Surgical Treatment After Neoadjuvant Systemic Therapy in Young Women With Breast Cancer: Results From a Prospective Cohort Study.

OBJECTIVE: We aimed to investigate eligibility for breast conserving surgery (BCS) pre- and post-neoadjuvant systemic therapy (NST), and trends in the surgical treatment of young breast cancer patients. BACKGROUND: Young women with breast cancer are more likely to present with larger tumors and aggressive phenotypes, and may benefit from NST. Little is known about how response to NST influences surgical decisions in young women. METHODS: The Young Women's Breast Cancer Study, a multicenter prospective cohort of women diagnosed with breast cancer at age ≤40, enrolled 1302 patients from 2006 to 2016. Disease characteristics, surgical recommendations, and reasons for choosing mastectomy among BCS-eligible patients were obtained through the medical record. Trends in use of NST, rate of clinical and pathologic complete response, and surgery were also assessed. RESULTS: Of 1117 women with unilateral stage I-III breast cancer, 315 (28%) received NST. Pre-NST, 26% were BCS eligible, 17% were borderline eligible, and 55% were ineligible. After NST, BCS eligibility increased from 26% to 42% (P < 0.0001). Among BCS-eligible patients after NST (n = 133), 41% chose mastectomy with reasons being patient preference (53%), BRCA or TP53 mutation (35%), and family history (5%). From 2006 to 2016, the rates of NST (P = 0.0012), clinical complete response (P < 0.0001), and bilateral mastectomy (P < 0.0001) increased, but the rate of BCS did not increase (P = 0.34). CONCLUSION: While the proportion of young women eligible for BCS increased after NST, many patients chose mastectomy, suggesting that surgical decisions are often driven by factors beyond extent of disease and treatment response.

Clinicopathological features and BRCA1 and BRCA2 mutation status in a prospective cohort of young women with breast cancer.

BACKGROUND: Breast cancer in young women is more likely to have higher risk features and be associated with germline BRCA1/BRCA2 mutations. We present the clinicopathologic features of breast cancers in a prospective cohort of young women, and associations between surrogate molecular subtype and BRCA1/BRCA2 mutation status. METHODS: Histopathological features, biomarker status, tumour stage and BRCA status were collected. Invasive tumours were categorised as luminal A-like (ER + and/or PR + , HER2-, grade 1/2), luminal B-like (ER + and/or PR + , HER2 + , or ER + and/or PR + , HER2-, and grade 3), HER2-enriched (ER/PR-, HER2 + ) or triple-negative. RESULTS: In all, 57.3% (654/1143) of invasive tumours were high grade. In total, 32.9% were luminal A-like, 42.4% luminal B-like, 8.3% HER2-enriched, and 16.4% triple-negative. Among different age groups, there were no differences in molecular phenotype, stage, grade or histopathology. 11% (131) of tumours were from BRCA mutation carriers; 64.1% BRCA1 (63.1% triple-negative), and 35.9% BRCA2 (55.3% luminal B-like). DISCUSSION: The opportunity to provide comparisons across young age groups, BRCA mutation status, surrogate molecular phenotype, and the identification of more aggressive hormone receptor-positive phenotypes in this population provides direction for future work to further understand and improve disparate outcomes for young women with luminal B-like cancers, particularly BRCA2-associated cancers, with potential implications for tailored prevention and treatment.

Correction: Evaluation of significant genome-wide association studies risk-SNPs in young breast cancer patients.

[This corrects the article DOI: 10.1371/journal.pone.0216997.].

Evaluation of significant genome-wide association studies risk - SNPs in young breast cancer patients.

PURPOSE: Genome-wide-association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) that are associated with an increased risk of breast cancer. Most of these studies were conducted primarily in postmenopausal breast cancer patients. Therefore, we set out to assess whether or not these breast cancer variants are also associated with an elevated risk of breast cancer in young premenopausal patients. METHODS: In 451 women of European ancestry who had prospectively enrolled in a longitudinal cohort study for women diagnosed with breast cancer at or under age 40, we genotyped 44 SNPs that were previously associated with breast cancer risk. A control group was comprised of 1142 postmenopausal healthy women from the Nurses' Health Study (NHS). We assessed if the frequencies of the adequately genotyped SNPs differed significantly (p≤0.05) between the cohort of young breast cancer patients and postmenopausal controls, and then we corrected for multiple testing. RESULTS: Genotyping of the controls or cases was inadequate for comparisons between the groups for seven of the 44 SNPs. 9 of the remaining 37 were associated with breast cancer risk in young women with a p-value <0.05: rs10510102, rs1219648, rs13387042, rs1876206, rs2936870, rs2981579, rs3734805, rs3803662 and rs4973768. The directions of these associations were consistent with those in postmenopausal women. However, after correction for multiple testing (Benjamini Hochberg) none of the results remained statistically significant. CONCLUSION: After correction for multiple testing, none of the alleles for postmenopausal breast cancer were clearly associated with risk of premenopausal breast cancer in this relatively small study.

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins.

The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.

Deficiency of fibroblast growth factor 21 (FGF21) promotes hepatocellular carcinoma (HCC) in mice on a long term obesogenic diet.

OBJECTIVE: Non-alcoholic fatty liver (NAFL) associated with obesity is a major cause of liver diseases which can progress to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) plays an important role in liver metabolism and is also a potential marker for NAFL. Here we aimed to test the effect of FGF21 deficiency on liver pathology in mice consuming a conventional high fat, high sucrose (HFHS) obesogenic diet for up to 52 weeks. METHODS: C57BL6 WT and FGF21 KO mice were fed a conventional obesogenic diet and were evaluated at 16 and 52 weeks. Evaluation included metabolic assessment, liver pathology, and transcriptomic analysis. RESULTS: With consumption of HFHS diet, FGF21 deficient mice (FGF21 KO) develop excess fatty liver within 16 weeks. Hepatic pathology progresses and at 52 weeks FGF21 KO mice show significantly worse fibrosis and 78% of mice develop HCC; in contrast only 6% of WT mice develop HCC. Well differentiated hepatocellular carcinomas in FGF21 KO mice were characterized by expanded hepatic plates, loss of reticulin network, cytologic atypia, and positive immunostaining for glutamine synthetase. Microarray analysis reveals enrichment of several fibroblast growth factor signaling pathways in the tumors. CONCLUSIONS: In addition to attenuating inflammation and fibrosis in mice under a number of dietary challenges, we show here that FGF21 is required to limit the progression from NAFL to HCC in response to prolonged exposure to an obesogenic diet. The induction of hepatic FGF21 in response to the high fat, high sucrose obesogenic diet may play an important role in limiting progression of liver pathology from NAFL to HCC.

Rapid histopathological imaging of skin and breast cancer surgical specimens using immersion microscopy with ultraviolet surface excitation.

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.

Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope.

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.

Fibroblast growth factor 21 (FGF21) is robustly induced by ethanol and has a protective role in ethanol associated liver injury.

OBJECTIVE: Excess ethanol consumption has serious pathologic consequences. In humans, repeated episodes of binge drinking can lead to liver damage and have adverse effects on other organs such as pancreas and brain. Long term chronic consumption of ethanol can also result in progressive alcoholic liver disease and cirrhosis. Fibroblast growth factor 21 (FGF21) is a metabolic regulator with multiple physiologic functions. FGF21 is a novel biomarker for non-alcoholic fatty liver disease (NAFLD) in humans and limits hepatotoxicity in mice. Therefore, we explored the possibility that FGF21 plays a role in response to ethanol consumption in both humans and mice. METHODS: We used a binge drinking paradigm in humans to examine the effect of acute ethanol consumption on circulating FGF21. We adapted this paradigm to evaluate the acute response to ethanol in mice. We then examined the role of FGF21 on liver pathology in two models of chronic ethanol consumption in both wild type (WT) mice and mice lacking FGF21 (FGF21-KO). RESULTS: Acute ethanol consumption resulted in a robust induction of serum FGF21 after 6 h in both humans and mice. Serum ethanol peaked at 1 h in both species and was cleared by 6 h. Ethanol clearance was the same in WT and FGF21-KO mice, indicating that FGF21 does not play a major role in ethanol metabolism in a binge paradigm. When FGF21-KO mice were fed the Lieber-DeCarli diet, a high fat diet supplemented with ethanol, a higher mortality was observed compared to WT mice after 16 days on the diet. When FGF21-KO mice consumed 30% ethanol in drinking water, along with a normal chow diet, there was no mortality observed even after 16 weeks, but the FGF21-KO mice had significant liver pathology compared to WT mice. CONCLUSIONS: Acute or binge ethanol consumption significantly increases circulating FGF21 levels in both humans and mice. However, FGF21 does not play a role in acute ethanol clearance. In contrast, chronic ethanol consumption in the absence of FGF21 is associated with significant liver pathology alone or in combination with excess mortality, depending on the type of diet consumed with ethanol. This suggests that FGF21 protects against long term ethanol induced hepatic damage and may attenuate progression of alcoholic liver disease. Further study is required to assess the therapeutic potential of FGF21 in the treatment of alcoholic liver disease.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue.

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging.

We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.

Rapid imaging of surgical breast excisions using direct temporal sampling two photon fluorescent lifetime imaging.

Two photon fluorescent lifetime imaging is a modality that enables depth-sectioned, molecularly-specific imaging of cells and tissue using intrinsic contrast. However, clinical applications have not been well explored due to low imaging speed and limited field of view, which make evaluating large pathology samples extremely challenging. To address these limitations, we have developed direct temporal sampling two photon fluorescent lifetime imaging (DTS-FLIM), a method which enables a several order of magnitude increase in imaging speed by capturing an entire lifetime decay in a single fluorescent excitation. We use this greatly increased speed to perform a preliminary study using gigapixel-scale imaging of human breast pathology surgical specimens.