Novel Treatment Strategies for Hormone Receptor (HR)-Positive, HER2-Negative Metastatic Breast Cancer.

Estrogen receptor (ER)-positive breast cancer (BC) is the most common BC subtype. Endocrine therapy (ET) targeting ER signaling still remains the mainstay treatment option for hormone receptor (HR)-positive BC either in the early or in advanced setting, including different strategies, such as the suppression of estrogen production or directly blocking the ER pathway through SERMs-selective estrogen receptor modulators-or SERDs-selective estrogen receptor degraders. Nevertheless, the development of de novo or acquired endocrine resistance still remains challenging for oncologists. The use of novel ET combined with targeted drugs, such as cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, has significantly improved long-term outcome rates, thus changing the therapeutic algorithm for metastatic BC (MBC) and recently the therapeutic strategy in the adjuvant setting for early high-risk BC. Eluding the resistance to CDK4/6 inhibitors combined with ET is currently an unmet medical need, and there is disagreement concerning the best course of action for patients who continue to progress after this combination approach. Genetic changes in the tumor along its growth uncovered by genomic profiling of recurrent and/or metastatic lesions through tumor and/or liquid biopsies may predict the response or resistance to specific agents, suggesting the best therapeutic strategy for each patient by targeting the altered ER-dependent pathway (novel oral SERDs and a new generation of anti-estrogen agents) or alternative ER-independent signaling pathways such as PI3K/AKT/mTOR or tyrosine kinase receptors (HER2 mutations or HER2 low status) or by inhibiting pathways weakened through germline BRCA1/2 mutations. These agents are being investigated as single molecules and in combination with other target therapies, offering promising weapons to overcome or avoid treatment failure and propose increasingly more personalized treatment approaches. This review presents novel insights into ET and other targeted therapies for managing metastatic HR+/HER2- BC by exploring potential strategies based on clinical evidence and genomic profiling following the failure of the CDK4/6i and ET combination.

Secondary Metabolites and Antioxidant Activity against Moko Disease as a Defense Mechanism of Musa spp. from the Ecuadorian Coast Area.

The Musa spp. represents the most commonly produced, transitioned, and consumed fruit around the globe, with several important applications in the biotechnology, pharmaceutical, and food industries. Moko disease is produced by Ralstonia solanacearum-a factor with a high impact on all crops in Ecuador, representing one of the biggest phytosanitary problems. Four of the most common varieties of Musa spp. were tested to identify the metabolic reaction of plants facing Moko disease. The phenolic and flavonoid content has been evaluated as a defense system, and the α-diphenyl-α-picrylhydrazyl free-radical-scavenging method (DPPH), free-radical-scavenging activity (ABTS), ferric-reducing antioxidant power (FRAP) assays, and liquid chromatography and mass spectrometry (LC-MS) have been adapted to analyze the active compounds with the antioxidant capacity necessary to counteract the pathogenic attack. Our results indicate that all the studied varieties of Musa spp. react in the same way, such that the diseased samples showed a higher accumulation of secondary metabolites with antioxidant capacity compared with the healthy ones, with high active compound synthesis identified during the appearance of Moko disease symptoms. More than 40 compounds and their derivatives (from kaempferol and quercetin glycosides) with protective roles demonstrate the implication of the Musa spp. defense system against R. solanacearum infection.

Intraoperative spectroscopic evaluation of sentinel lymph nodes in breast cancer surgery.

BACKGROUND AND OBJECTIVES: Sentinel lymph node (SLN) biopsy is a standard procedure for patients with breast cancer and normal axilla on imaging. Positive SLNs on histological examination can lead to a subsequent surgery for axillary lymph node clearance (ALNC). Here we report a non-destructive technique based on autofluorescence (AF) imaging and Raman spectroscopy for intra-operative assessment of SLNs excised in breast cancer surgery. METHODS: A microscope integrating AF imaging and Raman spectroscopy modules was built to allow scanning of lymph node biopsy samples. During AF-Raman measurements, AF imaging determined optimal sampling locations for Raman spectroscopy measurements. After optimisation of the AF image analysis and training of classification models based on data from 85 samples, the AF-Raman technique was tested on an independent set of 81 lymph nodes comprising 58 fixed and 23 fresh specimens. The sensitivity and specificity of AF-Raman were calculated using post-operative histology as a standard of reference. RESULTS: The independent test set contained 66 negative lymph nodes and 15 positive lymph nodes according to the reference standard, collected from 78 patients. For this set of specimens, the area under the receiver operating characteristic (ROC) curve for the AF-Raman technique was 0.93 [0.83-0.98]. AF-Raman was then operated in a regime that maximised detection specificity, producing a 94% detection accuracy: 80% sensitivity and 97% specificity. The main confounders for SLN metastasis were areas rich in histiocytes clusters, for which only few Raman spectra had been included in the training dataset. DISCUSSION: This preliminary study indicates that with further development and extension of the training dataset by inclusion of additional Raman spectra of histiocytes clusters and capsule, the AF-Raman may become a promising technique for intra-operative assessment of SLNs. Intra-operative detection of positive biopsies could avoid second surgery for axillary clearance.

The Fungal, Nutritional, and Metabolomic Diagnostics of the Oil Palm Elaeis guineensis Affected by Bud Rot Disease in Esmeraldas, Ecuador.

The oil palm Elaeis guineensis represents one of the most important crops in Ecuador. Considering that bud rot disease is deadly in Ecuador, more attention has been given to identifying possible causes for palm debility from this disease. We studied the involvement of fungi and nutrients in triggering bud rot disease in E. guineensis. Special emphasis was given to the molecules synthesized by the plant to protect against this devastating disease. Techniques like Diagnosis and Recommendation Integrated System (DRIS) and metagenomic analysis were used to understand the possible implications of biotic and abiotic factors in the development of bud rot disease in oil palm in Ecuador. Liquid chromatography-mass spectrometry (LC-MS) analysis was used to identify the phenolic protection barrier of the palm facing the disease. Our results indicate that fungi from Ascomyceta phylum were found in the tested samples. The species directly involved are different in soil compared with plants. The results indicate a deficiency of chemical elements, such as Ca, Mn, Mg, and Fe, which are responsible for palm debility from bud rot disease. More than 30 compounds with protective roles were identified in the leaves of symptomatic plants from the first stage of the infection.

Defining the area of mitoses counting in invasive breast cancer using whole slide image.

Although counting mitoses is part of breast cancer grading, concordance studies showed low agreement. Refining the criteria for mitotic counting can improve concordance, particularly when using whole slide images (WSIs). This study aims to refine the methodology for optimal mitoses counting on WSI. Digital images of 595 hematoxylin and eosin stained sections were evaluated. Several morphological criteria were investigated and applied to define mitotic hotspots. Reproducibility, representativeness, time, and association with outcome were the criteria used to evaluate the best area size for mitoses counting. Three approaches for scoring mitoses on WSIs (single and multiple annotated rectangles and multiple digital high-power (×40) screen fields (HPSFs)) were evaluated. The relative increase in tumor cell density was the most significant and easiest parameter for identifying hotspots. Counting mitoses in 3 mm2 area was the most representative regarding saturation and concordance levels. Counting in area <2 mm2 resulted in a significant reduction in mitotic count (P = 0.02), whereas counting in area ≥4 mm2 was time-consuming and did not add a significant rise in overall mitotic count (P = 0.08). Using multiple HPSF, following calibration, provided the most reliable, timesaving, and practical method for mitoses counting on WSI. This study provides evidence-based methodology for defining the area and methodology of visual mitoses counting using WSI. Visual mitoses scoring on WSI can be performed reliably by adjusting the number of monitor screens.

Assessment of mitotic activity in breast cancer: revisited in the digital pathology era.

The assessment of cell proliferation is a key morphological feature for diagnosing various pathological lesions and predicting their clinical behaviour. Visual assessment of mitotic figures in routine histological sections remains the gold-standard method to evaluate the proliferative activity and grading of cancer. Despite the apparent simplicity of such a well-established method, visual assessment of mitotic figures in breast cancer (BC) remains a challenging task with low concordance among pathologists which can lead to under or overestimation of tumour grade and hence affects management. Guideline recommendations for counting mitoses in BC have been published to standardise methodology and improve concordance; however, the results remain less satisfactory. Alternative approaches such as the use of the proliferation marker Ki67 have been recommended but these did not show better performance in terms of concordance or prognostic stratification. The advent of whole slide image technology has brought the issue of mitotic counting in BC into the light again with more challenges to develop objective criteria for identifying and scoring mitotic figures in digitalised images. Using reliable and reproducible morphological criteria can provide the highest degree of concordance among pathologists and could even benefit the further application of artificial intelligence (AI) in breast pathology, and this relies mainly on the explicit description of these figures. In this review, we highlight the morphology of mitotic figures and their mimickers, address the current caveats in counting mitoses in breast pathology and describe how to strictly apply the morphological criteria for accurate and reliable histological grade and AI models.

Visual assessment of mitotic figures in breast cancer: a comparative study between light microscopy and whole slide images.

BACKGROUND AND AIMS: Visual assessment of mitotic figures in breast cancer (BC) remains a challenge. This is expected to be more pronounced in the digital pathology era. This study aims to refine the criteria of mitotic figure recognition, particularly in whole slide images (WSI). METHOD AND RESULTS: Haematoxylin and eosin (H&E)-stained BC sections (n = 506) were examined using light microscopy (LM) and WSI. A set of features for identifying mitosis in WSI and to distinguish true figures from mimickers was developed. Changes in the mitotic count between the two platforms was explored. Morphological features of mitoses were recorded separately, including absence of nuclear membrane, chromatin hairy-like projections, shape, cytoplasmic features, mitotic cell size and relationship to surrounding cells. Each mitotic phase has its own mimickers. Fifty-eight per cent of mitoses showed absent hairy-like projection in WSI; however, 89% retained their ragged nuclear border, which distinguished them from mimickers including apoptotic cells, lymphocytes and dark elongated hyperchromatic structures. Mitosis in WSI showed loss of fine details, and there was a 20% average reduction rate of mitotic counts when compared to the same area on LM. Using refined mitosis recognition criteria in WSI resulted in a twofold improvement of interobserver concordance. However, when compared to LM, 19% of cases were underscored in WSIs. CONCLUSIONS: All morphological features of mitosis should be considered to enable recognition and differentiation from their mimickers, particularly in WSI, to ensure reliable BC grading. Refining mitotic cut-offs per specific area when using WSI, based on the degree of reduction and association with outcome, is warranted.

Diagnostic concordance of phyllodes tumour of the breast.

AIMS: Phyllodes tumours (PT) are rare and distinct breast tumours, which span a morphological continuum. Classification into benign, borderline and malignant categories reflects their biology and clinical behaviour and is essential to guide management. This study aims to assess the diagnostic agreement of PT using the UK National Health Service Breast Screening Programme (NHSBSP) breast pathology external quality assurance (EQA) scheme data. METHODS AND RESULTS: Twenty-six PTs were identified in the EQA scheme, which were diagnosed by an average of 607 participants/circulation. Data on diagnostic categories were collected and representative slides were reviewed. The level of concordance between reporting pathologists was assessed. There were 14 benign, six borderline and six malignant PT. The overall rate of diagnosis agreement was 86% when analysed as benign lesions, borderline PT and malignant lesions, which decreased to 79% when diagnosed as PT (irrespective of grade) and to 63% when the diagnosis was further refined to PT categories (benign, borderline and malignant PTs). The highest agreement rate was observed in malignant PT (86%) and the lowest in borderline PT (42%). Malignant heterologous elements, stromal overgrowth and leaf-like architecture are features associated with higher concordance rates. Lower-priority features were stromal expansion, clefting and multinodularity. CONCLUSION: The concordance of PT diagnosis, as an entity, is high, but its classification into benign, borderline and malignant has variable agreement levels, with borderline tumours having the lowest concordance rate. More research to refine the diagnostic criteria for categorisation of PT is warranted to improve concordance between pathologists.

Combined total internal reflection AF spectral-imaging and Raman spectroscopy for fast assessment of surgical margins during breast cancer surgery.

The standard treatment for breast cancer is surgical removal mainly through breast-conserving surgery (BCS). We developed a new technique based on auto-fluorescence (AF) spectral imaging and Raman spectroscopy for fast intraoperative assessment of excision margins in BCS. A new wide-field AF imaging unit based on total internal reflection (TIR) was combined with a Raman spectroscopy microscope equipped with a 785 nm laser. The wavelength of the AF excitation was optimized to 365 nm in order to maximize the discrimination of adipose tissue. This approach allows for the non-adipose regions of tissue, which are at a higher risk of containing a tumor, to be targeted more efficiently by the Raman spectroscopy measurements. The integrated TIR-AF-Raman was tested on small tissue samples as well as fresh wide local excisions, delivering the analysis of the entire cruciate surface of BCS specimens (5.1 × 7.6 cm2) in less than 45 minutes and also providing information regarding the location of the tumor in the specimen. Full automation of the instrument and selection of a faster translation stage would allow for the measurement of BCS specimens within an intraoperative time scale (20 minutes). This study demonstrates that the TIR-AF Raman microscope represents a feasible step towards the development of a technique for intraoperative assessment of large WLE within intraoperative timescales.

Metaplastic carcinomas of the breast without evidence of epithelial differentiation: a diagnostic approach for management.

AIMS: Although rare, malignant sarcomatoid breast tumours without evidence of epithelial differentiation comprise a diagnostic challenge with management implications. Earlier studies have generally considered these to be primary breast sarcomas; however, supporting evidence is lacking and management remains variable. This study aimed to provide an evidence-based approach to improve the consistency of diagnosis and management for such cases. METHODS AND RESULTS: A large series (n = 140) of metaplastic breast carcinoma (MBC) diagnosed in Nottingham over 18 years was analysed. Only cases with available data on immunohistochemical expression of cytokeratins (CKs) were included. The prevalence and pattern of expression for various CKs were assessed and details of tumours negative for CKs were collected. A diagnostic approach based on our experience is provided. Forty-seven cases (34%) showed foci of conventional type invasive breast carcinoma or ductal carcinoma in situ (DCIS), while 93 cases (66%) were diagnosed as MBC based on morphology and/or CK expression. Ninety-seven cases (69%) were negative for one or more CKs, with 18 cases (13%) negative for five or more CKs. Eight cases (6%) lacked expression of all CKs tested. Further examination showed evidence of carcinomatous nature in five cases, and three were diagnosed as MBC following extensive diagnostic work-up and based on our experience. CONCLUSION: This study suggests that MBC represents a spectrum of neoplasms, with some lacking CK expression. Sarcomatoid neoplasms of the breast lacking evidence of carcinomatous morphology and CK expression may represent an extreme end of differentiation that can be considered as carcinomas rather than sarcomas for management purposes (following extensive work-up).

Correlations of morphological features and surgical management with clinical outcome in a multicentre study of 241 phyllodes tumours of the breast.

AIMS: Phyllodes tumours (PTs) represent an unusual but complex group of breast lesions with a tendency to recur locally and, less commonly, metastasise. On core biopsies, their appearances can be difficult to discriminate from those of other fibroepithelial lesions, which may compromise their surgical management. The aims of this study were to assess the preoperative diagnosis of PTs and to evaluate the impacts of surgical management and morphological features on their behaviour. METHODS AND RESULTS: We combined datasets from three centres over two decades, including core biopsies, excision specimens, and follow-up. Core biopsy results were compared with final excision specimens. The relationships of surgical procedure and morphological features with local recurrence (LR) and metastasis were assessed. Two hundred and forty-one PTs were studied. Core biopsy resulted in a diagnosis of possible or definite PT in 76% of cases. Malignant tumours were more likely to be larger, occurred at an older age, and were surgically more challenging, with difficulties being encountered in achieving negative margins. There were 12 cases (5%) that showed LR alone, and another six cases (2.5%) that had distant metastases. Morphological features associated with adverse outcome were grade of PT, increased mitotic counts, necrosis, infiltrative margins, stromal atypia, and heterologous components. Both LR and metastatic behaviour correlated with larger size and distance to margins. CONCLUSIONS: Our results suggest that excision margins have a significant impact on LR of PT, whereas metastatic behaviour is influenced by tumour biology. We add to the evidence base on histological features of tumours that contribute to long-term outcomes of PT patients.

Visual histological assessment of morphological features reflects the underlying molecular profile in invasive breast cancer: a morphomolecular study.

AIMS: Tumour genotype and phenotype are related and can predict outcome. In this study, we hypothesised that the visual assessment of breast cancer (BC) morphological features can provide valuable insight into underlying molecular profiles. METHODS AND RESULTS: The Cancer Genome Atlas (TCGA) BC cohort was used (n = 743) and morphological features, including Nottingham grade and its components and nucleolar prominence, were assessed utilising whole-slide images (WSIs). Two independent scores were assigned, and discordant cases were utilised to represent cases with intermediate morphological features. Differentially expressed genes (DEGs) were identified for each feature, compared among concordant/discordant cases and tested for specific pathways. Concordant grading was observed in 467 of 743 (63%) of cases. Among concordant case groups, eight common DEGs (UGT8, DDC, RGR, RLBP1, SPRR1B, CXorf49B, PSAPL1 and SPRR2G) were associated with overall tumour grade and its components. These genes are related mainly to cellular proliferation, differentiation and metabolism. The number of DEGs in cases with discordant grading was larger than those identified in concordant cases. The largest number of DEGs was observed in discordant grade 1:3 cases (n = 1185). DEGs were identified for each discordant component. Some DEGs were uniquely associated with well-defined specific morphological features, whereas expression/co-expression of other genes was identified across multiple features and underlined intermediate morphological features. CONCLUSION: Morphological features are probably related to distinct underlying molecular profiles that drive both morphology and behaviour. This study provides further evidence to support the use of image-based analysis of WSIs, including artificial intelligence algorithms, to predict tumour molecular profiles and outcome.

Immunohistochemical assessment of HRAS Q61R mutations in breast adenomyoepitheliomas.

AIMS: Breast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)-positive AMEs have mutations in phosphoinositide 3-kinase (PI3K) pathway genes, whereas ER-negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs. METHODS AND RESULTS: Twenty-six AMEs (14 ER-positive; 12 ER-negative) previously subjected to massively parallel sequencing (n = 21) or Sanger sequencing (n = 5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R-specific antibody, in addition to detailed histopathological analysis. Nine ER-negative AMEs harboured HRAS mutations, including Q61R (n = 7) and Q61K (n = 2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n = 17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P < 0.001), necrosis (P < 0.01) and mitotic index in the epithelial (P < 0.05) and myoepithelial (P < 0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2). CONCLUSIONS: IHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER-negative AMEs.