Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing.

Triple-negative breast cancer (TNBC) is an aggressive subtype that frequently develops resistance to chemotherapy. An unresolved question is whether resistance is caused by the selection of rare pre-existing clones or alternatively through the acquisition of new genomic aberrations. To investigate this question, we applied single-cell DNA and RNA sequencing in addition to bulk exome sequencing to profile longitudinal samples from 20 TNBC patients during neoadjuvant chemotherapy (NAC). Deep-exome sequencing identified 10 patients in which NAC led to clonal extinction and 10 patients in which clones persisted after treatment. In 8 patients, we performed a more detailed study using single-cell DNA sequencing to analyze 900 cells and single-cell RNA sequencing to analyze 6,862 cells. Our data showed that resistant genotypes were pre-existing and adaptively selected by NAC, while transcriptional profiles were acquired by reprogramming in response to chemotherapy in TNBC patients.

The role of serum thymidine kinase 1 activity in neoadjuvant-treated HER2-positive breast cancer: biomarker analysis from the Swedish phase II randomized PREDIX HER2 trial.

BACKGROUND: Thymidine kinase 1 (TK1) plays a pivotal role in DNA synthesis and cellular proliferation. TK1 has been studied as a prognostic marker and as an early indicator of treatment response in human epidermal growth factor 2 (HER2)-negative early and metastatic breast cancer (BC). However, the prognostic and predictive value of serial TK1 activity in HER2-positive BC remains unknown. METHODS: In the PREDIX HER2 trial, 197 HER2-positive BC patients were randomized to neoadjuvant trastuzumab, pertuzumab, and docetaxel (DPH) or trastuzumab emtansine (T-DM1), followed by surgery and adjuvant epirubicin and cyclophosphamide. Serum samples were prospectively collected from all participants at multiple timepoints: at baseline, after cycle 1, 2, 4, and 6, at end of adjuvant therapy, annually for a total period of 5 years and/or at the time of recurrence. The associations of sTK1 activity with baseline characteristics, pathologic complete response (pCR), event-free survival (EFS), and disease-free survival (DFS) were evaluated. RESULTS: No association was detected between baseline sTK1 levels and all the baseline clinicopathologic characteristics. An increase of TK1 activity from baseline to cycle 2 was seen in all cases. sTK1 level at baseline, after 2 and 4 cycles was not associated with pCR status. After a median follow-up of 58 months, 23 patients had EFS events. There was no significant effect between baseline or cycle 2 sTK1 activity and time to event. A non-significant trend was noted among patents with residual disease (non-pCR) and high sTK1 activity at the end of treatment visit, indicating a potentially worse long-term prognosis. CONCLUSION: sTK1 activity increased following neoadjuvant therapy for HER2-positive BC but was not associated with patient outcomes or treatment benefit. However, the post-surgery prognostic value in patients that have not attained pCR warrants further investigation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02568839. Registered on 6 October 2015.

Off-tumor targets compromise antiangiogenic drug sensitivity by inducing kidney erythropoietin production.

Anti-VEGF drugs are commonly used for treatment of a variety of cancers in human patients, and they often develop resistance. The mechanisms underlying anti-VEGF resistance in human cancer patients are largely unknown. Here, we show that in mouse tumor models and in human cancer patients, the anti-VEGF drug-induced kidney hypoxia augments circulating levels of erythropoietin (EPO). Gain-of-function studies show that EPO protects tumor vessels from anti-VEGF treatment and compromises its antitumor effects. Loss of function by blocking EPO function using a pharmacological approach markedly increases antitumor activity of anti-VEGF drugs through inhibition of tumor angiogenesis. Similarly, genetic loss-of-function data shows that deletion of EpoR in nonerythroid cells significantly increases antiangiogenic and antitumor effects of anti-VEGF therapy. Finally, in a relatively large cohort study, we show that treatment of human colorectal cancer patients with bevacizumab augments circulating EPO levels. These findings uncover a mechanism of desensitizing antiangiogenic and anticancer effects by kidney-produced EPO. Our work presents conceptual advances of our understanding of mechanisms underlying antiangiogenic drug resistance.

Separation of breast cancer and organ microenvironment transcriptomes in metastases.

BACKGROUND: The seed and soil hypothesis was proposed over a century ago to describe why cancer cells (seeds) grow in certain organs (soil). Since then, the genetic properties that define the cancer cells have been heavily investigated; however, genomic mediators within the organ microenvironment that mediate successful metastatic growth are less understood. These studies sought to identify cancer- and organ-specific genomic programs that mediate metastasis. METHODS: In these studies, a set of 14 human breast cancer patient-derived xenograft (PDX) metastasis models was developed and then tested for metastatic tropism with two approaches: spontaneous metastases from mammary tumors and intravenous injection of PDX cells. The transcriptomes of the cancer cells when growing as tumors or metastases were separated from the transcriptomes of the microenvironment via species-specific separation of the genomes. Drug treatment of PDX spheroids was performed to determine if genes activated in metastases may identify targetable mediators of viability. RESULTS: The experimental approaches that generated metastases in PDX models were identified. RNA sequencing of 134 tumors, metastases, and normal non-metastatic organs identified cancer- and organ-specific genomic properties that mediated metastasis. A common genomic response of the liver microenvironment was found to occur in reaction to the invading PDX cells. Genes within the cancer cells were found to be either transiently regulated by the microenvironment or permanently altered due to clonal selection of metastatic sublines. Gene Set Enrichment Analyses identified more than 400 gene signatures that were commonly activated in metastases across basal-like PDXs. A Src signaling signature was found to be extensively upregulated in metastases, and Src inhibitors were found to be cytotoxic to PDX spheroids. CONCLUSIONS: These studies identified that during the growth of breast cancer metastases, there were genomic changes that occurred within both the cancer cells and the organ microenvironment. We hypothesize that pathways upregulated in metastases are mediators of viability and that simultaneously targeting changes within different cancer cell pathways and/or different tissue compartments may be needed for inhibition of disease progression.

Assessment of early response biomarkers in relation to long-term survival in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy plus bevacizumab: Results from the Phase II PROMIX trial.

Pathologic complete response (pCR) is a predictor for favorable outcome after neoadjuvant treatment in early breast cancer. Modulation of gene expression may also provide early readouts of biological activity and prognosis, offering the possibility for timely response-guided treatment adjustment. The role of early transcriptional changes in predicting response to neoadjuvant chemotherapy plus bevacizumab was investigated. One-hundred-and-fifty patients with large, operable and locally advanced HER2-negative breast cancer received epirubicin and docetaxel, with the addition of bevacizumab. Patients underwent tumor biopsies at baseline, after Cycle 2 and at the time of surgery. The primary end point, pCR, and its relation with the secondary endpoints event-free survival (EFS), overall survival (OS) and gene expression profiles, are reported. The pCR rate was 13% (95% CI 8.6-20.2), with significantly more pCRs among triple-negative [28% (95% CI 14.8-45.4)] than among hormone receptor positive (HR+) tumors [9% (95% CI 4.6-16.3); (OR = 3.9 [CI = 1.5-10.3])]. pCR rates were not associated with EFS or OS. PAM50 subtypes significantly changed after Cycle 2 (p = 0.03) and an index of absolute changes in PAM50 correlations between these time-points was associated with EFS [HR = 0.62 (CI = 0.3-1.1)]. In univariable analyses, signatures for angiogenesis, proliferation, estrogen receptor signaling, invasion and metastasis, and immune response, measured after Cycle 2, were associated with pCR in HR+ tumors. Evaluation of changes in molecular subtypes and other signatures early in the course of neoadjuvant treatment may be predictive of pCR and EFS. These factors may help guide further treatment and should be considered when designing neoadjuvant trials.

Immune gene expression and response to chemotherapy in advanced breast cancer.

BACKGROUND: Transcriptomic profiles have shown promise as predictors of response to neoadjuvant chemotherapy in breast cancer (BC). This study aimed to explore their predictive value in the advanced BC (ABC) setting. METHODS: In a Phase 3 trial of first-line chemotherapy in ABC, a fine needle aspiration biopsy (FNAB) was obtained at baseline. Intrinsic molecular subtypes and gene modules related to immune response, proliferation, oestrogen receptor (ER) signalling and recurring genetic alterations were analysed for association with objective response to chemotherapy. Gene-set enrichment analysis (GSEA) of responders vs non-responders was performed independently. Lymphocytes were enumerated in FNAB smears and the absolute abundance of immune cell types was calculated using the Microenvironment Cell Populations counter method. RESULTS: Gene expression data were available for 109 patients. Objective response to chemotherapy was statistically significantly associated with an immune module score (odds ratio (OR)=1.62; 95% confidence interval (CI), 1.03-2.64; P=0.04). Subgroup analysis showed that this association was restricted to patients with ER-positive or luminal tumours (OR=3.54; 95%, 1.43-10.86; P=0.012 and P for interaction=0.04). Gene-set enrichment analysis confirmed that in these subgroups, immune-related gene sets were enriched in responders. CONCLUSIONS: Immune-related transcriptional signatures may predict response to chemotherapy in ER-positive and luminal ABC.

Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs).

BACKGROUND: Tumor heterogeneity in breast cancer tumors is today widely recognized. Most of the available knowledge in genetic variation however, relates to the primary tumor while metastatic lesions are much less studied. Many studies have revealed marked alterations of standard prognostic and predictive factors during tumor progression. Characterization of paired primary- and metastatic tissues should therefore be fundamental in order to understand mechanisms of tumor progression, clonal relationship to tumor evolution as well as the therapeutic aspects of systemic disease. METHODS: We performed full exome sequencing of primary breast cancers and their metastases in a cohort of ten patients and further confirmed our findings in an additional cohort of 20 patients with paired primary and metastatic tumors. Furthermore, we used gene expression from the metastatic lesions and a primary breast cancer data set to study the gene expression of the AKAP gene family. RESULTS: We report that somatic mutations in A-kinase anchoring proteins are enriched in metastatic lesions. The frequency of mutation in the AKAP gene family was 10% in the primary tumors and 40% in metastatic lesions. Several copy number variations, including deletions in regions containing AKAP genes were detected and showed consistent patterns in both investigated cohorts. In a second cohort containing 20 patients with paired primary and metastatic lesions, AKAP mutations showed an increasing variant allele frequency after multiple relapses. Furthermore, gene expression profiles from the metastatic lesions (n = 120) revealed differential expression patterns of AKAPs relative to the tumor PAM50 intrinsic subtype, which were most apparent in the basal-like subtype. This pattern was confirmed in primary tumors from TCGA (n = 522) and in a third independent cohort (n = 182). CONCLUSION: Several studies from primary cancers have reported individual AKAP genes to be associated with cancer risk and metastatic relapses as well as direct involvement in cellular invasion and migration processes. Our findings reveal an enrichment of mutations in AKAP genes in metastatic breast cancers and suggest the involvement of AKAPs in the metastatic process. In addition, we report an AKAP gene expression pattern that consistently follows the tumor intrinsic subtype, further suggesting AKAP family members as relevant players in breast cancer biology.

Swedish prospective multicenter trial evaluating sentinel lymph node biopsy after neoadjuvant systemic therapy in clinically node-positive breast cancer.

PURPOSE: Patients with clinically node-positive breast cancer planned for neoadjuvant systemic therapy (NAST) may draw advantages from the nodal downstaging effect and reduce the extent of axillary surgery with sentinel lymph node biopsy (SLNB) performed after NAST. Since there are concerns about lower sentinel lymph node (SLN) detection and higher false-negative rates (FNR) in this setting, our aim was to define the accuracy of SLNB after NAST. METHODS: This Swedish national multicenter trial prospectively recruited 195 breast cancer patients from ten hospitals with T1-T4d biopsy-proven node-positive disease planned for NAST between October 1, 2010 and December 31, 2015. Clinically node-negative axillary status after NAST was not mandatory. SLNB was always attempted and followed by a completion axillary lymph node dissection (ALND). RESULTS: The SLN identification rate was 77.9% (152/195) but improved to 80.7% (138/171) with dual mapping. The median number of SLNs was two (range 1-5). A positive SLNB was found in 52% (79/152), almost 66% (52/79) of whom had additional positive non-sentinel lymph nodes. The overall pathologic nodal response rate was 33.3% (66/195). The overall FNR was 14.1% (13/92) but decreased to 4% (2/50) when only patients with two or more sentinel nodes were analyzed. CONCLUSIONS: In biopsy-proven node-positive breast cancer, SLNB after NAST is feasible even though the identification rate is lower than in clinically node-negative patients. Since the overall FNR is unacceptably high, the omission of ALND should only be considered if two or more SLNs are identified.

Long-term effects on the incidence of second primary cancers in a randomized trial of two and five years of adjuvant tamoxifen.

BACKGROUND: Tamoxifen is a well established treatment for breast cancer, but its long-term effects on the incidence of secondary cancers are not fully evaluated. MATERIAL AND METHODS: We have studied 4128 postmenopausal patients with early stage breast cancer who were alive and free of breast cancer recurrence after two years of tamoxifen, and who were randomized to receive totally two or five years of therapy. RESULTS: Compared to patients randomized to two years of tamoxifen the incidence of contralateral breast cancer [hazard ratio (HR) 0.73; 95% CI 0.56-0.96] and of lung cancer (HR 0.45; 95% CI 0.27-0.77), especially squamous cell and small cell lung cancer, were reduced in the five-year group, and similar results were seen when restricting the analysis to the 10-year period after treatment stopped. An increased incidence of endometrial cancer was observed in the five-year group, but the excess risk decreased over time. CONCLUSION: Further studies of the effects of tamoxifen on the risk of different histological types of lung cancer are needed.

Contrast-enhanced ultrasound (CEUS) in assessing early response among patients with invasive breast cancer undergoing neoadjuvant chemotherapy.

Background One of the big challenges in onco-radiology is to find a reliable imaging method that may predict early response during the first cycles of any neoadjuvant chemotherapy. Purpose To evaluate the use of real-time harmonic contrast-enhanced ultrasound (CEUS) in predicting early response in breast cancer tumors under neoadjuvant chemotherapy (NAC) treatment. Material and Methods Nineteen consecutive patients with invasive breast cancer were evaluated with a bolus dose of 2.4 mL contrast agent using CEUS, before and after two cycles of epirubicin and docetaxel. The lognormal function was used for quantitative analysis of kinetic data to evaluate early response. Results There was statistically significant difference in time-to-peak ( tp) between responders and non-responders (two sample t-test, P = 0.027) where tp was significantly longer at the week 5 than at the baseline scan among responders when compared to non-responders. Conclusion In-flow of intravascular contrast agent in tumors is significantly slower in responders at real-time harmonic CEUS, and might be effectively used for the evaluation of early response to chemotherapy in invasive breast cancer. However, further investigations in a larger and more heterogeneous population should be performed to corroborate the reliability of the method.

Pronounced Interindividual But Not Intraindividual Variation in Tamoxifen and Metabolite Levels in Plasma During Adjuvant Treatment of Women With Early Breast Cancer.

BACKGROUND: Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor-positive tumors. More than 20% of patients relapse despite treatment. The drug is usually dosed 20 mg/d irrespective of interindividual variation in drug clearance. To study interindividual and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma on 2 occasions, with at least 4 weeks in between, of 39 women (19 premenopausal and 20 postmenopausal women) on adjuvant treatment (20 mg/d) of early breast cancer. METHODS: We used an ultra-performance liquid chromatography with a mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen. Follicle-stimulating hormone, luteinizing hormone, and estradiol levels were also measured. RESULTS: The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation, whereas intraindividual concentrations were rather stable. Despite the same dosage, interindividual tamoxifen concentrations varied from 51 to 307 ng/mL (124 ± 57, mean ± SD) and endoxifen values showed a range from 3.2 to 19 ng/mL (10.4 ± 5.2, mean ± SD), that is, 6-fold variation for both. CONCLUSIONS: Large interindividual variation of tamoxifen and endoxifen with stable intraindividual levels, and too low levels of endoxifen in a considerable proportion of patients strongly support that therapeutic drug monitoring and individualized dosing could lead to optimal exposure and hopefully better outcome. A randomized outcome study between conventional dosing and therapeutic drug monitoring-guided dosing is needed to show whether this approach works.

Audio-recorded information to patients considering participation in cancer clinical trials - a randomized study.

BACKGROUND: Patient information in cancer clinical trial is challenging. The value of audio-recording interventions for patients considering participating in clinical trials is unclear. The primary aim of this randomized study was to investigate effects of audio-recorded information on knowledge and understanding in patients considering participation in a clinical trial. MATERIAL AND METHODS: Patients scheduled for information about a phases 2 or 3 trial by one of the 13 participating oncologists at the Department of Oncology during the study period (2008-2013) were eligible. The intervention consisted of an audio-recording on compact disc (CD) of the information at the medical consultation in which the patients were informed about a trial. Knowledge and understanding was measured by the questionnaire, Quality of Informed Consent. RESULTS: A total of 130 patients were randomized, 70% of the calculated sample size (n = 186). Sixty-seven patients were randomized to the intervention. In total, 101 patients (78%) completed questionnaires. No statistical significant differences were found between the groups with respect to knowledge and understanding. The level of knowledge was relatively high, with the exceptions of the risks associated with, and the unproven nature of, the trial. Overall, patients who declined participation scored statistically significant lower on knowledge. CONCLUSION: The present study was underpowered and the results should therefore be interpreted with caution. Still, 130 patients were included with a response rate of 78%. A CD including the oral information about a clinical trial did not show any effects on knowledge or understanding. However, the levels of knowledge were high, possible due to the high levels of education in the study group. Information on risks associated with the trial is still an area for improvement.

When is the deep inferior epigastric artery flap indicated for breast reconstruction in patients not treated with radiotherapy?

BACKGROUND: It is controversial whether breast reconstruction with a microvascular free flap should be done without restrictions in patients who have not had radiotherapy. Many regard it as too expensive, but some consider it better and more economically advantageous than an implant reconstruction. METHODS: Databases of publications were searched to find out under what conditions is it suitable to offer a deep inferior epigastric perforator (DIEP) or a transverse rectus abdominis myocutaneous flap to normalize the body's appearance in a woman whose breast(s) had been removed for cancer or to prevent the development of breast cancer. The effect of breast reconstruction with DIEP flaps was analyzed, taking account of the following factors: general satisfaction (quality of life), aesthetic satisfaction (cosmesis), and morbidity. To find out which factors were of potential importance, we recorded age, hypertension, whether scars from previous abdominal surgery were present, microcirculation, whether the patient was overweight or obese, and costs of the procedure. RESULTS: Patients planning to have DIEP flaps should be willing to stop smoking at least 4 weeks before and after the procedure and have a body mass index of less than 30 kg/m to avoid a higher risk of complications. Because of the paucity of papers, it is difficult to recommend one approach over the other when considering general satisfaction, aesthetic satisfaction, and health economics. However, economical long-term outcome is highly dependent on the initial costs of each procedure and the cumulative costs of complications for each reconstruction method. CONCLUSIONS: The scientific foundation of assessment of methods of techniques of breast reconstruction is weak. Therefore, it is important that future studies should present more comparable series, highlight the long-term effects in high-quality studies, to provide the patients with optimal results without undue risks and to avoid financial burdens on society.

An integrated omics approach highlights how epigenetic events can explain and predict response to neoadjuvant chemotherapy and bevacizumab in breast cancer.

Treatment with the anti-angiogenic drug bevacizumab in addition to chemotherapy has shown efficacy for breast cancer in some clinical trials, but better biomarkers are needed to optimally select patients for treatment. Here, we present an omics approach where DNA methylation profiles are integrated with gene expression and results from proteomic data in breast cancer patients to predict response to therapy and pinpoint response-related epigenetic events. Fresh-frozen tumor biopsies taken before, during, and after treatment from human epidermal growth factor receptor 2 negative non-metastatic patients receiving neoadjuvant chemotherapy with or without bevacizumab were subjected to molecular profiling. Here, we report that DNA methylation at enhancer CpGs related to cell cycle regulation can predict response to chemotherapy and bevacizumab for the estrogen receptor positive subset of patients (AUC = 0.874), and we validated this observation in an independent patient cohort with a similar treatment regimen (AUC = 0.762). Combining the DNA methylation scores with the scores from a previously published protein signature resulted in a slight increase in the prediction performance (AUC = 0.784). We also show that tumors receiving the combination treatment underwent more extensive epigenetic alterations. Finally, we performed an integrative expression-methylation quantitative trait loci analysis on alterations in DNA methylation and gene expression levels, showing that the epigenetic alterations that occur during treatment are different between responders and non-responders and that these differences may be explained by the proliferation-epithelial-to-mesenchymal transition axis through the activity of grainyhead like transcription factor 2. Using tumor purity computed from copy number data, we developed a method for estimating cancer cell-specific methylation to confirm that the association to response reflects DNA methylation in cancer cells. Taken together, these results support the potential for clinical benefit of the addition of bevacizumab to chemotherapy when administered to the correct patients.

Longitudinal molecular profiling elucidates immunometabolism dynamics in breast cancer.

Although metabolic reprogramming within tumor cells and tumor microenvironment (TME) is well described in breast cancer, little is known about how the interplay of immune state and cancer metabolism evolves during treatment. Here, we characterize the immunometabolic profiles of tumor tissue samples longitudinally collected from individuals with breast cancer before, during and after neoadjuvant chemotherapy (NAC) using proteomics, genomics and histopathology. We show that the pre-, on-treatment and dynamic changes of the immune state, tumor metabolic proteins and tumor cell gene expression profiling-based metabolic phenotype are associated with treatment response. Single-cell/nucleus RNA sequencing revealed distinct tumor and immune cell states in metabolism between cold and hot tumors. Potential drivers of NAC based on above analyses were validated in vitro. In summary, the study shows that the interaction of tumor-intrinsic metabolic states and TME is associated with treatment outcome, supporting the concept of targeting tumor metabolism for immunoregulation.

Effects of adjuvant tamoxifen therapy on cardiac disease: results from a randomized trial with long-term follow-up.

Tamoxifen is associated with a reduced risk of coronary heart disease (CHD). However, there are few reports on long-term effects. Using data from a large Swedish randomized trial of 5 and 2 years of adjuvant tamoxifen in women with early breast cancer, we here present results on morbidity and mortality from cardiac diseases during treatment and long-term after treatment. A total of 4,150 patients were breast cancer recurrence-free after 2 years. Data from the Swedish National Hospital Discharge Registry combined with information from the Swedish Cause of Death Registry were used to define events of disease. Hazard ratios were estimated using Cox regression. Patients assigned to 5 years in comparison with 2 years of postoperative tamoxifen experienced a reduced incidence of CHD [hazard ratio (HR), 0.83; 95 % CI 0.70-1.00], especially apparent during the active treatment period (HR 0.65; 95 % CI 0.43-1.00). The mortality from CHD was significantly reduced (HR 0.72; 95 % CI 0.53-0.97). During the active treatment, the morbidity of other heart diseases was also significantly reduced (HR 0.40; 95 % CI 0.25-0.64) but not after treatment stopped (HR 1.06; 95 % CI 0.87-1.30). Similar results were seen for both heart failure and atrial fibrillation/flutter. As compared to 2 years of therapy, 5 years of postoperative tamoxifen therapy prevents CHD as well as other heart diseases. The risk reduction is most apparent during the active treatment period, and later tends to diminish.

CD169+ Macrophages in Primary Breast Tumors Associate with Tertiary Lymphoid Structures, Tregs and a Worse Prognosis for Patients with Advanced Breast Cancer.

The presence of CD169+ macrophages in the draining lymph nodes of cancer patients is, for unknown reasons, associated with a beneficial prognosis. We here investigated the prognostic impact of tumor-infiltrating CD169+ macrophages in primary tumors (PTs) and their spatial relation to tumor-infiltrating B and T cells. Using two breast cancer patient cohorts, we show that CD169+ macrophages were spatially associated with the presence of B and T cell tertiary lymphoid-like structures (TLLSs) in both PTs and lymph node metastases (LNMs). While co-infiltration of CD169+/TLLS in PTs correlated with a worse prognosis, the opposite was found when present in LNMs. RNA sequencing of breast tumors further confirmed that SIGLEC1 (CD169) expression was associated with mature tertiary lymphoid structure (TLS), and Treg and Breg signatures. We propose that the negative prognostic value related to CD169+ macrophages in PTs is a consequence of an immunosuppressive tumor environment rich in TLSs, Tregs and Bregs.

Multiresolution Self-Supervised Feature Integration via Attention Multiple Instance Learning for Histopathology Analysis.

Digital histopathology image analysis of tumor tissue sections has seen great research interest for automating standard diagnostic tasks, but also for developing novel prognostic biomarkers. However, research has mainly been focused on developing uniresolution models, capturing either high-resolution cellular features or low-resolution tissue architectural features. In addition, in the patch-based weakly-supervised training of deep learning models, the features which represent the intratumoral heterogeneity are lost. In this study, we propose a multiresolution attention-based multiple instance learning framework that can capture cellular and contextual features from the whole tissue for predicting patient-level outcomes. Several basic mathematical operations were examined for integrating multiresolution features, i.e. addition, mean, multiplication and concatenation. The proposed multiplication-based multiresolution model performed the best (AUC=0.864), while all multiresolution models outperformed the uniresolution baseline models (AUC=0.669, 0.713) for breast-cancer grading. (Implementation: https://github.com/tsikup/multiresolution-clam).

Breast cancer survival and incidence of second primary cancers after 30 years in a randomized study of two versus five years of adjuvant tamoxifen therapy.

BACKGROUND: Tamoxifen is an established treatment for breast cancer, but its long-term effects on survival and on secondary cancers are not fully evaluated. MATERIAL AND METHODS: We studied 30 years outcome of 4124 postmenopausal patients who were randomized to receive (totally) two or five years of adjuvant tamoxifen. RESULTS: After 5 years of follow-up, when tamoxifen treatment was finished in both groups, until 15 years of follow-up, overall mortality (HR 0.80, 95% CI 0.72-0.90, p < 0.001), breast cancer mortality for all patients (HR 0.80, 95% CI 0.68-0.94, p = 0.006) and breast cancer mortality for patients with estrogen receptor positive disease (HR 0.67, 95% CI 0.55-0.83, p < 0.001) were significantly reduced in the five-year group as compared to the two-year group. After 15 years, the difference remained but did not further increase. In the five-year group, the incidence of contralateral breast cancer was gradually reduced during the entire period of observation. The incidence of lung cancer was also reduced in the five-year group. In contrast there was an increased endometrial cancer incidence in the five-year group and for those receiving 40 mg of tamoxifen this incidence was further increased. CONCLUSION: Three more years of tamoxifen therapy reduced the risk of breast cancer mortality. The difference was established during the first 15 years after randomization. Moreover, the incidence of contralateral breast cancer gradually decreased for 30 years. The incidence of lung cancer was reduced in the five-year group. In contrast the incidence of endometrial cancer was increased.

Survival Outcomes, Digital TILs, and On-treatment PET/CT During Neoadjuvant Therapy for HER2-positive Breast Cancer: Results from the Randomized PREDIX HER2 Trial.

PURPOSE: PREDIX HER2 is a randomized Phase II trial that compared neoadjuvant docetaxel, trastuzumab, and pertuzumab (THP) with trastuzumab emtansine (T-DM1) for HER2-positive breast cancer. Rates of pathologic complete response (pCR) did not differ between the two groups. Here, we present the survival outcomes from PREDIX HER2 and investigate metabolic response and tumor-infiltrating lymphocytes (TIL) as prognostic factors. PATIENTS AND METHODS: In total, 202 patients with HER2-positive breast cancer were enrolled and 197 patients received six cycles of either THP or T-DM1. Secondary endpoints included event-free survival (EFS), recurrence-free survival (RFS), and overall survival (OS). Assessment with PET/CT was performed at baseline, after two and six treatment cycles. TILs were assessed manually at baseline biopsies, while image-based evaluation of TILs [digital TILs (DTIL)] was performed in digitized full-face sections. RESULTS: After a median follow-up of 5.21 years, there was no difference between the two treatment groups in terms of EFS [HR = 1.26; 95% confidence interval (CI), 0.54-2.91], RFS (HR = 0.69; 95% CI, 0.24-1.93), or OS (HR = 0.52; 95% CI, 0.09-2.82). Higher SUVmax at cycle 2 (C2) predicted lower pCR (ORadj = 0.65; 95% CI, 0.48-0.87; P = 0.005) and worse EFS (HRadj = 1.27; 95% CI, 1.12-1.41; P < 0.001). Baseline TILs and DTILs provided additional prognostic information to clinical parameters and C2 SUVmax. CONCLUSIONS: Long-term outcomes following neoadjuvant T-DM1 were similar to neoadjuvant THP. SUVmax after two cycles of neoadjuvant therapy for HER2-positive breast cancer may be an independent predictor of both short- and long-term outcomes. Combined assessment with TILs may facilitate early selection of poor responders for alternative treatment strategies.