Clinicopathologic Features of Breast Carcinoma With 2018 American Society of Clinical Oncology/College of American Pathologists Fluorescence In Situ Hybridization Group 3 (Human Epidermal Growth Factor Receptor 2 Chromosome 17 Centromere Ratio <2.0 and Average Human Epidermal Growth Factor Receptor 2 Copy Number ≥6.0) Breast Cancers.

CONTEXT.­: The American Society of Clinical Oncology/College of American Pathologists 2018 update of the human epidermal growth factor receptor 2 (HER2) testing guideline includes a fluorescence in situ hybridization (FISH) group with a HER2 to chromosome 17 centromere (CEP17) ratio less than 2.0 and HER2 copy number 6.0 or greater (group 3), which requires integrated review of HER2 immunohistochemistry (IHC). OBJECTIVE.­: To assess the clinicopathologic features of group 3 patients and determine features associated with HER2-positive status after workup. DESIGN.­: Cases submitted for HER2 FISH between January 2019 and June 2022 were identified, and relevant clinicopathologic information was obtained. RESULTS.­: One hundred forty-two HER2 FISH cases (1.6%) were group 3. In 52 cases (36.6%) IHC was negative (0/1+), in 3 (2.8%) IHC was positive (3+), and in 86 (60.6%) IHC was 2+. Annotated IHC 2+ slides were recounted by a second reviewer in targeted areas, where 16 of 86 (18.6%) had a HER2:CEP17 ratio less than 2.0 and a HER2 copy number of 4.0 or greater to less than 6.0 (HER2 negative). After combined IHC/FISH review, 74 of 142 (52.1%) were classified as HER2 positive. HER2 copy number/cell was higher in HER2-positive compared with HER2-negative cases after the workup. The extent and intensity of staining in IHC 2+ cases did not correlate with the level of gene amplification. Twenty percent of HER2-positive patients achieved pathologic complete response. CONCLUSIONS.­: About half of group 3 cases were classified as HER2 positive after additional workup. Pathologic complete response rates in HER2-positive cases were lower than expected for group 1 (HER2:CEP17 ratio ≥2.0; HER2 copy number ≥4.0) patients. IHC targeted FISH recounts may be redundant and may potentially lead to classification of some patients as HER2 negative, resulting in withholding of targeted therapy.

Metaplastic Breast Carcinoma: Clinicopathologic Features and Recurrence Score Results From a Population-based Database.

OBJECTIVES: Metaplastic breast carcinoma (MBC) is a rare, aggressive form of cancer comprising epithelial and mesenchymal elements. The purpose of this study was to use population-based data to review the clinicopathologic, molecular features, and outcomes of MBC. METHODS: Surveillance, Epidemiology, and End Results Program (SEER) data were used to identify MBC and invasive ductal carcinoma (IDC), no special type (NOS) between 2004 and 2015. Results from Oncotype DX's 21-gene assay linked to SEER registries were included for hormone receptor (HR)-positive tumors. χ 2 analysis was performed to determine the differences between MBC and IDC. Kaplan-Meier curves and multivariate Cox proportional hazards models were used for breast cancer specific death (BCSD). RESULTS: Compared with IDC, NOS (n=509,864), MBC (n=3876) were more likely to present at an older age, be black, have negative lymph nodes, be >2 cm, grade 3, and triple negative (TN). All subtypes [HR-positive/human epidermal growth receptor 2 (HER2)-negative, HR-positive/HER2-positive, HR-negative/HER2-positive, and TN] had higher BCSD than IDC, NOS. 22.3% of MBC cases were HR-positive. HR-positive MBCs tested for a recurrence score (RS) 65% were high-risk compared with 16.8% of IDC, NOS. Within the MBC cohort, no significant differences in BCSD were identified with respect to different molecular subtypes. In a fully adjusted model, TN or HER2-positive status did not adversely affect BCSD compared with HR-positive MBC. CONCLUSIONS: All molecular subtypes of MBC had a poorer prognosis compared with IDC, NOS. The different molecular subtypes of MBC did not affect the BCSD. HR-positive MBC patients had a significantly higher high-risk RS than IDC, NOS patients.

Quantitative Imaging Analysis Fluorescence In Situ Hybridization Validation for Clinical HER2 Testing in Breast Cancer.

CONTEXT.­: Quantitative imaging is a promising tool that is gaining wide use across several areas of pathology. Although there has been increasing adoption of morphologic and immunohistochemical analysis, the adoption of evaluation of fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue has been limited because of complexity and lack of practice guidelines. OBJECTIVE.­: To perform human epidermal growth factor receptor 2 (HER2) FISH validation in breast carcinoma in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guideline. DESIGN.­: Clinical validation of HER2 FISH was performed using the US Food and Drug Administration-approved dual-probe HER2 IQFISH (Dako, Carpinteria, California) with digital scanning performed on a PathFusion (Applied Spectral Imaging, Carlsbad, California) system. Validation parameters evaluated included z-stacking, classifier, accuracy, precision, software, and hardware settings. Finally, we evaluated the performance of digital enumeration on clinical samples in a real-world setting. RESULTS.­: The accuracy samples showed a final concordance of 95.3% to 100% across HER2 groups 1 to 5. During clinical implementation for HER2 groups 2, 3, and 4, we achieved a final concordance of 76% (95 of 125). Of these cases, only 8% (10 of 125) had discordances with clinical impact that could be identified algorithmically and triaged for manual review. CONCLUSIONS.­: Digital FISH enumeration is a useful tool to improve the efficacy of HER2 FISH enumeration and capture genetic heterogeneity across HER2 signals. Excluding cases with high background or poor image quality and manual review of cases with ASCO/CAP group discordances can further improve the efficiency of digital HER2 FISH enumeration.

The Critical Role of Breast Specimen Gross Evaluation for Optimal Personalized Cancer Care.

Gross examination is the foundation for the pathologic evaluation of all surgical specimens. The rapid identification of cancers is essential for intraoperative assessment and preservation of biomolecules for molecular assays. Key components of the gross examination include the accurate identification of the lesions of interest, correlation with clinical and radiologic findings, assessment of lesion number and size, relationship to surgical margins, documenting the extent of disease spread to the skin and chest wall, and the identification of axillary lymph nodes. Although the importance of gross evaluation is undeniable, current challenges include the difficulty of teaching grossing well and its possible perceived undervaluation compared with microscopic and molecular studies. In the future, new rapid imaging techniques without the need for tissue processing may provide an ideal melding of gross and microscopic pathologic evaluation.

Immunotherapy in Breast Cancer- Paving New Roads?

Significant advances have been made in our understanding of the mechanisms of innate and adaptive immunity and the mechanisms by which tumors escape from the normal process of immune surveillance. However, effectively harnessing the power of the immune system to specifically target the tumor cells has proven quite difficult, in large part due to the fact that tumors are composed of patient's own cells, expressing antigens recognized as "self" and thus escaping the immune surveillance. For breast cancer treatment, the first successful inroads in immunotherapy came with the introduction of monoclonal antibodies against a membrane receptor for the epidermal growth factor receptor family (HER2). In 1998 the first monoclonal antibody against the Her 2 antigen was approved for clinical use (trastuzumab). Clinical trials combining trastuzumab with various chemotherapy regimens have consistently shown improvement in survival in both metastatic as well as early stage breast cancer. Another promising approach has been the development of anti HER 2 vaccines. More recently the discovery of immune modulating antibodies (checkpoint inhibitors) and their success in the treatment of cancers such as melanoma and renal cell carcinoma has lead to renewed interest in immune therapies in cancer in general and new clinical trials exploring the role of immune therapies in breast cancer.

Tumour cell heterogeneity maintained by cooperating subclones in Wnt-driven mammary cancers.

Cancer genome sequencing studies indicate that a single breast cancer typically harbours multiple genetically distinct subclones. As carcinogenesis involves a breakdown in the cell-cell cooperation that normally maintains epithelial tissue architecture, individual subclones within a malignant microenvironment are commonly depicted as self-interested competitors. Alternatively, breast cancer subclones might interact cooperatively to gain a selective growth advantage in some cases. Although interclonal cooperation has been shown to drive tumorigenesis in fruitfly models, definitive evidence for functional cooperation between epithelial tumour cell subclones in mammals is lacking. Here we use mouse models of breast cancer to show that interclonal cooperation can be essential for tumour maintenance. Aberrant expression of the secreted signalling molecule Wnt1 generates mixed-lineage mammary tumours composed of basal and luminal tumour cell subtypes, which purportedly derive from a bipotent malignant progenitor cell residing atop a tumour cell hierarchy. Using somatic Hras mutations as clonal markers, we show that some Wnt tumours indeed conform to a hierarchical configuration, but that others unexpectedly harbour genetically distinct basal Hras mutant and luminal Hras wild-type subclones. Both subclones are required for efficient tumour propagation, which strictly depends on luminally produced Wnt1. When biclonal tumours were challenged with Wnt withdrawal to simulate targeted therapy, analysis of tumour regression and relapse revealed that basal subclones recruit heterologous Wnt-producing cells to restore tumour growth. Alternatively, in the absence of a substitute Wnt source, the original subclones often evolve to rescue Wnt pathway activation and drive relapse, either by restoring cooperation or by switching to a defector strategy. Uncovering similar modes of interclonal cooperation in human cancers may inform efforts aimed at eradicating tumour cell communities.

CCAAT/enhancer binding protein-delta expression is increased in fast skeletal muscle by food deprivation and regulates myostatin transcription in vitro.

We recently demonstrated that mRNA levels of three members of the CCAAT/enhancer binding factor (C/EBP) family of transcription factors are increased in skeletal muscle following 12 days of spaceflight. In the present study, we further explored the expression of C/EBP-δ in atrophying fast skeletal muscle by examining its expression in muscle from food-deprived (FD) mice, and investigated its role in regulating the expression of the secreted antigrowth factor myostatin. C/EBP-δ mRNA and protein levels were significantly increased by 2 days of food deprivation in the tibialis anterior (TA) muscle, and expression of both myostatin and C/EBP-δ mRNA during food deprivation was attenuated by injection with the glucocorticoid inhibitor RU486. The increase in myostatin mRNA levels with food deprivation appears to be at least partially transcriptionally driven, since levels of myostatin pre-mRNA were significantly increased in the TA muscle. C/EBP-δ mRNA levels and promoter activity were significantly increased by transfection of C(2)C(12) myotubes with a glucocorticoid receptor construct and 24 h of treatment with the synthetic glucocorticoid dexamethasone. Furthermore, activity of the C/EBP-δ promoter was significantly increased with as little as 1 h of dexamethasone treatment, while activity of the mouse myostatin promoter was only significantly increased with longer treatment periods of 24 h or more. Activity of the myostatin promoter-reporter construct was significantly increased in C(2)C(12) myotubes by cotransfection with expression constructs for C/EBP-α, -ß, and -δ, with C/EBP-δ having the greatest effect. The myostatin promoter contains two potential C/EBP binding sequences, a CCAAT box, and a C/EBP binding element (CBE). Mutation of the CCAAT box attenuated basal myostatin promoter activity but potentiated C/EBP-δ-activated myostatin promoter activity in C(2)C(12) myotubes in vitro, while mutation of the CBE abolished glucocorticoid receptor and C/EBP-δ responsiveness. The present results support a model in which glucocorticoid-induced increases in C/EBP-δ expression may contribute to myostatin transcription during atrophic states.

Myostatin expression is increased by food deprivation in a muscle-specific manner and contributes to muscle atrophy during prolonged food deprivation in mice.

During food deprivation (FD), skeletal muscle protein is broken down to produce amino acids for hepatic gluconeogenesis to maintain blood glucose levels. However, it is unclear what role, if any, the secreted antigrowth factor myostatin (MSTN) plays in the muscle atrophy induced by FD. We therefore examined expression and function of MSTN in FD in mice. Two days of FD significantly decreased muscle mass and protein content and increased mRNA levels of ubiquitin ligases MuRF-1 and atrogin-1 in fast-twitch tibialis anterior (TA) muscle but not slow-twitch soleus (Sol) muscle, while 2 days of refeeding returned these to fed values in TA. MSTN mRNA levels were significantly increased approximately threefold by 2 days, but not 1 day, of FD and returned to fed levels with 2 days of refeeding in TA but were not significantly affected by FD or refeeding in Sol. TA mass decreased to a similar amount after 1 day of FD in wild-type mice and mice null for the MSTN gene but was decreased to a greater amount in wild-type than MSTN-null mice by 2 days of FD. In addition, blood glucose levels decreased and corticosterone levels increased to a greater extent in MSTN-null mice after 2 days of FD, but surprisingly muscle MuRF-1 and atrogin-1 mRNA levels were not affected by the lack of MSTN during FD. Similarly, changes in hepatic enzyme expression in response to FD were identical between wild-type and MSTN-null mice. Our data are consistent with the hypothesis that MSTN is dispensable for the initial atrophy occurring in response to FD but attenuates the decrease in fast-twitch muscle mass during prolonged FD.

Skeletal muscle expression of bone morphogenetic protein-1 and tolloid-like-1 extracellular proteases in different fiber types and in response to unloading, food deprivation and differentiation.

Members of the bone morphogenetic protein-1/mammalian tolloid (BMP-1/mTLD) family of proteases cleave diverse extracellular proteins, including the growth inhibitor myostatin. The purpose of this work was to examine the expression of BMP-1/mTLD, tolloid-like-1 and -2 (TLL1 and TLL2) in hindlimb muscles of the mouse in vivo and in C(2)C(12) muscle cells in vitro. Quantitative real-time polymerase chain reaction revealed that neither BMP-1/mTLD nor TLL1 mRNA levels differed between the predominantly fast-twitch tibialis anterior (TA) and gastrocnemius (GAST) muscles and the more slow-twitch soleus (SOL) muscle; TLL2 mRNA levels were not detectable in any of the muscles examined. Interestingly, however, immunohistochemical analysis revealed that BMP-1 protein was expressed in type I and IIa but not in IIb fibers. TLL1 mRNA levels significantly increased in the TA but not the SOL with 3 days of hindlimb suspension and significantly decreased in both TA and SOL in response to 2 days of food deprivation. In contrast, BMP-1/mTLD mRNA levels were unaffected in either muscle by either condition. In addition, BMP-1/mTLD and TLL1 mRNA levels significantly decreased during C(2)C(12) myoblast differentiation in vitro, and activity of a 1,200-bp mouse TLL1 promoter construct was significantly decreased in C(2)C(12) myotubes by differentiation, by mutation of an nuclear factor kappa-beta (NF-kappaB) site, or deletion of a sma/mothers against decapentaplegic (SMAD) site. Together, these data demonstrate that TLL1 mRNA levels are altered by loading, energy status, and differentiation, and thus its expression may be regulated so as to modulate activity of myostatin or other extracellular substrates during these adaptive states.

Calcineurin activates interleukin-6 transcription in mouse skeletal muscle in vivo and in C2C12 myotubes in vitro.

Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2-10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel-running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C(2)C(12) myotubes with the calcium ionophore A23187. Cotransfection of C(2)C(12) myotubes with a constitutively active calcineurin construct significantly increased while cotransfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Cotransfection with a myocyte enhancer-factor-2 (MEF-2) expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin cotransfection, while cotransfection with the MEF-2 antagonist MITR repressed calcineurin-activated IL-6 promoter activity in vitro. Surprisingly, cotransfection with a dominant-negative form of another calcineurin-activated transcription factor, nuclear factor activator of T cells (NFAT), greatly potentiated both basal and calcineurin-stimulated IL-6 promoter activity in C(2)C(12) myotubes. Mutation of the MEF-2 DNA binding sites attenuated, while mutation of the NFAT DNA binding sites potentiated basal and calcineurin-activated IL-6 promoter activity. Finally, CREB and C/EBP were necessary for basal IL-6 promoter activity and sufficient to increase IL-6 promoter activity but had minimal roles in calcineurin-activated IL-6 promoter activity. Together, these results suggest that IL-6 transcription in skeletal muscle cells can be activated by a calcineurin-MEF-2 axis which is antagonized by NFAT.

Myostatin, activin receptor IIb, and follistatin-like-3 gene expression are altered in adipose tissue and skeletal muscle of obese mice.

Myostatin (MSTN) is a secreted growth inhibitor expressed in muscle and adipose. We sought to determine whether expression of MSTN, its receptor activin RIIb (ActRIIb), or its binding protein follistatin-like-3 (FSTL3) are altered in subcutaneous or visceral adipose or in skeletal muscle in response to obesity. MSTN and ActRIIb mRNA levels were low in subcutaneous (SQF) and visceral fat (VF) from wild-type mice but were 50- to 100-fold higher in both SQF and VF from ob/ob compared with wild-type mice. FSTL3 mRNA levels were increased in SQF but decreased in VF in ob/ob compared with wild-type mice. Moreover, MSTN mRNA levels were twofold greater in tibialis anterior (TA) from ob/ob mice, whereas ActRIIb and FSTL3 mRNA levels were unchanged. MSTN mRNA levels were also increased in TA and SQF from mice on a high-fat diet. Injection of ob/ob mice with recombinant leptin caused FSTL3 mRNA levels to decrease in both VF and SQF in ob/ob mice; MSTN and ActRIIb mRNA levels tended to decrease only in VF. Finally, MSTN mRNA levels and promoter activity were low in adipogenic 3T3-L1 cells, but an MSTN promoter-reporter construct was activated in 3T3-L1 cells by cotransfection with the adipogenic transcription factors SREBP-1c, C/EBPalpha, and PPARgamma. These results demonstrate that expression of MSTN and its associated binding proteins can be modulated in adipose tissue and skeletal muscle by chronic obesity and suggest that alterations in their expression may contribute to the changes in growth and metabolism of lean and fat tissues occurring during obesity.

Loaded wheel running and muscle adaptation in the mouse.

Voluntary cage wheel exercise has been used extensively to determine the physiological adaptation of cardiac and skeletal muscle in mice. In this study, we tested the effect of different loading conditions on voluntary cage wheel performance and muscle adaptation. Male C57Bl/6 mice were exposed to a cage wheel with no-resistance (NR), low-resistance (LR), or high-resistance (HR) loads for 7 wk. Power output was elevated (3-fold) under increased loading (LR and HR) conditions compared with unloaded (NR) exercise training. Only unloaded (NR) exercise induced an increase in heart mass, whereas only loaded (LR and HR) exercise training induced an increase in skeletal (soleus) muscle mass. Moreover, unloaded and loaded exercise training had a differential impact on the cross-sectional area of muscle fibers, depending on the type of myosin heavy chain expressed by each fiber. The biochemical adaptation of the heart was characterized by a decrease in genes associated with pathological (but not physiological) cardiac hypertrophy and a decrease in calcineurin expression in all exercise groups. In addition, transcriptional activity of myocyte enhancer factor-2 (MEF-2) was significantly decreased in the hearts of the LR group as determined by a MEF-2-dependent transgene driving the expression of beta-galactosidase. Phosphorylation of glycogen synthase kinase-3beta, protein kinase B (Akt), and p70 S6 kinase was increased only in the hearts of the NR group, consistent with the significant increase in cardiac mass. In conclusion, unloaded and loaded cage wheel exercise have a differential impact on cage wheel performance and muscle (cardiac and skeletal) adaptation.