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.

Retrospective genetic testing (Traceback) in women with early-onset breast cancer after revised national guidelines: a clinical implementation study.

PURPOSE: This study focused on identifying a hereditary predisposition in women previously diagnosed with early-onset breast cancer through a retrospective outreach activity (Traceback). The objectives were to evaluate the possible clinical implementation of a simplified Traceback strategy and to identify carriers of pathogenic variants among previously untested women. METHODS: Three hundred and fifteen Traceback-eligible women diagnosed with breast cancer at 36-40 years in Southern Sweden between 2000 and 2019 were identified and offered an analysis of the genes ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C, and RAD51D through a standardized letter. Women who chose to participate were asked about their experiences through a questionnaire. The workload for the study personnel was measured and recorded. RESULTS: One hundred and seventy-six women underwent genetic testing and pathogenic variants were identified in 9.7%: ATM (n = 6), BARD1 (n = 1), BRCA1 (n = 3), CHEK2 (n = 5), and PALB2 (n = 2). Women with normal test results were informed through a standardized letter. Carriers of pathogenic variants were contacted by telephone and offered in-person genetic counseling. One hundred and thirty-four women returned the subsequent questionnaire. Most study participants were satisfied with both written pre- and post-test information and many expressed their gratitude. The extra workload as compared to routine clinical genetic counseling was modest (8 min per patient). CONCLUSION: The insights from the participants' perspectives and sentiments throughout the process support the notion that the Traceback procedure is a safe and an appreciated complement to routine genetic counseling. The genetic yield of almost 10% also suggests that the associated extra workload for genetic counselors could be viewed as acceptable in clinical implementation scenarios.

Whole-body MRI surveillance in TP53 carriers is perceived as beneficial with no increase in cancer worry regardless of previous cancer: Data from the Swedish TP53 Study.

BACKGROUND: To evaluate the psychosocial consequences of surveillance with whole-body MRI (WB-MRI) in individuals with the heritable TP53-related cancer (hTP53rc) syndrome, also known as the Li-Fraumeni syndrome, with regard to cancer worry, perceived benefits and risks to surveillance and overall health. PATIENTS AND METHODS: Since 2016, the national Swedish TP53 Study (SWEP53) has offered surveillance with WB-MRI to all individuals with hTP53rc syndrome. Seventy-five individuals have been included in the study. Sixty consecutive participants fulfilled a base-line evaluation as well as an evaluation after 1 year with structured questionnaires including the Cancer Worry Scale (CWS), perceived benefits and risks of surveillance, and the 36-item Short Form Survey (SF-36). Individuals with or without previous personal cancer diagnosis were enrolled and results at baseline and after 1 year of surveillance were compared. For SF-36, a comparison with the normal population was also made. RESULTS: Participants with previous cancer tend to worry more about cancer, but both individuals with and without cancer had a positive attitude toward surveillance with no differences regarding perceived benefits and barriers to surveillance. Participants with a previous cancer scored significantly lower on some of the SF-36 subscales, but between-group differences were found only for social functioning after 1 year. CONCLUSIONS: Surveillance with WB-MRI is feasible from a psychosocial point of view both among TP53 carriers with as well as without a previous history of cancer and does not increase cancer worry in any of the groups. PLAIN LANGUAGE SUMMARY: Individuals with heritable TP53-related cancer syndrome (also known as the Li-Fraumeni syndrome) have a high lifetime risk of developing cancer. These TP53 carriers are offered surveillance with whole-body MRI to detect cancer early. There are few reports of the psychosocial impact of surveillance. In this study, we wanted to evaluate cancer worry, benefits and barriers to participation, and perceived overall health. Our study shows no increase in cancer worry after 1 year of surveillance, regardless of previous cancer.

Adjuvant trastuzumab emtansine in HER2-positive breast cancer patients with HER2-negative residual invasive disease in KATHERINE.

Following chemotherapy and human epidermal growth factor 2 (HER2)-targeted neoadjuvant therapy for HER2-positive early breast cancer, residual invasive breast cancer at surgery may be HER2-negative on retesting in some patients. We evaluated outcomes with T-DM1 and trastuzumab in patients randomized in the phase III KATHERINE trial based on HER2-positive central testing of the pre-treatment core biopsy with HER2-negative central testing on their corresponding surgical specimen after neoadjuvant treatment. In the 70/845 (8.3%) patients with HER2-negative residual disease on retesting at surgery, there were 11 IDFS events in the 42 trastuzumab-treated patients (26.2%) and none in the 28 T-DM1-treated patients, suggesting that T-DM1 should not be withheld in this patient population.

How Reliable Are Gene Expression-Based and Immunohistochemical Biomarkers Assessed on a Core-Needle Biopsy? A Study of Paired Core-Needle Biopsies and Surgical Specimens in Early Breast Cancer.

In early breast cancer, a preoperative core-needle biopsy (CNB) is vital to confirm the malignancy of suspected lesions and for assessing the expression of treatment predictive and prognostic biomarkers in the tumor to choose the optimal treatments, emphasizing the importance of obtaining reliable results when biomarker status is assessed on a CNB specimen. This study aims to determine the concordance between biomarker status assessed as part of clinical workup on a CNB compared to a medically untreated surgical specimen. Paired CNB and surgical specimens from 259 patients that were part of the SCAN-B cohort were studied. The concordance between immunohistochemical (IHC) and gene expression (GEX) based biomarker status was investigated. Biomarkers of interest included estrogen receptor (ER; specifically, the alpha variant), progesterone receptor (PgR), Ki67, HER2, and tumor molecular subtype. In general, moderate to very good correlation in biomarker status between the paired CNB and surgical specimens was observed for both IHC assessment (83-99% agreement, kappa range 0.474-0.917) and GEX assessment (70-97% agreement, kappa range 0.552-0.800), respectively. However, using IHC, 52% of cases with low Ki67 status in the CNB shifted to high Ki67 status in the surgical specimen (McNemar's p = 0.011). Similarly, when using GEX, a significant shift from negative to positive ER (47%) and from low to high Ki67 (16%) was observed between the CNB and surgical specimen (McNemar's p = 0.027 and p = 0.002 respectively). When comparing biomarker status between different techniques (IHC vs. GEX) performed on either CNBs or surgical specimens, the agreement in ER, PgR, and HER2 status was generally over 80% in both CNBs and surgical specimens (kappa range 0.395-0.708), but Ki67 and tumor molecular subtype showed lower concordance levels between IHC and GEX (48-62% agreement, kappa range 0.152-0.398). These results suggest that both the techniques used for collecting tissue samples and analyzing biomarker status have the potential to affect the results of biomarker assessment, potentially also impacting treatment decisions and patient survival outcomes.

RNA sequencing-based single sample predictors of molecular subtype and risk of recurrence for clinical assessment of early-stage breast cancer.

Multigene assays for molecular subtypes and biomarkers can aid management of early invasive breast cancer. Using RNA-sequencing we aimed to develop single-sample predictor (SSP) models for clinical markers, subtypes, and risk of recurrence (ROR). A cohort of 7743 patients was divided into training and test set. We trained SSPs for subtypes and ROR assigned by nearest-centroid (NC) methods and SSPs for biomarkers from histopathology. Classifications were compared with Prosigna in two external cohorts (ABiM, n = 100 and OSLO2-EMIT0, n = 103). Prognostic value was assessed using distant recurrence-free interval. Agreement between SSP and NC for PAM50 (five subtypes) was high (85%, Kappa = 0.78) for Subtype (four subtypes) very high (90%, Kappa = 0.84) and for ROR risk category high (84%, Kappa = 0.75, weighted Kappa = 0.90). Prognostic value was assessed as equivalent and clinically relevant. Agreement with histopathology was very high or high for receptor status, while moderate for Ki67 status and poor for Nottingham histological grade. SSP and Prosigna concordance was high for subtype (OSLO-EMIT0 83%, Kappa = 0.73 and ABiM 80%, Kappa = 0.72) and moderate and high for ROR risk category (68 and 84%, Kappa = 0.50 and 0.70, weighted Kappa = 0.70 and 0.78). Pooled concordance for emulated treatment recommendation dichotomized for chemotherapy was high (85%, Kappa = 0.66). Retrospective evaluation suggested that SSP application could change chemotherapy recommendations for up to 17% of postmenopausal ER+/HER2-/N0 patients with balanced escalation and de-escalation. Results suggest that NC and SSP models are interchangeable on a group-level and nearly so on a patient level and that SSP models can be derived to closely match clinical tests.

Preexisting Somatic Mutations of Estrogen Receptor Alpha (ESR1) in Early-Stage Primary Breast Cancer.

Background: More than three-quarters of primary breast cancers are positive for estrogen receptor alpha (ER; encoded by the gene ESR1), the most important factor for directing anti-estrogenic endocrine therapy (ET). Recently, mutations in ESR1 were identified as acquired mechanisms of resistance to ET, found in 12% to 55% of metastatic breast cancers treated previously with ET. Methods: We analyzed 3217 population-based invasive primary (nonmetastatic) breast cancers (within the SCAN-B study, ClinicalTrials.gov NCT02306096), sampled from initial diagnosis prior to any treatment, for the presence of ESR1 mutations using RNA sequencing. Mutations were verified by droplet digital polymerase chain reaction on tumor and normal DNA. Patient outcomes were analyzed using Kaplan-Meier estimation and a series of 2-factor Cox regression multivariable analyses. Results: We identified ESR1 resistance mutations in 30 tumors (0.9%), of which 29 were ER positive (1.1%). In ET-treated disease, presence of ESR1 mutation was associated with poor relapse-free survival and overall survival (2-sided log-rank test P < .001 and P = .008, respectively), with hazard ratios of 3.00 (95% confidence interval = 1.56 to 5.88) and 2.51 (95% confidence interval = 1.24 to 5.07), respectively, which remained statistically significant when adjusted for other prognostic factors. Conclusions: These population-based results indicate that ESR1 mutations at diagnosis of primary breast cancer occur in about 1% of women and identify for the first time in the adjuvant setting that such preexisting mutations are associated to eventual resistance to standard hormone therapy. If replicated, tumor ESR1 screening should be considered in ER-positive primary breast cancer, and for patients with mutated disease, ER degraders such as fulvestrant or other therapeutic options may be considered as more appropriate.

Protein Signature Predicts Response to Neoadjuvant Treatment With Chemotherapy and Bevacizumab in HER2-Negative Breast Cancers.

PURPOSE: Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer (BC), there is an unmet need to identify patients who benefit from such treatment. PATIENTS AND METHODS: In the NeoAva phase II clinical trial, patients (N = 132) with large (≥ 25 mm) human epidermal growth factor receptor 2 (HER2)-negative primary tumors were randomly assigned 1:1 to treatment with neoadjuvant chemotherapy (CTx) alone or in combination with bevacizumab (Bev plus CTx). The ratio of the tumor size after relative to before treatment was calculated into a continuous response scale. Tumor biopsies taken prior to neoadjuvant treatment were analyzed by reverse-phase protein arrays (RPPA) for expression levels of 210 BC-relevant (phospho-) proteins. Lasso regression was used to derive a predictor of tumor shrinkage from the expression of selected proteins prior to treatment. RESULTS: We identified a nine-protein signature score named vascular endothelial growth factor inhibition response predictor (ViRP) for use in the Bev plus CTx treatment arm able to predict with accuracy pathologic complete response (pCR) (area under the curve [AUC] = 0.85; 95% CI, 0.74 to 0.97) and low residual cancer burden (RCB 0/I) (AUC = 0.80; 95% CI, 0.68 to 0.93). The ViRP score was significantly lower in patients with pCR (P < .001) and in patients with low RCB (P < .001). The ViRP score was internally validated on mRNA data and the resultant surrogate mRNA ViRP score significantly separated the pCR patients (P = .016). Similarly, the mRNA ViRP score was validated (P < .001) in an independent phase II clinical trial (PROMIX). CONCLUSION: Our ViRP score, integrating the expression of nine proteins and validated on mRNA data both internally and in an independent clinical trial, may be used to increase the likelihood of benefit from treatment with bevacizumab combined with chemotherapy in patients with HER2-negative BC.

Breast cancer survival in Nordic BRCA2 mutation carriers-unconventional association with oestrogen receptor status.

BACKGROUND: The natural history of breast cancer among BRCA2 carriers has not been clearly established. In a previous study from Iceland, positive ER status was a negative prognostic factor. We sought to identify factors that predicted survival after invasive breast cancer in an expanded cohort of BRCA2 carriers. METHODS: We studied 608 women with invasive breast cancer and a pathogenic BRCA2 mutation (variant) from four Nordic countries. Information on prognostic factors and treatment was retrieved from health records and by analysis of archived tissue specimens. Hazard ratios (HR) were estimated for breast cancer-specific survival using Cox regression. RESULTS: About 77% of cancers were ER-positive, with the highest proportion (83%) in patients under 40 years. ER-positive breast cancers were more likely to be node-positive (59%) than ER-negative cancers (34%) (P < 0.001). The survival analysis included 584 patients. Positive ER status was protective in the first 5 years from diagnosis (multivariate HR = 0.49; 95% CI 0.26-0.93, P = 0.03); thereafter, the effect was adverse (HR = 1.91; 95% CI 1.07-3.39, P = 0.03). The adverse effect of positive ER status was limited to women who did not undergo endocrine treatment (HR = 2.36; 95% CI 1.26-4.44, P = 0.01) and patients with intact ovaries (HR = 1.99; 95% CI 1.11-3.59, P = 0.02). CONCLUSIONS: The adverse effect of a positive ER status in BRCA2 carriers with breast cancer may be contingent on exposure to ovarian hormones.

Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer.

Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients co-transplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.

The mutational landscape of the SCAN-B real-world primary breast cancer transcriptome.

Breast cancer is a disease of genomic alterations, of which the panorama of somatic mutations and how these relate to subtypes and therapy response is incompletely understood. Within SCAN-B (ClinicalTrials.gov: NCT02306096), a prospective study elucidating the transcriptomic profiles for thousands of breast cancers, we developed a RNA-seq pipeline for detection of SNVs/indels and profiled a real-world cohort of 3,217 breast tumors. We describe the mutational landscape of primary breast cancer viewed through the transcriptome of a large population-based cohort and relate it to patient survival. We demonstrate that RNA-seq can be used to call mutations in genes such as PIK3CA, TP53, and ERBB2, as well as the status of molecular pathways and mutational burden, and identify potentially druggable mutations in 86.8% of tumors. To make this rich dataset available for the research community, we developed an open source web application, the SCAN-B MutationExplorer (http://oncogenomics.bmc.lu.se/MutationExplorer). These results add another dimension to the use of RNA-seq as a clinical tool, where both gene expression- and mutation-based biomarkers can be interrogated in real-time within 1 week of tumor sampling.

Prognostic implications of the expression levels of different immunoglobulin heavy chain-encoding RNAs in early breast cancer.

The extent and composition of the immune response in a breast cancer is one important prognostic factor for the disease. The aim of the current work was to refine the analysis of the humoral component of an immune response in breast tumors by quantifying mRNA expression of different immunoglobulin classes and study their association with prognosis. We used RNA-Seq data from two local population-based breast cancer cohorts to determine the expression of IGJ and immunoglobulin heavy (IGH) chain-encoding RNAs. The association with prognosis was investigated and public data sets were used to corroborate the findings. Except for IGHE and IGHD, mRNAs encoding heavy chains were generally detected at substantial levels and correlated with other immune-related genes. High IGHG1 mRNA was associated with factors related to poor prognosis such as estrogen receptor negativity, HER2 amplification, and high grade, whereas high IGHA2 mRNA levels were primarily associated with lower age at diagnosis. High IGHA2 and IGJ mRNA levels were associated with a more favorable prognosis both in univariable and multivariable Cox models. When adjusting for other prognostic factors, high IGHG1 mRNA levels were positively associated with improved prognosis. To our knowledge, these results are the first to demonstrate that expression of individual Ig class types has prognostic implications in breast cancer.

Comprehensive molecular comparison of BRCA1 hypermethylated and BRCA1 mutated triple negative breast cancers.

Homologous recombination deficiency (HRD) is a defining characteristic in BRCA-deficient breast tumors caused by genetic or epigenetic alterations in key pathway genes. We investigated the frequency of BRCA1 promoter hypermethylation in 237 triple-negative breast cancers (TNBCs) from a population-based study using reported whole genome and RNA sequencing data, complemented with analyses of genetic, epigenetic, transcriptomic and immune infiltration phenotypes. We demonstrate that BRCA1 promoter hypermethylation is twice as frequent as BRCA1 pathogenic variants in early-stage TNBC and that hypermethylated and mutated cases have similarly improved prognosis after adjuvant chemotherapy. BRCA1 hypermethylation confers an HRD, immune cell type, genome-wide DNA methylation, and transcriptional phenotype similar to TNBC tumors with BRCA1-inactivating variants, and it can be observed in matched peripheral blood of patients with tumor hypermethylation. Hypermethylation may be an early event in tumor development that progress along a common pathway with BRCA1-mutated disease, representing a promising DNA-based biomarker for early-stage TNBC.

Whole-genome sequencing of triple-negative breast cancers in a population-based clinical study.

Whole-genome sequencing (WGS) brings comprehensive insights to cancer genome interpretation. To explore the clinical value of WGS, we sequenced 254 triple-negative breast cancers (TNBCs) for which associated treatment and outcome data were collected between 2010 and 2015 via the population-based Sweden Cancerome Analysis Network-Breast (SCAN-B) project (ClinicalTrials.gov ID:NCT02306096). Applying the HRDetect mutational-signature-based algorithm to classify tumors, 59% were predicted to have homologous-recombination-repair deficiency (HRDetect-high): 67% explained by germline/somatic mutations of BRCA1/BRCA2, BRCA1 promoter hypermethylation, RAD51C hypermethylation or biallelic loss of PALB2. A novel mechanism of BRCA1 abrogation was discovered via germline SINE-VNTR-Alu retrotransposition. HRDetect provided independent prognostic information, with HRDetect-high patients having better outcome on adjuvant chemotherapy for invasive disease-free survival (hazard ratio (HR) = 0.42; 95% confidence interval (CI) = 0.2-0.87) and distant relapse-free interval (HR = 0.31, CI = 0.13-0.76) compared to HRDetect-low, regardless of whether a genetic/epigenetic cause was identified. HRDetect-intermediate, some possessing potentially targetable biological abnormalities, had the poorest outcomes. HRDetect-low cancers also had inadequate outcomes: ~4.7% were mismatch-repair-deficient (another targetable defect, not typically sought) and they were enriched for (but not restricted to) PIK3CA/AKT1 pathway abnormalities. New treatment options need to be considered for now-discernible HRDetect-intermediate and HRDetect-low categories. This population-based study advocates for WGS of TNBC to better inform trial stratification and improve clinical decision-making.

Agreement between molecular subtyping and surrogate subtype classification: a contemporary population-based study of ER-positive/HER2-negative primary breast cancer.

PURPOSE: Oestrogen receptor-positive (ER+) and human epidermal receptor 2-negative (HER2-) breast cancers are classified as Luminal A or B based on gene expression, but immunohistochemical markers are used for surrogate subtyping. The aims of this study were to examine the agreement between molecular subtyping (MS) and surrogate subtyping and to identify subgroups consisting mainly of Luminal A or B tumours. METHODS: The cohort consisted of 2063 patients diagnosed between 2013-2017, with primary ER+/HER2- breast cancer, analysed by RNA sequencing. Surrogate subtyping was performed according to three algorithms (St. Gallen 2013, Maisonneuve and our proposed Grade-based classification). Agreement (%) and kappa statistics (κ) were used as concordance measures and ROC analysis for luminal distinction. Ki67, progesterone receptor (PR) and histological grade (HG) were further investigated as surrogate markers. RESULTS: The agreement rates between the MS and St. Gallen 2013, Maisonneuve and Grade-based classifications were 62% (κ = 0.30), 66% (κ = 0.35) and 70% (κ = 0.41), respectively. PR did not contribute to distinguishing Luminal A from B tumours (auROC = 0.56). By classifying HG1-2 tumours as Luminal A-like and HG3 as Luminal B-like, agreement with MS was 80% (κ = 0.46). Moreover, by combining HG and Ki67 status, a large subgroup of patients (51% of the cohort) having > 90% Luminal A tumours could be identified. CONCLUSIONS: Agreement between MS and surrogate classifications was generally poor. However, a post hoc analysis showed that a combination of HG and Ki67 could identify patients very likely to have Luminal A tumours according to MS.

Cross comparison and prognostic assessment of breast cancer multigene signatures in a large population-based contemporary clinical series.

Multigene expression signatures provide a molecular subdivision of early breast cancer associated with patient outcome. A gap remains in the validation of such signatures in clinical treatment groups of patients within population-based cohorts of unselected primary breast cancer representing contemporary disease stages and current treatments. A cohort of 3520 resectable breast cancers with RNA sequencing data included in the population-based SCAN-B initiative (ClinicalTrials.gov ID NCT02306096) were selected from a healthcare background population of 8587 patients diagnosed within the years 2010-2015. RNA profiles were classified according to 19 reported gene signatures including both gene expression subtypes (e.g. PAM50, IC10, CIT) and risk predictors (e.g. Oncotype DX, 70-gene, ROR). Classifications were analyzed in nine adjuvant clinical assessment groups: TNBC-ACT (adjuvant chemotherapy, n = 239), TNBC-untreated (n = 82), HER2+/ER- with anti-HER2+ ACT treatment (n = 110), HER2+/ER+ with anti-HER2 + ACT + endocrine treatment (n = 239), ER+/HER2-/LN- with endocrine treatment (n = 1113), ER+/HER2-/LN- with endocrine + ACT treatment (n = 243), ER+/HER2-/LN+ with endocrine treatment (n = 423), ER+/HER2-/LN+ with endocrine + ACT treatment (n = 433), and ER+/HER2-/LN- untreated (n = 200). Gene signature classification (e.g., proportion low-, high-risk) was generally well aligned with stratification based on current immunohistochemistry-based clinical practice. Most signatures did not provide any further risk stratification in TNBC and HER2+/ER- disease. Risk classifier agreement (low-, medium/intermediate-, high-risk groups) in ER+ assessment groups was on average 50-60% with occasional pair-wise comparisons having <30% agreement. Disregarding the intermediate-risk groups, the exact agreement between low- and high-risk groups was on average ~80-95%, for risk prediction signatures across all assessment groups. Outcome analyses were restricted to assessment groups of TNBC-ACT and endocrine treated ER+/HER2-/LN- and ER+/HER2-/LN+ cases. For ER+/HER2- disease, gene signatures appear to contribute additional prognostic value even at a relatively short follow-up time. Less apparent prognostic value was observed in the other groups for the tested signatures. The current study supports the usage of gene expression signatures in specific clinical treatment groups within population-based breast cancer. It also stresses the need of further development to reach higher consensus in individual patient classifications, especially for intermediate-risk patients, and the targeting of patients where current gene signatures and prognostic variables provide little support in clinical decision-making.

Prediction of Lymph Node Metastasis in Breast Cancer by Gene Expression and Clinicopathological Models: Development and Validation within a Population-Based Cohort.

PURPOSE: More than 70% of patients with breast cancer present with node-negative disease, yet all undergo surgical axillary staging. We aimed to define predictors of nodal metastasis using clinicopathological characteristics (CLINICAL), gene expression data (GEX), and mixed features (MIXED) and to identify patients at low risk of metastasis who might be spared sentinel lymph node biopsy (SLNB).Experimental Design: Breast tumors (n = 3,023) from the population-based Sweden Cancerome Analysis Network-Breast initiative were profiled by RNA sequencing and linked to clinicopathologic characteristics. Seven machine-learning models present the discriminative ability of N0/N+ in development (n = 2,278) and independent validation cohorts (n = 745) stratified as ER+HER2-, HER2+, and TNBC. Possible SLNB reduction rates are proposed by applying CLINICAL and MIXED predictors. RESULTS: In the validation cohort, the MIXED predictor showed the highest area under ROC curves to assess nodal metastasis; AUC = 0.72. For the subgroups, the AUCs for MIXED, CLINICAL, and GEX predictors ranged from 0.66 to 0.72, 0.65 to 0.73, and 0.58 to 0.67, respectively. Enriched proliferation metagene and luminal B features were noticed in node-positive ER+HER2- and HER2+ tumors, while upregulated basal-like features were observed in node-negative TNBC tumors. The SLNB reduction rates in patients with ER+HER2- tumors were 6% to 7% higher for the MIXED predictor compared with the CLINICAL predictor accepting false negative rates of 5% to 10%. CONCLUSIONS: Although CLINICAL and MIXED predictors of nodal metastasis had comparable accuracy, the MIXED predictor identified more node-negative patients. This translational approach holds promise for development of classifiers to reduce the rates of SLNB for patients at low risk of nodal involvement.

Refinement of breast cancer molecular classification by miRNA expression profiles.

BACKGROUND: Accurate classification of breast cancer using gene expression profiles has contributed to a better understanding of the biological mechanisms behind the disease and has paved the way for better prognostication and treatment prediction. RESULTS: We found that miRNA profiles largely recapitulate intrinsic subtypes. In the case of HER2-enriched tumors a small set of miRNAs including the HER2-encoded mir-4728 identifies the group with very high specificity. We also identified differential expression of the miR-99a/let-7c/miR-125b miRNA cluster as a marker for separation of the Luminal A and B subtypes. High expression of this miRNA cluster is linked to better overall survival among patients with Luminal A tumors. Correlation between the miRNA cluster and their precursor LINC00478 is highly significant suggesting that its expression could help improve the accuracy of present day's signatures. CONCLUSIONS: We show here that miRNA expression can be translated into mRNA profiles and that the inclusion of miRNA information facilitates the molecular diagnosis of specific subtypes, in particular the clinically relevant sub-classification of luminal tumors.

Detection of circulating tumor cells and circulating tumor DNA before and after mammographic breast compression in a cohort of breast cancer patients scheduled for neoadjuvant treatment.

PURPOSE: It is not known if mammographic breast compression of a primary tumor causes shedding of tumor cells into the circulatory system. Little is known about how the detection of circulating biomarkers such as circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) is affected by breast compression intervention. METHODS: CTCs and ctDNA were analyzed in blood samples collected before and after breast compression in 31 patients with primary breast cancer scheduled for neoadjuvant therapy. All patients had a central venous access to allow administration of intravenous neoadjuvant chemotherapy, which enabled blood collection from superior vena cava, draining the breasts, in addition to sampling from a peripheral vein. RESULTS: CTC and ctDNA positivity was seen in 26% and 65% of the patients, respectively. There was a significant increase of ctDNA after breast compression in central blood (p = 0.01), not observed in peripheral testing. No increase related with breast compression was observed for CTC. ctDNA positivity was associated with older age (p = 0.05), and ctDNA increase after breast compression was associated with high Ki67 proliferating tumors (p = 0.04). CTCs were more abundant in central compared to peripheral blood samples (p = 0.04). CONCLUSIONS: There was no significant release of CTCs after mammographic breast compression but more CTCs were present in central compared to peripheral blood. No significant difference between central and peripheral levels of ctDNA was observed. The small average increase in ctDNA after breast compression is unlikely to be clinically relevant. The results give support for mammography as a safe procedure from the point of view of CTC and ctDNA shedding to the blood circulation. The results may have implications for the standardization of sampling procedures for circulating tumor markers.

Maximum Tolerated Dose and Pharmacokinetics of Paclitaxel Micellar in Patients with Recurrent Malignant Solid Tumours: A Dose-Escalation Study.

INTRODUCTION: A water-soluble Cremophor EL-free formulation of paclitaxel, in which retinoic acid derivates solubilize paclitaxel by forming micelles (paclitaxel micellar), was studied for the first time in man to establish the maximum tolerated dose (MTD) and to characterize the pharmacokinetics (PK). METHODS: This was an open-label, one-arm, dose-escalating study in patients with advanced solid malignant tumours, for which no standard therapy was available or had failed. Paclitaxel micellar was given as 1-h intravenous infusion every 21 days for 3 cycles, mainly without premedication. Plasma samples were collected during 24 h at the first cycle and paclitaxel concentrations were assayed by high-performance liquid chromatography. PK was evaluated using a two-compartment model. RESULTS: Thirty-four patients received paclitaxel micellar at doses ranging between 90 and 275 mg/m2. MTD was established as 250 mg/m2. Fatigue and neuropathy were the most frequent dose-limiting toxicities. No hypersensitivity reactions were observed. PK of paclitaxel was evaluated in 25 data sets. Paclitaxel micellar had a rapid initial distribution phase, mean half-life 0.55 h, estimated to be completed 3 h after dosing and a mean terminal half-life of 8.8 h. Mean clearance was 13.4 L/h/m2 with fivefold interindividual variability. The residual areas after 10 h and 24 h were 15.7 ± 8.6% and 5.7 ± 3.9% of the area under the plasma concentration-time curve to infinite time (AUCinf), respectively. CONCLUSION: No new side effects unknown for paclitaxel were observed. Maximum plasma concentration (Cmax) and AUCinf showed a tendency to increase linearly with dose within the 150-275 mg/m2 dose range. The possibility to administer paclitaxel micellar without steroid premedication makes it an attractive candidate for further studies in combination with immunotherapy. TRIAL REGISTRATION: EudraCT no: 2004-001821-54. FUNDING: Oasmia Pharmaceutical AB.