and the findings were compared with the two groups (HR+/HER2- and HR-/HER2+) following nuclear cytomorphological features: nuclear/cytoplasmic ratio, difference of nuclear size, nuclear pleomorphism, chromatin feature, nuclear membrane and nucleoli, and Nottingham grading. RESULTS: Two groups were significantly associated with the difference of nuclear size, nuclear pleomorphism, and nucleoli (P < 0.001) and consistent with histological grading (P < 0.001). Moreover, nucleolar characteristics of size and number had obviously statistical significance (P < 0.001). Multiple micro-nucleoli were frequently seen in the HR+/HER2- group compared with the HR-/HER2+ group which mostly were observed centered medium-large nucleoli. We described four interesting nuclear morphologies in the experiment. CONCLUSIONS: There were significant differences in nuclear characteristics between two groups. HR and HER2 status not only might be predicted in cytological samples, but some specific nuclear morphological features might have potential value to help us understand molecular function and predict more information.

Clinicopathological Characteristics and Prognosis of Squamous Cell Carcinoma of the Breast: A Population-Based Analysis.

BACKGROUND: To better understand the clinicopathological features and prognostic profiles of squamous cell carcinoma (SCC) of the breast. METHODS: Information on breast cancer was obtained from the Surveillance, Epidemiology, and End Results database (2004-2016). Comparative analyses were carried out to investigate the heterogeneity in the clinicopathological characteristics and survival outcomes between SCC and invasive ductal carcinoma (IDC), while propensity score matching was conducted to analyze the variations among baseline characteristics. Prognostic factors for SCC of the breast were successively identified using Cox regression analysis. RESULTS: A total of 382 SCC patients and 561477 IDC patients were identified in this study. Comparatively, the SCC cohort exhibited a higher proportion of male individuals, poor differentiation, an advanced TNM stage, an increasing percentage of triple-negative (TN) subtype, an increasing rate of organ involvement, and less access to therapeutics. The aggressive profile was consistent in the TN subgroup, with a significantly higher proportion in SCC than in IDC (25.7% vs 6.8%). Prognosis of SCC was profoundly poorer than that of IDC (mOS, 78.6 months and 121.6 months, P < .0001; mBCSS 91.9 months vs 135.6 months, P < .0001), of which the inferior tendency remained stable among disease stage and therapeutic options, while no difference was detected in the 2 subgroups with the TN subtype. The 2-year survival rate was 66.9% and the 5-year survival rate was 51.4%, with the risk factors being older age, bilateral disease, advanced TNM stage, bone and visceral involvement, surgical intervention, radiation treatment, and chemotherapy. CONCLUSIONS: This study systematically analyzed the heterogeneous characteristics of SCC of the breast in comparison with IDC. Squamous cell breast cancer presented with increasing aggressive behavior and inferior prognosis. Prospective studies should focus on this subgroup and introduce individualized therapeutic protocols in clinical practice.

HER2 Testing: Insights From Pathologists' Perspective on Technically Challenging HER2 FISH Cases.

OBJECTIVE: College of American Pathologists and the American Society of Clinical Oncology guidelines provide straightforward criteria for HER2 interpretation in breast carcinomas; however, a subset of cases present unusual diagnostic dilemmas. MATERIALS AND METHODS: Ten challenging HER2 fluorescence in situ hybridization (FISH) cases were selected for analysis. The study included a variety of problematic cases such as those with discordant immunohistochemistry (IHC) and FISH results, cases with high intratumoral variability in HER2 copy number, a case with a highly amplified clone in 5% to 10% of the tumor sample, and a case with tumor cells containing tightly clumped HER2 signals. Six high volume HER2 FISH laboratories performed and interpreted HER2 FISH (adding HER2 IHC if necessary). Interpretation strategies were discussed. RESULTS: There was 100% concordance between laboratories in 4/10 cases. Tumors with increased intratumoral variability (tumors with high variability in HER2 copy number per cell but which otherwise do not fulfill College of American Pathologists and the American Society of Clinical Oncology criteria for heterogeneity) exhibited 100% concordance in 3/4 cases, but 1 case had only 50% agreement. Low positive HER2 cases (group 1 cases with <6 average HER2 copies/cell) had 1 laboratory disagreeing with the majority in 4/4 cases, and this was the only category with discordance between IHC and FISH methodologies. All laboratories identified the case with heterogeneity and interpreted it as positive. Five of the 6 laboratories interpreted the case with tightly clustered HER2 signals as positive. CONCLUSIONS: This study offers specific observations and interpretation strategies that laboratories can use when confronted with difficult HER 2 cases. It then highlights communication strategies a laboratory may use to discuss these unusual HER2 results with the clinical team.

The mechanisms involved in the resistance of estrogen receptor-positive breast cancer cells to palbociclib are multiple and change over time.

PURPOSE: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used for the treatment of advanced estrogen receptor (ER)-positive breast cancer. To develop a treatment strategy for cancers resistant to CDK4/6 inhibitors, here, we established palbociclib-resistant sublines and analyzed their resistance mechanisms. METHODS: Palbociclib-resistant sublines were established from T47D and MCF7 cells. Sensitivity to other drugs was assessed via the WST assay. Altered expression/phosphorylation of proteins related to signal transduction and cell cycle regulation was examined using western blotting. Copy number alterations and mutations in the retinoblastoma (RB1) gene were also analyzed. RESULTS: Although an increase in CDK6 and decrease in retinoblastoma protein (Rb) expression/phosphorylation were commonly observed in the resistant sublines, changes in other cell cycle-related proteins were heterogeneous. Upon extended exposure to palbociclib, the expression/phosphorylation of these proteins became altered, and the long-term removal of palbociclib did not restore the Rb expression/phosphorylation patterns. Consistently a copy number decrease, as well as RB1 mutations were detected. Moreover, although the resistant sublines exhibited cross-resistance to abemaciclib, their response to dinaciclib was the same as that of wild-type cells. Of note, the cell line exhibiting increased mTOR phosphorylation also showed a higher sensitivity to everolimus. However, the sensitivity to chemotherapeutic agents was unchanged in palbociclib-resistant sublines. CONCLUSION: ER-positive breast cancer cells use multiple molecular mechanisms to survive in the presence of palbociclib, suggesting that targeting activated proteins may be an effective strategy to overcome resistance. Additionally, palbociclib monotherapy induces mutations and copy number alterations in the RB1 gene.

Antibody-drug nanoparticle induces synergistic treatment efficacies in HER2 positive breast cancer cells.

Chemotherapeutic drugs suffer from non-specific binding, undesired toxicity, and poor blood circulation which contribute to poor therapeutic efficacy. In this study, antibody-drug nanoparticles (ADNs) are engineered by synthesizing pure anti-cancer drug nanorods (NRs) in the core of nanoparticles with a therapeutic monoclonal antibody, Trastuzumab on the surface of NRs for specific targeting and synergistic treatments of human epidermal growth factor receptor 2 (HER2) positive breast cancer cells. ADNs were designed by first synthesizing ~ 95 nm diameter × ~ 500 nm long paclitaxel (PTX) NRs using the nanoprecipitation method. The surface of PTXNRs was functionalized at 2' OH nucleophilic site using carbonyldiimidazole and conjugated to TTZ through the lysine residue interaction forming PTXNR-TTZ conjugates (ADNs). The size, shape, and surface charge of ADNs were characterized using scanning electron microscopy (SEM), SEM, and zeta potential, respectively. Using fluorophore labeling and response surface analysis, the percentage conjugation efficiency was found > 95% with a PTX to TTZ mass ratio of 4 (molar ratio ≈ 682). In vitro therapeutic efficiency of PTXNR-TTZ was evaluated in two HER2 positive breast cancer cell lines: BT-474 and SK-BR-3, and a HER2 negative MDA-MB-231 breast cancer cell using MTT assay. PTXNR-TTZ inhibited > 80% of BT-474 and SK-BR-3 cells at a higher efficiency than individual PTX and TTZ treatments alone after 72 h. A combination index analysis indicated a synergistic combination of PTXNR-TTZ compared with the doses of single-drug treatment. Relatively lower cytotoxicity was observed in MCF-10A human breast epithelial cell control. The molecular mechanisms of PTXNR-TTZ were investigated using cell cycle and Western blot analyses. The cell cycle analysis showed PTXNR-TTZ arrested > 80% of BT-474 breast cancer cells in the G2/M phase, while > 70% of untreated cells were found in the G0/G1 phase indicating that G2/M arrest induced apoptosis. A similar percentage of G2/M arrested cells was found to induce caspase-dependent apoptosis in PTXNR-TTZ treated BT-474 cells as revealed using Western blot analysis. PTXNR-TTZ treated BT-474 cells showed ~ 1.3, 1.4, and 1.6-fold higher expressions of cleaved caspase-9, cytochrome C, and cleaved caspase-3, respectively than untreated cells, indicating up-regulation of caspase-dependent activation of apoptotic pathways. The PTXNR-TTZ ADN represents a novel nanoparticle design that holds promise for targeted and efficient anti-cancer therapy by selective targeting and cancer cell death via apoptosis and mitotic cell cycle arrest.

Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Nectin-4, upregulated in various cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2∆Ex16, enhancing DNA synthesis through the PI3K-AKT signaling in human breast cancer T47D cells in an adherent culture. We found here that nectin-4 and p95-ErbB2, but not nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced SOX2 gene expression and cell proliferation in a suspension culture. This enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2 was dependent on the SOX2 gene expression. In T47D cells, nectin-4 and any one of p95-ErbB2, ErbB2, or ErbB2∆Ex16 cooperatively activated the PI3K-AKT signaling, known to induce the SOX2 gene expression, to similar extents. However, only a combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced the SOX2 gene expression. Detailed studies revealed that only nectin-4 and p95-ErbB2 cooperatively activated the Hippo signaling. YAP inhibited the SOX2 gene expression in this cell line and thus the MST1/2-LATS1/2 signaling-mediated YAP inactivation increased the SOX2 gene expression. These results indicate that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively regulates the Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Cost-effectiveness analysis of ribociclib plus fulvestrant for hormone receptor-positive/human EGF receptor 2-negative breast cancer.

Aim: To evaluate the cost-effectiveness of ribociclib plus fulvestrant versus fulvestrant in hormone receptor-positive/human EGF receptor 2-negative advanced breast cancer. Materials & methods: A three-state Markov model was developed to evaluate the costs and effectiveness over 10 years. Direct costs and utility values were obtained from previously published studies. We calculated incremental cost-effectiveness ratio to evaluate the cost-effectiveness at a willingness-to-pay threshold of $150,000 per additional quality-adjusted life year. Results: The incremental cost-effectiveness ratio was $1,073,526 per quality-adjusted life year of ribociclib plus fulvestrant versus fulvestrant. Conclusions: Ribociclib plus fulvestrant is not cost-effective versus fulvestrant in the treatment of advanced hormone receptor-positive/human EGF receptor 2-negative breast cancer. When ribociclib is at 10% of the full price, ribociclib plus fulvestrant could be cost-effective.

Clinical and Immunologic Responses to a B-Cell Epitope Vaccine in Patients with HER2/neu-Overexpressing Advanced Gastric Cancer-Results from Phase Ib Trial IMU.ACS.001.

PURPOSE: HER2/neu is overexpressed in up to 30% of gastroesophageal adenocarcinomas (GEA) and linked to poor prognosis. Recombinant mAbs to treat HER2/neu-overexpressing cancers are effective with limitations, including resistance and toxicity. Therefore, we developed a therapeutic B-cell epitope vaccine (IMU-131/HER-Vaxx) consisting of three fused B-cell epitopes from the HER2/neu extracellular domain coupled to CRM197 and adjuvanted with Montanide. This phase Ib study aimed to evaluate the optimal/safe dose leading to immunogenicity and clinical responses (https//clinicaltrials.gov/ct2/show/NCT02795988). PATIENTS AND METHODS: A total of 14 patients with HER2/neu-overexpressing GEA were enrolled, and dose escalation (10, 30, 50 µg) was performed in three cohorts (C). Immunogenicity was evaluated by HER2-specific Abs and cellular responses, clinical responses by CT scans according to RECIST version 1.1. RESULTS: IMU-131 was safe without vaccine-related significant local/systemic reactions or serious adverse events. A total of 11 of 14 patients were evaluable for changes in tumor size and vaccine-specific immune responses. One patient showed complete, 5 partial responses, and 4 stable diseases as their best response. HER2-specific IgG levels were dose dependent. In contrast to patients in C1 and C2, all patients in C3 mounted substantial HER2-specific Ab levels. In addition, cellular vaccine responses, such as Th1-biased cytokine ratios and reduced regulatory T cell numbers, were generated. Progression-free survival was prolonged in C3, correlating with the vaccine-specific humoral and cellular responses. CONCLUSIONS: IMU-131 was well tolerated and safe. The induced HER2-specific Abs and cellular responses were dose dependent and correlated with clinical responses. The highest dose (50 µg) was recommended for further evaluation in a phase II trial, with chemotherapy + IMU-131 or chemotherapy alone, which is currently ongoing.