Dynamics of breast-cancer relapse reveal late-recurring ER-positive genomic subgroups.

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis1-6. It is therefore essential to identify patients who have a high risk of late relapse7-9. Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns10,11; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression12,13). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47-62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials.

Stromal lymphocytes are associated with upgrade of B3 breast lesions.

Various histopathological, clinical and imaging parameters have been evaluated to identify a subset of women diagnosed with lesions with uncertain malignant potential (B3 or BIRADS 3/4A lesions) who could safely be observed rather than being treated with surgical excision, with little impact on clinical practice. The primary reason for surgery is to rule out an upgrade to either ductal carcinoma in situ or invasive breast cancer, which occurs in up to 30% of patients. We hypothesised that the stromal immune microenvironment could indicate the presence of carcinoma associated with a ductal B3 lesion and that this could be detected in biopsies by counting lymphocytes as a predictive biomarker for upgrade. A higher number of lymphocytes in the surrounding specialised stroma was observed in upgraded ductal and papillary B3 lesions than non-upgraded (p < 0.01, negative binomial model, n = 307). We developed a model using lymphocytes combined with age and the type of lesion, which was predictive of upgrade with an area under the curve of 0.82 [95% confidence interval 0.77-0.87]. The model can identify some patients at risk of upgrade with high sensitivity, but with limited specificity. Assessing the tumour microenvironment including stromal lymphocytes may contribute to reducing unnecessary surgeries in the clinic, but additional predictive features are needed.

Delineation of the adult phenotype of Coffin-Siris syndrome in 35 individuals.

Coffin-Siris syndrome (CSS) is a rare multisystemic autosomal dominant disorder. Since 2012, alterations in genes of the SWI/SNF complex were identified as the molecular basis of CSS, studying largely pediatric cohorts. Therefore, there is a lack of information on the phenotype in adulthood, particularly on the clinical outcome in adulthood and associated risks. In an international collaborative effort, data from 35 individuals ≥ 18 years with a molecularly ascertained CSS diagnosis (variants in ARID1B, ARID2, SMARCA4, SMARCB1, SMARCC2, SMARCE1, SOX11, BICRA) using a comprehensive questionnaire was collected. Our results indicate that overweight and obesity are frequent in adults with CSS. Visual impairment, scoliosis, and behavioral anomalies are more prevalent than in published pediatric or mixed cohorts. Cognitive outcomes range from profound intellectual disability (ID) to low normal IQ, with most individuals having moderate ID. The present study describes the first exclusively adult cohort of CSS individuals. We were able to delineate some features of CSS that develop over time and have therefore been underrepresented in previously reported largely pediatric cohorts, and provide recommendations for follow-up.

Expression of Cathepsin D in early-stage breast cancer and its prognostic and predictive value.

PURPOSE: Cathepsin D is a proteolytic enzyme that is normally localized in the lysosomes and is involved in the malignant progression of breast cancer. There are conflicting results regarding Cathepsin D significance as prognostic and predictor marker in breast cancer. This study aimed to evaluate the expression and prognostic significance of Cathepsin D in early-stage breast cancer. METHODS: Expression of Cathepsin D was assessed by immunohistochemical staining of tissue microarrays, in a large well-characterized series of early-stage operable breast cancer (n = 954) from Nottingham Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. Correlation of Cathepsin D expression with clinicopathological parameters and prognosis was evaluated. RESULTS: Cathepsin D expression was positive in 71.2% (679/954) of breast cancer tumours. Positive expression of Cathepsin D was significantly associated with high histological grade (p = 0.007), pleomorphism (p = 0.002), poor Nottingham Prognostic Index (NPI) score (p < 0.002), recurrence (p = 0.005) and distant metastasis (p < 0.0001). Kaplan-Meier analysis showed that Cathepsin D expression was significantly associated with shorter breast cancer-specific survival (p = 0.001), higher risk of recurrence (p = 0.001) and distant metastasis (p < 0.0001). ER-positive tumours expressing Cathepsin D and treated with tamoxifen demonstrated a significantly higher risk of distant metastasis. CONCLUSION: Cathepsin D expression significantly predicts poor prognosis in breast cancer and is associated with variables of poor prognosis and shorter outcome. The strong association of Cathepsin D with aggressive tumour characteristics and poor outcomes warrants further research of its potential as a therapeutic target The results also suggest a possible interaction between Cathepsin D and tamoxifen therapy in ER-positive breast cancer which needs further investigation to elucidate the underlying mechanisms.

Performance of a novel spectroscopy-based tool for adjuvant therapy decision-making in hormone receptor-positive breast cancer: a validation study.

PURPOSE: Digistain Index (DI), measured using an inexpensive mid-infrared spectrometer, reflects the level of aneuploidy in unstained tissue sections and correlates with tumor grade. We investigated whether incorporating DI with other clinicopathological variables could predict outcomes in patients with early breast cancer. METHODS: DI was calculated in 801 patients with hormone receptor-positive, HER2-negative primary breast cancer and ≤ 3 positive lymph nodes. All patients were treated with systemic endocrine therapy and no chemotherapy. Multivariable proportional hazards modeling was used to incorporate DI with clinicopathological variables to generate the Digistain Prognostic Score (DPS). DPS was assessed for prediction of 5- and 10-year outcomes (recurrence, recurrence-free survival [RFS] and overall survival [OS]) using receiver operating characteristics and Cox proportional hazards regression models. Kaplan-Meier analysis evaluated the ability of DPS to stratify risk. RESULTS: DPS was consistently highly accurate and had negative predictive values for all three outcomes, ranging from 0.96 to 0.99 at 5 years and 0.84 to 0.95 at 10 years. DPS demonstrated statistically significant prognostic ability with significant hazard ratios (95% CI) for low- versus high-risk classification for RFS, recurrence and OS (1.80 [CI 1.31-2.48], 1.83 [1.32-2.52] and 1.77 [1.28-2.43], respectively; all P < 0.001). CONCLUSION: DPS showed high accuracy and predictive performance, was able to stratify patients into low or high-risk, and considering its cost and rapidity, has the potential to offer clinical utility.

Novel 2 Gene Signatures Associated With Breast Cancer Proliferation: Insights From Predictive Differential Gene Expression Analysis.

The use of proliferation markers provides valuable information about the rate of tumor growth, which can guide treatment decisions. However, there is still a lack of consensus regarding the optimal molecular markers or tests to use in clinical practice. Integrating gene expression data with clinical and histopathologic parameters enhances our understanding of disease processes, facilitates the identification of precise prognostic predictors, and supports the development of effective therapeutic strategies. The purpose of this study was to apply an integrated approach that combines morphologic, clinical, and bioinformatic data to reveal effective regulators of proliferation. Whole-slide images generated from hematoxylin-and-eosin-stained sections of The Cancer Genome Atlas (TCGA) breast cancer (BC) database (n = 1053) alongside their transcriptomic and clinical data were used to identify genes differentially expressed between tumors with high and low mitotic scores. Genes enriched in the cell-cycle pathway were used to predict the protein-protein interaction (PPI) network. Ten hub genes (ORC6, SKP2, SMC1B, CDKN2A, CDC25B, E2F1, E2F2, ORC1, PTTG1, and CDC25A) were identified using CytoHubba a Cytoscape plugin. In a multivariate Cox regression model, ORC6 and SKP2 were predictors of survival independent of existing methods of proliferation assessment including mitotic score and Ki67. The prognostic ability of these genes was validated using the Molecular Taxonomy of Breast Cancer International Consortium, Nottingham cohort, Uppsala cohort, and a combined multicentric cohort. The protein expression of these 2 genes was investigated on a large cohort of BC cases, and they were significantly associated with poor prognosis and patient outcome. A positive correlation between ORC6 and SKP2 mRNA and protein expression was observed. Our study has identified 2 gene signatures, ORC6 and SKP2, which play a significant role in BC proliferation. These genes surpassed both mitotic scores and Ki67 in multivariate analysis. Their identification provides potential opportunities for the development of targeted treatments for patients with BC.

Photon counting fibre optic distributed temperature sensing with a CMOS SPAD array.

Time-resolved fibre optic Raman distributed temperature sensing (DTS) measurements experience long measurement times due to a weak backscattered Raman signal inside optical fibres or limited detector count rates. Here, improvements to previous work based on individual detectors are demonstrated using a 512 pixel complementary-metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) line sensor array with integrated (on-chip) timing electronics. Multiplexed single photon counting increases count rate and decreases measurement time for practical applications. This allows temperature to be measured every 0.5 m with 0.7 °C accuracy and a 10 s measurement time using a 13.0 m optical fibre, performance over longer distance is also investigated.

Nottingham prognostic x (NPx): a risk stratification tool in ER-positive HER2-negative breast cancer: a validation study.

AIMS: In this study, we validate the use of Nottingham Prognostic x (NPx), consisting of tumour size, tumour grade, progesterone receptor (PR) and Ki67 in luminal BC. MATERIALS AND METHODS: Two large cohorts of luminal early-stage BC (n = 2864) were included. PR and Ki67 expression were assessed using full-face resection samples using immunohistochemistry. NPx was calculated and correlated with clinical variables and outcome, together with Oncotype DX recurrence score (RS), that is frequently used as a risk stratifier in luminal BC. RESULTS: In the whole cohort, 38% of patients were classified as high risk using NPx which showed significant association with parameters characteristics of aggressive tumour behaviour and shorter survival (P < 0.0001). NPx classified the moderate Nottingham Prognostic Index (NPI) risk group (n = 1812) into two distinct prognostic subgroups. Of the 82% low-risk group, only 3.8% developed events. Contrasting this, 14% of the high-risk patients developed events during follow-up. A strong association was observed between NPx and Oncotype Dx RS (P < 0.0001), where 66% of patients with intermediate risk RS who had subsequent distant metastases also had a high-risk NPx. CONCLUSION: NPx is a reliable prognostic index in patients with luminal early-stage BC, and in selected patients may be used to guide adjuvant chemotherapy recommendations.

ATF4 as a Prognostic Marker and Modulator of Glutamine Metabolism in Oestrogen Receptor-Positive Breast Cancer.

INTRODUCTION: ATF4, a stress-responsive transcription factor that upregulates adaptive genes, is a potential prognostic marker and modulator of glutamine metabolism in breast cancer. However, its exact role remains to be elucidated. METHODS: ATF4 expression was evaluated at genomic and transcriptomic levels using METABRIC (n = 1,980), GeneMiner (n = 4,712), and KM-Plotter datasets. Proteomic expression was assessed via immunohistochemistry (n = 2,225) in the Nottingham Primary Breast Cancer Series. ATF4 genomic copy number (CN) variation and mRNA/protein in association with clinicopathological parameters, amino acid transporters (AATs), and patient outcome were investigated. RESULTS: Genomic, transcriptomic, and proteomic overexpression of ATF4 was associated with more aggressive ER-negative tumours. ATF4 mRNA and protein expression were significantly associated with increased expression of glutamine related AATs including SLC1A5 (p < 0.01) and SLC7A11 (p < 0.02). High ATF4 and SLC1A5 protein expression was significantly associated with shorter breast cancer-specific survival (p < 0.01), especially in ER+ tumours (p < 0.01), while high ATF4 and SLC7A11 protein expression was associated with shorter survival (p < 0.01). CONCLUSION: These findings suggest a complex interplay between ATF4 and AATs in breast cancer biology and underscore the potential role for ATF4 as a prognostic marker in ER+ breast cancer, offering a unique opportunity for risk stratification and personalized treatment strategies.

An unusual case of nephrotic syndrome.

BACKGROUND: Alport syndrome is a genetically heterogenous disorder resulting from variants in genes coding for alpha-3/4/5 chains of Collagen IV, which results in defective basement membranes in the kidney, cochlea and eye. The syndrome has different inheritance patterns and historically, was thought of as a disease affecting solely males. CASE: A 15-year-old female presented with pedal oedema, hypertension and proteinuria. She underwent a kidney biopsy which showed findings in keeping with focal segmental glomerulosclerosis. Her condition was refractory to steroids. Steroid-resistant nephrotic syndrome genetics were sent, revealing a rare pathogenic variant in the COL4A5 gene. CONCLUSION: Heterozygous females with X-linked Alport syndrome can develop chronic kidney disease and hearing loss. Clinicians should be mindful when reviewing kidney histology to include Alport syndrome as a differential for female patients. COL4A3-5 genes should be included in all steroid-resistant nephrotic syndrome genetic panels.

Evaluation of Ergot Resistance in Hard Red Spring Wheat in North Dakota.

Higher levels of ergot (Claviceps purpurea (Fr.)) Tul. were reported in North Dakota hard red spring wheat (HRSW) in 2018, leading to questions pertaining to management and cultivar resistance. To better understand pathogen and HRSW cultivar responses, greenhouse experiments were conducted from 2020 to 2021 to evaluate aggressiveness of nine C. purpurea isolates and ergot resistance in 21 HRSW cultivars. Results from the aggressiveness assay indicated significant cultivar by isolate interactions for total weight of sclerotia produced and ergot incidence. Mean data across all cultivar by isolate combinations suggested isolates CC-3 and IA-Tim were the most aggressive and subsequently used in ergot resistance experiments. Results from ergot resistance screening indicated none of the HRSW cultivars were immune to C. purpurea as all cultivars produced sclerotia. However, differences in ergot incidence, kernel incidence, aborted kernel incidence, total sclerotia weight, sclerotia length, and sclerotia width occurred among cultivars. Both 'ND-Frohberg' and 'TCG-Spitfire' had the lowest ergot incidence values and were among the lowest in total sclerotia weight. 'Waldron' and 'LCS-Trigger' had the highest ergot incidence and the highest total sclerotia weight. Given that most concerns with ergot occur post-harvest, we suggest two categories to describe ergot resistance: host resistance (fate of inoculation for a stigma) and logistical resistance (size characteristics of a sclerotium that influence its ability to remain with a seed lot after harvest and cleaning). This research provides a strong foundation on our understanding of HRSW resistance to ergot that will influence variety decisions in ergot-prone areas in North Dakota.

The effect of concurrent cervical spine degenerative disease on the outcome of rotator cuff repair: a national database study.

BACKGROUND: Cervical spine degenerative disease (CSD) can cause shoulder pain, potentially confounding the management of patients with rotator cuff tears. This study aimed to investigate the relationships between CSD and rotator cuff repair (RCR). METHODS: A national administrative database (PearlDiver) was used to study 4 patient cohorts: (1) RCR only (RCRo), (2) RCR with concurrent CSD (RCRC), (3) RCR after a cervical spine procedure (RCRA), and (4) RCR before a cervical spine procedure (RCRB). The outcomes of RCR were compared using multivariable logistic regression, controlling for age, sex, and Elixhauser Comorbidity Index, as well as preoperative opioid utilization in the analysis of opioid use. RESULTS: Between 2010 and 2021, a total of 889,977 patients underwent RCR. Of these patients, 784,230 (88%) underwent RCRo whereas 105,747 (12%) underwent RCRC, of whom 21,585 (2.4%) underwent cervical spine procedures (RCRA in 9670 [1.1%] and RCRB in 11,915 [1.3%]). At 2 years after RCR, compared with RCRo patients, RCRC patients had an increased risk of surgical-site infection (adjusted odds ratio [aOR] = 1.25, P = .0004), deep vein thrombosis (aOR = 1.17, P = .0002), respiratory complications (aOR = 1.19, P = .0164), and ipsilateral shoulder reoperations (débridement [aOR = 1.66, P < .0001], manipulation under anesthesia or arthroscopic lysis of adhesions [aOR = 1.23, P < .0001], distal clavicle excision [aOR = 1.78, P < .0001], subacromial decompression [aOR = 1.72, P < .0001], biceps tenodesis [aOR = 1.76, P < .0001], incision and drainage [aOR = 1.34, P = .0020], synovectomy [aOR = 1.48, P = .0136], conversion to shoulder arthroplasty [aOR = 1.62, P < .0001], revision RCR [aOR = 1.77, P < .0001], and subsequent contralateral RCR [aOR = 1.71, P < .0001]). At 2 years, compared with RCRC patients who did not undergo cervical spine procedures, RCRC patients who underwent cervical spine procedures had an increased risk of incision and drainage (aOR = 1.50, P = .0255), conversion to arthroplasty (aOR = 1.40, P < .0001), and revision RCR (aOR = 1.11, P = .0374), as well as a lower risk of contralateral RCR (aOR = 0.89, P = .0469). The sequence of cervical spine procedures did not affect the risk of shoulder reoperations. At 1 year, the risk of opioid use after RCR was less for RCRA patients compared with RCRB patients (aOR = 1.71 [95% confidence interval, 1.61-1.80; P < .0001] vs. aOR = 2.01 [95% confidence interval, 1.92-2.12; P < .0001]). CONCLUSION: Concurrent CSD has significant detrimental effects on RCR outcomes. Patients with concurrent CSD undergoing cervical spine procedures have a greater risk of ipsilateral shoulder reoperations but a decreased risk of contralateral RCR. The risk of prolonged opioid use was lower if RCR followed a cervical spine procedure. Concurrent CSD must be considered and possibly treated to optimize the outcomes of RCR.

Latarjet procedure: biomechanical evaluation of 2-screw coracoid fixation.

BACKGROUND: Coracoid nonunion is a relevant complication following the Latarjet procedure and is influenced by multiple factors, including the method of graft fixation. The purpose of this study was to evaluate and characterize the biomechanical properties of various two-screw fixation constructs used for coracoid graft fixation in the Latarjet procedure. METHODS: Forty model scapulae (Sawbones Inc., Vashon, WA, USA) were used for this study. A 15% anterior inferior glenoid bone defect was created. The coracoid was osteotomized at the juncture of the vertical and horizontal aspects, transferred to the anterior-inferior edge of the glenoid, and fixed with either two 3.5 mm fully threaded cannulated cortical screws, two 3.5 mm fully threaded solid cortical screws, two 3.5 mm partially threaded cannulated screws, or two 4.5 mm partially threaded malleolar screws (MS). Biomechanical testing was performed with an Instron material testing machine (Instron Corp., Norwood, MA, USA) by applying loads to the lateral aspect of the transferred coracoid graft. The constructs were preconditioned with nondestructive cyclical loading (0N-20N) to determine construct stiffness. After 100 cycles of dynamic loading, the construct was loaded to failure to determine ultimate failure load, yield displacement, and mode of failure. RESULTS: All failures were associated with plastic deformation of the screws and coracoid graft fracture. There was a significantly lower initial stiffness for partially threaded cannulated screws compared to MS (186 ± 49.3 N/mm vs. 280 ± 65.5 N/mm, P = .01) but no significant differences among the other constructs. There was no difference in ultimate failure load (P = .18) or yield displacement (P = .05) among constructs. CONCLUSION: Two screw coracoid fixation of the coracoid in a simulated classic Latarjet procedure with 3.5 mm fully threaded cortical and cannulated screws is comparable to 4.5 mm MS in strength, stiffness, and displacement at failure. On the other hand, partially threaded 3.5 mm cannulated screws provide inferior fixation stiffness and could potentially affect clinical outcomes.

How anatomic should anatomic total shoulder arthroplasty be? Evaluation of humeral head reconstruction with the best-fit circle.

BACKGROUND: Humeral implant designs for anatomic total shoulder arthroplasty (aTSA) focus on anatomic reconstruction of the articular segment. Likewise, the pathoanatomy of advanced glenohumeral osteoarthritis often results in humeral head deformity. We hypothesized the anatomic reconstruction of the humeral head in aTSA risks overstuffing the glenohumeral joint. METHODS: Ninety-seven cases (52 females) of primary glenohumeral osteoarthritis in patients treated with aTSA were evaluated. Preoperative computed tomography scans were used to classify glenoid morphology according to the Walch classification. Coronal plane images in the plane of the humerus were used to determine the anatomic best-fit circle as described by Youderian et al. Humeral head thinning was determined as the distance from the center of rotation of the best-fit circle to the nearest point along the humeral articular surface. aTSA was modeled with a predicted anatomic humeral head and a simulated 4-mm polyethylene glenoid component. The change in the position of the native humerus was determined. Wilcoxon Rank Sum tests were used to evaluate differences in humeral head thinning and humeral lateralization between monoconcave and biconcave glenoid morphologies. Spearman's rank correlation coefficients were used to assess the relationship between humeral head thinning with preoperative active forward elevation and external rotation. RESULTS: The mean radius of the best-fit circle was 25.0 ± 2.1 mm. There was a mean thinning of 2.4 ± 2.0 mm (range -1.7 to 8.3). The mean percent thinning of the humeral head was 9.4% ± 7.7%. The mean humeral lateralization was 6.4 ± 2.0 mm. Humeral head thinning was not significantly associated with active forward elevation (r = -0.15, P = .14) or active external rotation (r = -0.12, P = .25). There were no significant differences in the percentage of humeral head thinning (P = .324) or humeral lateralization (P = .350) between concentric and eccentric glenoid wear patterns. CONCLUSIONS: Utilization of the best-fit circle as a guide in aTSA may risk excessive lateralization of the humerus and overstuffing the glenohumeral joint. This may have implications for subscapularis repair and healing, as well as glenoid implant and rotator cuff longevity. These findings call into question whether recreation of normal glenohumeral anatomy in aTSA is appropriate for all patients. Humeral head reconstruction in aTSA should account for glenohumeral joint volume and soft tissue contracture.

Trends in reported outcomes and patient reported outcome measures (PROMs) in humeral shaft fracture literature: a systematic review.

PURPOSE: With a lack of standardization among outcome measures in fracture literature, cross-study comparisons remain limited. This systematic review aimed to identify trends in outcome measures reported by studies of the treatment of humeral shaft fractures. METHODS: A systematic review was performed of studies reporting clinical outcomes of humeral shaft fractures indexed in PubMed. Extracted data included demographics, fracture characteristics, treatment modalities, outcomes, patient reported outcome measures (PROMs), and journal characteristics. Cochran-Armitage tests and linear regressions were used to identify data trends. Pearson chi-square and Kruskal-Wallis tests were used for comparisons between studies. RESULTS: This review included 197 studies with outcomes of 15,445 humeral shaft fractures. 126 studies reported PROMs and 37 different PROMs were used. The Constant Score was most commonly reported (34% of studies), followed by ASES Score (21%), MEPS (21%), and DASH Score (20%). There was a significant increase in PROM usage over time (p = 0.016) and in articles using three or more PROMs (p = 0.005). The number of PROMs were significantly greater in prospective cohort studies and RCTs (p = 0.012) compared to retrospective cohort studies and case series (p = 0.044 for both). Post-treatment shoulder motion was reported in 43% of studies and 34% reported elbow motion. 86% of studies reported complications as an outcome parameter. Time to union and nonunion rate were published in 69% and 88% of studies, respectively. CONCLUSION: This study identified increasing PROM usage over time and disparities in the reporting of outcomes in humeral shaft fracture literature requiring further validation and standardization of available outcome measures.

The clinical value of progesterone receptor expression in luminal breast cancer: A study of a large cohort with long-term follow-up.

BACKGROUND: The routine assessment of progesterone receptor (PR) expression in breast cancer (BC) remains controversial. This study aimed to evaluate the role of PR expression in luminal BC, with emphasis on the definition of positivity and its prognostic significance as compared to Ki67 expression. METHODS: A large cohort (n = 1924) of estrogen receptor (ER)-positive/HER2-negative BC was included. PR was immunohistochemically (IHC) stained on full face sections and core needle biopsies (CNB) where the optimal scoring cutoff was evaluated. In addition, the association of PR with other clinicopathological factors, cellular proliferation, disease outcome, and response to adjuvant therapy were analyzed. RESULTS: Although several cutoffs showed prognostic significance, the optimal cutoff to categorize PR expression into two clinically distinct prognostic groups on CNB was 10%. PR negativity showed a significant association with features of aggressive tumor behavior and poor outcome. Multivariate analyses indicated that the association between PR negativity and poor outcome was independent of tumor grade, size, node stage, and Ki67. PR negativity showed independent association with shorter survival in patients who received endocrine therapy whereas Ki67did not. CONCLUSION: PR IHC expression provides independent prognostic value superior to Ki67. Routine assessment of PR expression in BC using 10% cutoff in the clinical setting is recommended. PLAIN LANGUAGE SUMMARY: In this study, we have established an optimal approach to determine the prognostic value of progesterone receptor expression in estrogen receptor-positive breast cancer patients. To do this, the levels of progesterone receptor were measured in a large cohort of estrogen receptor-positive breast cancer patients. We have refined the definition of progesterone receptor positivity in estrogen receptor-positive breast cancer. We show that progesterone receptor expression adds prognostic and predictive value of endocrine therapy in estrogen receptor-positive breast cancer patients, and our results show that the absence of progesterone receptor is associated with poorer outcomes independent of tumor grade, size, node stage, and Ki67 expression.

The clinical significance of cyclin B1 (CCNB1) in invasive breast cancer with emphasis on its contribution to lymphovascular invasion development.

BACKGROUND: Lymphovascular invasion (LVI) is regulated through complex molecular mechanisms. Cyclin B1 (CCNB1) was previously determined as being associated with LVI using large cohorts of breast cancer (BC) and artificial neural network (ANN) technique. In this study, we aimed to assess the association between CCNB1 and LVI, other clinicopathological and other LVI-related biomarkers at the molecular (RNA transcriptomic) and proteomic levels in BC. METHODS: Two transcriptomic BC cohorts (n = 2834) were used to assess the association between the expression of CCNB1 at the mRNA level and clinicopathological characteristics and patient outcome. Tissue microarrays (TMAs) from a well-characterised BC cohort (n = 2480) with long-term outcome were also used to assess the clinical significance of CCNB1 protein expression using immunohistochemistry. RESULTS: High CCNB1 mRNA expression was associated with aggressive tumour behaviour, including LVI, larger size, higher tumour grade, high lymph nodal stage, hormonal receptor negativity, HER2 positivity and poor clinical outcome (all p < 0.0001). Similarly, high CCNB1 protein expression was associated with higher tumour grade, hormonal receptor negativity and HER2 positivity (all p < 0.0001). Additionally, there was a significant association between CCNB1- and LVI-related biomarkers including N-cadherin, P-cadherin and TWIST2 at the transcriptomic and proteomic level. Multivariate analysis revealed that CCNB1 was an independent predictor of shorter BC-specific survival (HR = 1.3; 95% CI 1.2-1.5; p = 0.010). CONCLUSION: CCNB1 is a key gene associated with LVI in BC and has prognostic value. More functional studies are warranted to unravel the mechanistic role of CCNB1 in the development of LVI.

The Clinical and Biological Significance of Estrogen Receptor-Low Positive Breast Cancer.

Estrogen receptor (ER) status in breast cancer (BC) is determined using immunohistochemistry (IHC) with nuclear expression in ≥1% of cells defined as ER-positive. BC with 1%-9% expression (ER-low-positive), is a clinically and biologically unique subgroup. In this study, we hypothesized that ER-low-positive BC represents a heterogeneous group with a mixture of ER-positive and ER-negative tumor, which may explain their divergent clinical behavior. A large BC cohort (n = 8171) was investigated and categorized into 3 groups: ER-low-positive (1%-9%), ER-positive (≥10%), and ER-negative (<1%) where clinicopathological and outcome characteristics were compared. A subset of ER-low-positive cases was further evaluated using IHC, RNAscope, and RT-qPCR. PAM50 subtyping and ESR1 mRNA expression levels were assessed in ER-low-positive cases within The Cancer Genome Atlas data set. The reliability of image analysis software in assessment of ER expression in the ER-low-positive category was also assessed. ER-low-positive tumors constituted <2% of BC cases examined and showed significant clinicopathological similarity to ER-negative tumors. Most of these tumors were nonluminal types showing low ESR1 mRNA expression. Further validation of ER status revealed that 45% of these tumors were ER-negative with repeated IHC staining and confirmed by RNAscope and RT-qPCR. ER-low-positive tumors diagnosed on needle core biopsy were enriched with false-positive ER staining. BCs with 10% ER behaved similar to ER-positive, rather than ER-negative or low-positive BCs. Moderate concordance was found in assessment of ER-low-positive tumors, and this was not improved by image analysis. Routinely diagnosed ER-low-positive BC includes a proportion of ER-negative cases. We recommend repeat testing of BC showing 1%-9% ER expression and using a cutoff ≥10% expression to define ER positivity to help better inform treatment decisions.

Combined proliferation and apoptosis index provides better risk stratification in breast cancer.

AIMS: Breast cancer (BC) risk stratification is critical for predicting behaviour and guiding management decision-making. Despite the well-established prognostic value of cellular proliferation in BC, the interplay between proliferation and apoptosis remains to be defined. In this study, we hypothesised that the combined proliferation and apoptosis indices can provide a more accurate in-vivo growth rate measure and a precise prognostic predictor. METHODS AND RESULTS: Apoptotic and mitotic figures were counted in whole slide images (WSI) generated from haematoxylin and eosin-stained sections of 1545 BC cases derived from two well-defined BC cohorts. Counts were carried out visually within defined areas. There was a significant correlation between mitosis and apoptosis scores. High apoptotic counts were associated with features of aggressive behaviour, including high grade, high pleomorphism score and hormonal receptor negativity. Although the mitotic index (MI) and apoptotic index (AI) were independent prognostic indicators, the prognostic value was synergistically higher when combined. BC patients with a high combined AI and MI had the shortest survival. Replacing the mitosis score with the mitosis-apoptosis index in the Nottingham grading system revealed that the modified grade with the new score had a higher significant association with BC-specific survival with a higher hazard ratio. CONCLUSION: Apoptotic figures count provides additional prognostic value in BC when combined with MI; such a combination can be implemented to assess the behaviour of BC and provides an accurate prognostic indicator. This can be considered when using artificial intelligence algorithms to assess proliferation in BC.

Characteristics and prognostic significance of polo-like kinase-1 (PLK1) expression in breast cancer.

AIM: Polo-like kinase-1 (PLK1) plays a crucial role in cell cycle progression, and it is considered a potential therapeutic target in many cancers. Although the role of PLK1 is well established in triple-negative breast cancer (TNBC) as an oncogene, its role in luminal BC is still controversial. In this study, we aimed to evaluate the prognostic and predictive role of PLK1 in BC and its molecular subtypes. METHODS: A large BC cohort (n = 1208) were immunohistochemically stained for PLK1. The association with clinicopathological, molecular subtypes, and survival data was analysed. PLK1 mRNA was evaluated in the publicly available datasets (n = 6774), including The Cancer Genome Atlas and the Kaplan-Meier Plotter tool. RESULTS: 20% of the study cohort showed high cytoplasmic PLK1 expression. High PLK1 expression was significantly associated with a better outcome in the whole cohort, luminal BC. In contrast, high PLK1 expression was associated with a poor outcome in TNBC. Multivariate analyses indicated that high PLK1 expression is independently associated with longer survival in luminal BC, and in poorer prognosis in TNBC. At the mRNA levels, PLK1 expression was associated with short survival in TNBC consistent with the protein expression. However, in luminal BC, its prognostic value significantly varies between cohorts. CONCLUSION: The prognostic role of PLK1 in BC is molecular subtype-dependent. As PLK1 inhibitors are introduced to clinical trials for several cancer types, our study supports evaluation of the pharmacological inhibition of PLK1 as an attractive therapeutic target in TNBC. However, in luminal BC, PLK1 prognostic role remains controversial.