Integrated genomic characterization of ERBB2/HER2 alterations in invasive breast carcinoma: a focus on unusual FISH groups.

In patients with invasive breast cancer, fluorescence in situ hybridization (FISH) testing for HER2 typically demonstrates the clear presence or lack of ERBB2 (HER2) amplification (i.e., groups 1 or 5). However, a small subset of patients can present with unusual HER2 FISH patterns (groups 2-4), resulting in diagnostic confusion. To provide clarity, the 2018 CAP/ASCO HER2 testing guideline recommends additional testing using HER2 immunohistochemistry (IHC) for determining the final HER2 status. Despite this effort, the genomic correlates of unusual HER2 FISH groups remain poorly understood. Here, we used droplet digital PCR (ddPCR) and targeted next-generation sequencing (NGS) to characterize the genomic features of both usual and unusual HER2 FISH groups. In this study, 51 clinical samples were selected to represent FISH groups 1-5. Furthermore, group 1 was subdivided into two groups, with groups 1A and 1B corresponding to cases with HER2 signals/cell ≥6.0 and 4-6, respectively. Overall, our findings revealed a wide range of copy number alterations in HER2 across the different FISH groups. As expected, groups 1A and 5 showed the clear presence and lack of HER2 copy number gain, respectively, as measured by ddPCR and NGS. In contrast, group 1B and other uncommon FISH groups (groups 2-4) were characterized by a broader range of HER2 copy levels with only a few select cases showing high-level gain. Notably, these cases with increased HER2 copy levels also showed HER2 overexpression by IHC, thus highlighting the correlation between HER2 copy number and HER2 protein expression. Given the concordance between the genomic and protein results, our findings suggest that HER2 IHC may inform HER2 copy number status in patients with unusual FISH patterns. Hence, our results support the current recommendation for using IHC to resolve HER2 status in FISH groups 2-4.

'Non-classical' HER2 FISH results in breast cancer: a multi-institutional study.

The 2013 CAP/ASCO HER2 Testing Guidelines Update modified HER2 FISH categories such that some cases with 'monosomy', 'co-amplification/polysomy', low-level increased HER2 signals or clustered heterogeneity now are considered amplified or equivocal. This study examines the frequency and clinico-pathologic characteristics of breast cancers with equivocal or 'non-classical' HER2 FISH results. Breast cancers (2001-2014) with HER2 FISH results, HER2 immunohistochemistry, ER, grade, and age from three institutions (Stanford, UCSF, UWMC) were collected. HER2 FISH was interpreted using the updated recommendations. Amplified cases with non-classical results were grouped into the following categories: (1) 'monosomy' (ratio ≥2.0, mean HER2/cell<4.0); (2) 'co-amplified' (ratio<2.0, mean HER2/cell ≥6.0); (3) 'low amplified' (ratio ≥2.0, mean HER2/cell 4.0-5.9). Heterogeneous cases with clustered HER2-positive cells were also included. Of 8068 cases, 5.2% were equivocal and 4.6% had a 'non-classical' HER2 amplified result; 1.4% 'monosomy', 0.8% 'co-amplified', 2.1% 'low amplified', and 0.3% clustered heterogeneity. These cancers had a high frequency of ER positive (80.4%), Nottingham grade 3 (52.1%) results. The highest percentage of grade 3 cancers (66.7%) and positive HER2 immunohistochemistry (31.7%) was in the 'co-amplified' group. The 'monosomy' group had the highest percent grade 1 cancers (13.3%) and was most frequently HER2 immunohistochemistry negative (30.1%). Equivocal cases had very similar characteristics to the 'low-amplified' category. Cases with non-classical HER2 amplification or equivocal results are typically ER positive, higher grade cancers. 'Co-amplified' cases have the highest frequencies of aggressive characteristics and 'monosomy' cases the highest frequencies of lower risk features. With little clinical outcomes data currently available on these non-classical HER2 results, these results support the current classification scheme for HER2 FISH, with case-by-case correlation with additional clinical-pathologic factors when evaluating whether to offer HER2-targeted therapies in these non-classical cases.

Cystic Neutrophilic Granulomatous Mastitis: Association With Gram-Positive Bacilli and Corynebacterium.

OBJECTIVES: To determine whether cystic neutrophilic granulomatous mastitis (CNGM) can be associated with Gram-positive bacilli and Corynebacterium METHODS: We reviewed our experience with 35 granulomatous mastitis patients over a 10-year period, including histologic pattern, Gram stain and other microbiologic data, clinical presentation, treatment and outcome. RESULTS: Biopsies from 19 patients demonstrated CNGM, while 16 patients had other patterns of granulomatous mastitis. Gram-positive organisms were seen within microcystic spaces in 16/19 CNGM, but 0/16 non-CNGM patients (P = .000). Culture or molecular studies demonstrated Corynebacterium species in three, all CNGM. Patients with CNGM were more likely to be younger, of Hispanic ethnicity, and born outside of the United States. Granulomatous mastitis resolved after a protracted course with widely variable treatment (antibiotics, surgery, steroids). CONCLUSIONS: Our data further support CNGM as an infectious disease; further study of Corynebacterium-directed therapy in CNGM is needed.

Novel mutations in neuroendocrine carcinoma of the breast: possible therapeutic targets.

Primary neuroendocrine carcinoma of the breast is a rare variant, accounting for only 2% to 5% of diagnosed breast cancers, and may have relatively aggressive behavior. Mutational profiling of invasive ductal breast cancers has yielded potential targets for directed cancer therapy, yet most studies have not included neuroendocrine carcinomas. In a tissue microarray screen, we found a 2.4% prevalence (9/372) of neuroendocrine breast carcinoma, including several with lobular morphology. We then screened primary or metastatic neuroendocrine breast carcinomas (excluding papillary and mucinous) for mutations in common cancer genes using polymerase chain reaction-mass spectroscopy (643 hotspot mutations across 53 genes), or semiconductor-based next-generation sequencing analysis (37 genes). Mutations were identified in 5 of 15 tumors, including 3 with PIK3CA exon 9 E542K mutations, 2 of which also harbored point mutations in FGFR family members (FGFR1 P126S, FGFR4 V550M). Single mutations were found in each of KDR (A1065T) and HRAS (G12A). PIK3CA mutations are common in other types of breast carcinoma. However, FGFR and RAS family mutations are exceedingly rare in the breast cancer literature. Likewise, activating mutations in the receptor tyrosine kinase KDR (VEGFR2) have been reported in angiosarcomas and non-small cell lung cancers; the KDR A1065T mutation is reported to be sensitive to VEGFR kinase inhibitors, and fibroblast growth factor receptor inhibitors are in trials. Our findings demonstrate the utility of broad-based genotyping in the study of rare tumors such as neuroendocrine breast cancer.

Severe bleeding in a woman heterozygous for the fibrinogen gammaR275C mutation.

The dysfibrinogen gammaR275C can be a clinically silent mutation, with only two out of 17 cases in the literature reporting a hemorrhagic presentation and four cases reporting a thrombotic presentation. We describe here a particularly severe presentation in 54-year-old female patient who required a hysterectomy at 47 years of age due to heavy menstrual bleeding. Coagulation studies revealed a prolonged prothrombin time and thrombin time, a normal fibrinogen antigen level, and a low fibrinogen activity level. Molecular analysis of the patient's DNA revealed a gamma chain gene mutation resulting in an amino acid substitution at residue 275 (gammaR275C). Protein sequencing of the fibrinogen gamma chain confirmed this mutation, which was named Fibrinogen Portland I. This case demonstrates that the gammaR275C mutation can lead to a severe hemorrhagic phenotype.