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Abstract Neoadjuvant endocrine therapy (NET) for hormone receptor-positive (HR+) breast cancer might be as effective as chemotherapy, with a better toxicity profile. Blocking a crucial process such as angiogenesis with sunitinib may have a synergistic effect with NET. We aimed to assess the efficacy and safety of neoadjuvant sunitinib plus exemestane in early-stage HR+/HER2-negative breast cancer. In this phase I/II study, postmenopausal women with HR+/HER2− stage II-III breast cancer received neoadjuvant exemestane at conventional dose of 25mg plus sunitinib in a 3 + 3 design at 25mg (3/1weeks scheme) or 37.5mg continuous dose, for 6 months. Coprimary endpoints were the recommended dose of sunitinib combined with exemestane and objective response. Secondary endpoints included safety and biomarkers of early response. For 15 months, 18 patients were enrolled, 15 at sunitinib 25mg and 3 at 37.5mg. Median age was 73, 77% of patients had T2 tumors and 67% node-positive disease. The most common grade 2 toxicity was asthenia (44%), as was hypertension (22%) for grade 3. No grade 4–5 were reported. Twelve patients (66%) achieved an objective response. VEGFR-2 levels significantly decreased after one month of treatment. Differential gene expression analysis showed downregulation of ESR1, PGR and NAT1 in post-treatment samples and upregulation of EGFR, MYC, SFRP1, and FOXC1. PAM50 analysis on 83% of patients showed a prevalence of luminal A subtype, both in pre-treatment (63.6%) and post-treatment tumors (54.5%). Sunitinib plus exemestane was associated with substantial yet reversible toxicities, providing safety, efficacy and biological impact insights of combining an antiangiogenic drug with hormone therapy in early-stage breast cancer. Trial registration: Registered with ClinicalTrials.gov, NCT00931450. 02/07/2009

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of progesterone and estrogen receptors and low (or absent) HER2 expression. TNBC accounts for 15-20% of all breast cancers. It is associated with younger age, a higher mutational burden, and an increased risk of recurrence and mortality. Standard treatment for TNBC primarily relies on cytotoxic agents, such as taxanes, anthracyclines, and platinum compounds for both early and advanced stages of the disease. Several targeted therapies, including bevacizumab and sunitinib, have failed to demonstrate significant clinical benefit in TNBC. The emergence of immune checkpoint inhibitors (ICI) has revolutionized cancer treatment. By stimulating the immune system, ICIs induce a durable anti-tumor response across various solid tumors. TNBC is a particularly promising target for treatment with ICIs due to the higher levels of tumor-infiltrating lymphocytes (TIL), increased PD-L1 expression, and higher mutational burden, which generates tumor-specific neoantigens that activate immune cells. ICIs administered as monotherapy in advanced TNBC yields only a modest response; however, response rates significantly improve when ICIs are combined with cytotoxic agents, particularly in tumors expressing PD-L1. Pembrolizumab is approved for use in both early and advanced TNBC in combination with standard chemotherapy. However, more research is needed to identify more potent biomarkers, and to better elucidate the synergism of ICIs with other targeted agents. In this review, we explore the challenges of immunotherapy in TNBC, examining the mechanisms of tumor progression mediated by immune cells within the tumor microenvironment, and the signaling pathways involved in both primary and acquired resistance. Finally, we provide a comprehensive overview of ongoing clinical trials underway to investigate novel immune-targeted therapies for TNBC.

Purpose: To evaluate olaparib in advanced triple negative breast cancer (TNBC) patients with homologous recombination deficiency (HRD) and no germline BRCA1/2 mutations (gBRCA1/2mut). Methods: NOBROLA (NCT03367689) is a single-arm, open-label, multicenter, phase IIa trial, enrolling adult patients with advanced TNBC without gBRCA1/2mut and with HRD, who were treated with olaparib. The primary endpoint was clinical benefit rate (CBR) per RECIST v.1.1. Results: Six of 114 patients were eligible and received olaparib. Median follow up was 8.5 months. CBR and overall response rate (ORR) were 50 % (95 % CI, 11.8–88.2). Conclusions: The observed results could prompt further investigation. Trial: ClinicalTrials.gov identifier NCT03367689.

Purpose: To evaluate the efficacy and safety of the combination of olaparib plus trastuzumab in patients with HER2-positive advanced breast cancer (ABC) and germinal BRCA mutations (gBRCAm). Methods: OPHELIA (NCT03931551) was a single-arm, open-label, phase 2 clinical trial. Patients aged ≥18 years diagnosed with HER2-positive ABC with germinal deleterious mutations in BRCA1 or BRCA2 who had received at least one prior systemic regimen for advanced disease were enrolled. Patients received olaparib plus trastuzumab until disease progression, unacceptable toxicity, or consent withdrawal. The primary endpoint was investigator-assessed clinical benefit rate for at least 24 weeks as per RECIST v.1.1. Key secondary endpoints included overall response rate (ORR) and safety profile. Results: A total of 68 pre-treated HER2-positive ABC patients were screened. Due to slow accrual the trial was stopped after enrolling 5 patients instead of the planned sample size of 20. Four patients achieved clinical benefit (80.0 %, 95 % CI; 28.4–99.5, p

Background: The capsule formulation of CDK4/6 inhibitor palbociclib has reduced solubility at gastric pH > 4.5 and may have decreased activity when used with proton-pump inhibitors (PPI). Herein, we report the effect of PPI on palbociclib capsule activity and safety in the PARSIFAL study. Methods: First-line endocrine-sensitive, hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC) patients received palbociclib capsules plus fulvestrant or letrozole. The primary endpoint was progression-free survival (PFS). This post-hoc analysis compared PPI use. Patients were PPI-naïve (N-PPI) if not on PPI during the study, and either early (E-PPI) or long-term PPI (LT-PPI) if on PPI at study entry or for at least ≥⅔ of treatment, respectively. PPI groups were not mutually exclusive. Results: Among 486 patients, 66.9 % were N-PPI, 13.2 % E-PPI, 18.7 % LT-PPI, and 11.5 % of the PPI users were defined as neither. Median PFS (mPFS) was 29.6 months in the study population, 28.7 months in N-PPI, 23.0 months in E-PPI (Hazard Ratio [HR] 1.5; 95%Confidence Interval [CI] 1.1–2.2; p = 0.024), and 23.0 months in LT-PPI (HR 1.4; 95%CI 1.0–1.9; p = 0.035). By landmark analysis, PPI use was associated with poorer mPFS at 3 and 12 months. Grade ≥3 hematological adverse events occurred in 71.7 % of N-PPI, 57.8 % of E-PPI (p = 0.021), and 54.9 % of LT-PPI (p = 0.003). Dose reductions and dosing delays due to hematological toxicity occurred in 70.8 % of N-PPI, 56.3 % of E-PPI (p = 0.018), and 52.7 % of LT-PPI (p = 0.002). Conclusions: PPI use may reduce palbociclib capsule toxicity, dose modifications, and clinical activity in HR+/HER2- ABC.

Introduction: Advancements in monoclonal antibodies, tyrosine kinase inhibitors, and antibody drug conjugates (ADCs) have notably enhanced outcomes for metastatic HER2-positive breast cancer patients. Despite the expanding treatment options and clinical complexities, determining the optimal sequence of HER2-targeted therapies remains partly uncertain, influenced by various factors. Methods: To refine HER2-positive metastatic breast cancer management, particularly regarding tucatinib's position, a Steering Committee of leading oncologists in breast cancer care devised a panel of statements via a Delphi approach, focusing on five key topics: general clinical management, therapeutic approaches for patients with HER2-positive breast cancer and brain metastases, treatment sequence, and tucatinib's safety and efficacy. Results: A total of 29 statements were deliberated, with strong consensus achieved for most. However, no consensus emerged regarding the management of brain progression alongside stable extracranial disease: 48 % advocated for switching to tucatinib, while 53 % favored a stereotactic brain radiotherapy (SBRT) approach if feasible. Conclusion: The unanimous consensus attained in this Delphi panel, particularly regarding tucatinib's efficacy and safety, underscores oncologists' recognition of its clinical significance based on existing trial data. These findings align closely with current literature, shedding light on areas necessitating further investigation, not thoroughly explored in prior studies. Moreover, the results underscore the scarcity of data on managing brain progression alongside stable extracranial disease, emphasizing the imperative for dedicated research to address these gaps and yield definitive insights.

Summary: Background: Hyperglycaemia is an early and frequent adverse event during alpelisib treatment. METALLICA aimed to evaluate prophylactic metformin to prevent or reduce hyperglycaemia occurrence in patients with HR+/HER2−/PIK3CA-mutated advanced breast cancer (ABC). Methods: Between August 13th, 2020 and March 23rd, 2022, this 2-cohort, phase 2, multicentre, single-arm trial (NCT04300790) enrolled patients with HR+/HER2−/PIK3CA-mutated ABC: cohort A, normal glycaemia (fasting plasma glucose

Abstract Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD1 mRNA expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.

Abstract Stratification of recurrence risk is a cornerstone of early breast cancer diagnosis that informs a patient’s optimal treatment pathway. Several tools exist that combine clinicopathological and molecular information, including multigene assays, which can estimate risk of recurrence and quantify the potential benefit of different adjuvant treatment modalities. While the tools endorsed by treatment guidelines are supported by level I and II evidence and provide similar prognostic accuracy at the population level, they can yield discordant risk prediction at the individual patient level. This review examines the evidence for these tools in clinical practice and offers a perspective of potential future risk stratification strategies. Experience from clinical trials with cyclin D kinase 4/6 (CDK4/6) inhibitors in the setting of hormone receptor–positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) early breast cancer is provided as an illustrative example of risk stratification.

Background: The COVID-19 pandemic is the biggest global health problem in the last hundred years. The efficacy of the vaccine to protect against severe disease is estimated to be 70–95% according to the studies carried out, although there are aspects of the immune response to the vaccine that remain unclear. Methods: Humoral and cellular immunity after the administration of three doses of the Pfizer–BioNTech and Oxford AstraZeneca vaccines against SARS-CoV-2 over one year and the appearance of post-vaccination COVID-19 were studied. SARS-CoV-2 IgG and IgA antibodies, αβ and γδ T-cell subsets, and their differentiation stages and apoptosis were analyzed. Results: Anti-SARS-CoV-2 IgG and IgA antibodies showed a progressive increase throughout the duration of the study. This increase was the greatest after the third dose. The highest levels were observed in subjects who had anti-SARS-CoV-2 antibodies prior to vaccination. There was an increase in CD4+ αβ, CD8+ γδ and TEM CD8+ γδ T cells, and a decrease in apoptosis in CD4+ CD8+ and CD56+ αβ and γδ T cells. Post-vaccination SARS-CoV-2 infection was greater than 60%. The symptoms of COVID-19 were very mild and were related to a γδ T cell deficit, specifically CD8+ TEMRA and CD56+ γδ TEM, as well as lower pre-vaccine apoptosis levels. Conclusions: The results unveil the important role of γδ T cells in SARS-CoV-2-vaccine-mediated protection from the disease.

Objectives: Severe COVID-19 is associated with immune dysregulation and hyperinflammation (lymphocyte exhaustion and elevated interleukin 6. Pembrolizumab (P; immune-activating anti-programmed cell death-1 antibody) plus tocilizumab (TCZ; anti- interleukin 6 receptor antibody) might interrupt the hyperinflammation and restore cellular immunocompetence. We assessed the efficacy and safety of P + TCZ + standard of care (SOC) in high-risk, hospitalized patients with COVID-19 pneumonia without mechanical ventilation. Methods: Randomized, controlled, open-label, phase II trial in patients with severe SARS-CoV-2 infection to assess the hospitalization period to discharge. Results: A total of 12 patients were randomized (P + TCZ + SOC, n = 7; SOC, n = 5). Nine (75%) were males, with a median age of 68 (41-79) years. The median time to discharge for P + TCZ + SOC and SOC was 10 and 47.5 days (P = 0.03), with zero (n = 1 patient had P-related grade 5 myositis) and two COVID-19-related deaths, respectively. Conclusion: The addition of P and TCZ to SOC reduced the hospitalization period, with higher and faster discharges without sequelae than SOC alone.

De-escalation trials in oncology evaluate therapies that aim to improve the quality of life of patients with low-risk cancer by avoiding overtreatment. Non-inferiority randomized trials are commonly used to investigate de-intensified regimens with similar efficacy to that of standard regimens but with fewer adverse effects (ESMO evidence tier A). In cases where it is not feasible to recruit the number of patients needed for a randomized trial, single-arm prospective studies with a hypothesis of non-inferiority can be conducted as an alternative. Single-arm studies are also commonly used to evaluate novel treatment strategies (ESMO evidence tier B). A single-arm design that includes both non-inferiority and superiority primary objectives will enable the ranking of clinical activity and other parameters such as safety, pharmacokinetics, and pharmacodynamics data. Here, we describe the statistical principles and procedures to support such a strategy. The non-inferiority margin is calculated using the fixed margin method. Sample size and statistical analyses are based on the maximum likelihood method for exponential distributions. We present example analyses in metastatic and adjuvant settings to illustrate the usefulness of our methodology. We also explain its implementation with nonparametric methods. Single-arm designs with non-inferiority and superiority analyses are optimal for proof-of-concept and de-escalation studies in oncology.

Abstract Background Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. Results To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. Conclusions miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.

Abstract Eribulin prolongs overall survival in patients with pre-treated advanced breast cancer. However, no biomarker exists to prospectively select patients who will benefit the most from this drug. SOLTI-1007-NeoEribulin is a phase II, open-label, two-cohort, exploratory pharmacogenomic study in patients with clinical stage I–II HER2-negative breast cancer receiving neoadjuvant eribulin monotherapy treatment. Primary objective was to explore the association of baseline tumor gene expression with pathological complete response in the breast (pCRB) at surgery. Key secondary objectives were pCRB rates in all patients and according to HR status, gene expression changes during treatment and safety. One-hundred one hormonal receptor-positive (HR + ) and seventy-three triple-negative breast cancer (TNBC) patients were recruited. The pCRB rates were 6.4% in all patients, 4.9% in HR + disease and 8.2% in TNBC. The TNBC cohort was interrupted due to a progression disease rate of 30.1%. The pCRB rates differed according to intrinsic subtypes: 28.6% in HER2-enriched, 11.1% in Normal-like, 7.9% in Luminal B, 5.9% in Basal-like and 0% in Luminal A (HER2-enriched vs. others odds ratio = 7.05, 95% CI 1.80–42.14; p-value = 0.032). Intrinsic subtype changes at surgery occurred in 33.3% of cases, mostly (49.0%) Luminal B converting to Luminal A or Basal-like converting to Normal-like. Baseline tumor-infiltrating lymphocytes (TILs) were significantly associated with pCR. Eribulin showed a good safety profile with a low response and pCRB rates. Patients with HER2-negative disease with a HER2-enriched profile may benefit the most from eribulin. In addition, significant biological activity of eribulin is observed in Luminal B and Basal-like subtypes.

Breast cancer (BC) is the most frequently occurring malignancy and the second cancer-specific cause of mortality in women in developed countries. Over 70% of the total number of BCs are hormone receptor-positive (HR+), and elevated levels of circulating estrogen (E) in the blood have been shown to be a major risk factor for the development of HR+ BC. This is attributable to estrogen’s contribution to increased cancer cell proliferation, stimulation of angiogenesis and metastasis, and resistance to therapy. The E metabolism–gut microbiome axis is functional, with subjacent individual variations in the levels of E. It is conceivable that the estrobolome (bacterial genes whose products metabolize E) may contribute to the risk of malignant neoplasms of hormonal origin, including BC, and may serve as a potential biomarker and target. It has been suggested that β-glucuronidase (GUS) enzymes of the intestinal microbiome participate in the strobolome. In addition, it has been proposed that bacterial GUS enzymes from the gastrointestinal tract participate in hormone BC. In this review, we discuss the latest knowledge about the role of the GUS enzyme in the pathogenesis of BC, focusing on (i) the microbiome and E metabolism; (ii) diet, estrobolome, and BC development; (iii) other activities of the bacterial GUS; and (iv) the new molecular targets for BC therapeutic application.

The use of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) has proven to be a successful strategy in the treatment of advanced hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancer (BC), leading to a strong interest in their possible role in the treatment of early luminal BC. In this review we collect the most relevant and recent information on the use of CDK4/6i for the treatment of early BC in the neoadjuvant and adjuvant settings. Specifically, we evaluate the results of the large phase 3 adjuvant trials recently released, which have yielded apparently divergent results. We also examine the relevance of biomarkers as response predictive factors for CDI4/6i, the combination between radiotherapy and CDK4/6i, and provide a critical discussion on the evidence that we have so far and future directions of the role of these drugs in the treatment of early BC.

Background: This study evaluated efficacy and safety of palbociclib, a CDK4/6 inhibitor, in heavily-pretreated hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (mBC) patients during the compassionate use program in Spain from February 2015 to November 2017. Patients and methods: Patient data were collected retrospectively from 35 hospitals in Spain. Patients with HR+/HER2- mBC who had progressed on ≥4 treatments for advanced disease were eligible. Results: A total of 219 patients received palbociclib in combination with aromatase inhibitors (110; 50.2%), fulvestrant (87; 39.7%), tamoxifen (8; 3.6%) or as single agent (10; 4.6%). Mean age of the patients was 58 years; 31 patients (16.1%) were premenopausal and 162 (83.9%) were postmenopausal at the beginning of treatment with palbociclib. Patients had received a median of 3 previous lines of endocrine therapy (ET) for advanced disease. Real-world tumor response (rwTR) and clinical benefit rate were 5.9% (n = 13) and 46.2% (n = 101), respectively. The median real world progression-free survival (rwPFS) was 6.0 months (95% CI 5.7–7.0) and the median overall survival was 19.0 months (95% CI 16.4–21.7). Subgroup analysis revealed a significant difference in median rwPFS in patients treated with palbociclib plus fulvestrant depending on the duration of prior treatment with fulvestrant monotherapy (>6 versus ≤6 months; HR 1.93, 95% CI 1.37–2.73, p

Summary: Background: Both clinical and genomic data independently predict survival and treatment response in early-stage HER2-positive breast cancer. Here we present the development and validation of a new HER2DX risk score, and a new HER2DX pathological complete response (pCR) score, both based on a 27-gene expression plus clinical feature-based classifier. Methods: HER2DX is a supervised learning algorithm incorporating tumour size, nodal staging, and 4 gene expression signatures tracking immune infiltration, tumour cell proliferation, luminal differentiation, and the expression of the HER2 amplicon, into a single score. 434 HER2-positive tumours from the Short-HER trial were used to train a prognostic risk model; 268 cases from an independent cohort were used to verify the accuracy of the HER2DX risk score. In addition, 116 cases treated with neoadjuvant anti-HER2-based chemotherapy were used to train a predictive model of pathological complete response (pCR); two independent cohorts of 91 and 67 cases were used to verify the accuracy of the HER2DX pCR likelihood score. Five publicly available independent datasets with >1,000 patients with early-stage HER2-positive disease were also analysed. Findings: In Short-HER, HER2DX variables were associated with good risk outcomes (i.e., immune, and luminal) and poor risk outcomes (i.e., proliferation, and tumour and nodal staging). In an independent cohort, continuous HER2DX risk score was significantly associated with disease-free survival (DFS) (p=0·002); the 5-year DFS in the low-risk group was 97·4% (94·4-100·0%). For the neoadjuvant pCR predictor training cohort, HER2DX variables were associated with pCR (i.e., immune, proliferation and HER2 amplicon) and non-pCR (i.e., luminal, and tumour and nodal staging). In both independent test set cohorts, continuous HER2DX pCR likelihood score was significantly associated with pCR (p

Drug approval for early-stage breast cancer (EBC) has been historically granted in the context of registration trials based on adequate outcomes such as disease-free survival and overall survival. Improvements in long-term outcomes have made it more difficult to demonstrate the clinical benefit of a new cancer drug in large, randomized, comparative clinical trials. Therefore, the use of surrogate endpoints rather than traditional measures allows for cancer drug trials to proceed with smaller sample sizes and shorter follow-up periods, which reduces drug development time. Among surrogate endpoints for breast cancer, the increase in pathological complete response (pCR) rates was considered appropriate for accelerated drug approval. The association between pCR and long-term outcomes was strongest in patients with aggressive tumor subtypes, such as triple-negative and human epidermal growth factor receptor 2 (HER2)-positive/hormone receptor-negative breast cancers. Whereas in hormone receptor-positive/HER2-negative EBC, the most accepted surrogate markers for endocrine therapy–based trials include changes in Ki67 and the preoperative endocrine prognostic index. Beyond the classic endpoints, further prognostic tools are required to provide EBC patients with individualized and effective therapies, and the neoadjuvant setting provides an excellent platform for drug development and biomarker discovery. Nowadays, the availability of multigene signatures is offering a standardized quantitative and reproducible tool to potentiate the efficacy of standard treatment for high-risk patients and develop de-escalated treatments for patients at lower risk of relapse. In this article, we first evaluate the surrogacies used for long-term outcomes and the underlying evidence supporting the use of each surrogate endpoint for the accelerated or regular drug approval process in EBC. Next, we provide an overview of the most recent studies and innovative strategies in a (neo)adjuvant setting as a platform to accelerate new drug approval. Finally, we highlight some clinical trials aimed at tailoring systemic treatment of EBC using prognosis-related factors or early biomarkers of drug sensitivity or resistance.

In the global health emergency caused by the COVID-19, clinical trial management has proven to be critical for the pharmaceutical industry, sponsors, and healthcare professionals. Our experience as a sponsor managing interventional oncology clinical studies has provided us with some data and insights. Though limited by sample size, our data emphasize the importance of quickly adopting measures that first prioritize patient safety and data validity, then consider contingency measures such as telemedicine, virtual medical review, and remote monitoring. Successful adaptations of healthcare and patient management in response to COVID-19 have been fundamental to ensuring continuing clinical cancer research.

HER2-enriched breast cancers within the HER2-positive subtype are addicted to the HER2 pathway. Here, the authors analyse gene expression before, during, and after treatment with anti-HER2-based therapies in the phase II PAMELA clinical trial, finding phenotypic changes induced by treatment.

Abstract Purpose The primary aim of NSABP FB-7 was to determine the pathologic complete response (pCR) rate in locally advanced HER2-positive (HER2+) breast cancer patients treated with neoadjuvant trastuzumab or neratinib or the combination and weekly paclitaxel followed by standard doxorubicin plus cyclophosphamide. The secondary aims include biomarker analyses. Experimental design pCR was tested for association with treatment, gene expression, and a single nucleotide polymorphism (SNP) in the Fc fragment of the IgG receptor IIIa-158V/F (FCGR3A). Pre-treatment biopsies and residual tumors were also compared to identify molecular changes. Results The numerical pCR rate in the trastuzumab plus neratinib arm (50% [95%CI 34–66%]) was greater than that for single-targeted therapies with trastuzumab (38% [95%CI 24–54]) or neratinib (33% [95%CI 20–50]) in the overall cohort but was not statistically significant. Hormone receptor-negative (HR−) tumors had a higher pCR rate than HR+ tumors in all three treatment arms, with the highest pCR rate in the combination arm. Diarrhea was the most frequent adverse event and occurred in virtually all patients who received neratinib-based therapy. Grade 3 diarrhea was reported in 31% of patients; there were no grade 4 events. Our 8-gene signature, previously validated for trastuzumab benefit in two different clinical trials in the adjuvant setting, was correlated with pCR across all arms of NSABP FB-7. Specifically, patients predicted to receive no trastuzumab benefit had a significantly lower pCR rate than did patients predicted to receive the most benefit (P = 0.03). FCGR genotyping showed that patients who were homozygous for the Fc low-binding phenylalanine (F) allele for FCGR3A-158V/F were less likely to achieve pCR. Conclusions Combining trastuzumab plus neratinib with paclitaxel increased the absolute pCR rate in the overall cohort and in HR− patients. The 8-gene signature, which is validated for predicting trastuzumab benefit in the adjuvant setting, was associated with pCR in the neoadjuvant setting, but remains to be validated as a predictive marker in a larger neoadjuvant clinical trial. HR status, and the FCGR3A-158V/F genotype, also warrant further investigation to identify HER2+ patients who may benefit from additional anti-HER2 therapies beyond trastuzumab. All of these markers will require further validation in the neoadjuvant setting. Trials registration ClinicalTrials.gov, NCT01008150. Retrospectively registered on October 5, 2010.

Abstract Background A preliminary exploratory study shows solid agreement between the results of case reports and clinical study meta-analyses in mucopolysaccharidosis Type I (MPS-I) adult patients. The aim of the present study is to confirm previous results in another patient population, suffering from mucopolysaccharidosis Type II (MPS-II). Methods A systematic review and meta-analysis of case reports published by April 2018 was conducted for MPS-II patients treated with enzyme replacement therapy (ERT). The study is reported in accordance with PRISMA and MOOSE guidelines (PROSPERO database code CRD42018093408). The assessed population and outcomes were the same as previously analyzed in a meta-analysis of MPS-II clinical studies. The primary endpoint was the percent of clinical cases showing improvement in efficacy outcome, or no harm in safety outcome after ERT initiation. A restrictive procedure to aggregate case reports, by selecting standardized and well-defined outcomes, was proposed. Different sensitivity analyses were able to evaluate the robustness of results. Results Every outcome classified as “acceptable evidence group” in our case report meta-analysis had been graded as “moderate strength of evidence” in the aforementioned meta-analysis of clinical studies. Sensitivity, specificity, and positive-negative predictive values for results of both meta-analyses reached 100%, and were deemed equivalent. Conclusions Aggregating case reports quantitatively, rather than analyzing them qualitatively, may improve conclusions in rare diseases and personalized medicine. Additionally, we propose some methods to evaluate publication bias and heterogeneity of the included studies in a meta-analysis of case reports.

Nonrandomized studies are usually excluded from systematic reviews. This could lead to loss of a considerable amount of information on rare diseases. In this article, we explore the impact of excluding nonrandomized studies on the generalizability of meta-analyses results on mucopolysaccharidosis (MPS) disease. A comprehensive search of systematic reviews on MPS patients up to May 2020 was carried out (CRD42020191217). The primary endpoint was the rate of patients excluded from systematic reviews if only randomized studies were considered. Secondary outcomes included the differences in patient and study characteristics between randomized and nonrandomized studies, the methods used to combine data from studies with different designs, and the number of patients excluded from systematic reviews if case reports were not considered. More than 50% of the patients analyzed have been recruited in nonrandomized studies. Patient characteristics, duration of follow-up, and the clinical outcomes evaluated differ between the randomized and nonrandomized studies. There are feasible strategies to combine the data from different randomized and nonrandomized designs. The analyses suggest the relevance of including case reports in the systematic reviews, since the smaller the number of patients in the reference population, the larger the selection bias associated to excluding case reports. Our results recommend including nonrandomized studies in the systematic reviews of MPS to increase the representativeness of the results and to avoid a selection bias. The recommendations obtained from this study should be considered when conducting systematic reviews on rare diseases.

BackgroundLymphopenia is associated with various pathologies such as sepsis, burns, trauma, general anesthesia and major surgeries. All these pathologies are clinically expressed by the so-called Systemic Inflammatory Response Syndrome which does not include lymphopenia into defining criteria. The main objective of this work was to analyze the diagnosis of patients admitted to a hospital related to lymphopenia during hospital stay. In addition, we investigated the relationship of lymphopenia with the four levels of the Severity of Illness (SOI) and the Risk of Mortality (ROM).Method and findingsLymphopenia was defined as Absolute Lymphocyte Count (ALC) ConclusionsLymphopenia had a high prevalence in hospitalized patients with greater relevance in infectious pathologies. Lymphopenia was related and clearly predicts SOI and ROM at the time of admission, and should be considered as clinical diagnostic criteria to define SIRS.

Introduction: Non-inferiority (NI) analysis is not usually considered in the early phases of clinical development. In some negative phase II trials, a post-hoc NI analysis justified additional phase III trials that were successful. However, the risk of false positive achievements was not controlled in these early phase analyses. We propose to preplan NI analyses in superiority-based Simon's two-stage designs to control type I and II error rates. Methods: Simulations have been proposed to assess the control of type I and II errors rates with this method. A total of 12,768 two-stage Simon's design trials were constructed based on different assumptions of rejection response probability, desired response probability, type I and II errors, and NI margins. P-value and type II error were calculated with stochastic ordering using Uniformly Minimum Variance Unbiased Estimator. Type I and II errors were simulated using the Monte Carlo method. The agreement between calculated and simulated values was analyzed with Bland-Altman plots. Results: We observed the same level of agreement between calculated and simulated type I and II errors from both two-stage Simon's superiority designs and designs in which NI analysis was allowed. Different examples has been proposed to explain the utility of this method. Conclusion: Inclusion of NI analysis in superiority-based single-arm clinical trials may be useful for weighing additional factors such as safety, pharmacokinetics, pharmacodynamic, and biomarker data while assessing early efficacy. Implementation of this strategy can be achieved through simple adaptations to existing designs for one-arm phase II clinical trials.

Abstract Background The Opti-HER HEART trial aimed to optimize activity while minimizing cardiac risk by combining trastuzumab, pertuzumab, and paclitaxel with non-pegylated liposomal doxorubicin in the treatment of HER2-positive early breast cancer. Methods Patients with stage II–IIIB HER2-positive breast cancer received neoadjuvant trastuzumab, pertuzumab, paclitaxel, and a non-pegylated liposomal doxorubicin every three weeks for six cycles. The primary endpoint was cardiac safety during neoadjuvant therapy. Type A (symptomatic congestive heart failure) and B (asymptomatic reduction of left ventricular ejection fraction) cardiac events were evaluated. Secondary endpoints included the evaluation of the pathological complete response (pCR) rate and overall response rate, among others. As an ad-hoc exploratory analysis, the expression of 55 breast cancer-related genes, including the PAM50 genes, was measured in 58 baseline tumor samples and 60 surgical specimens. Results Eighty-three patients were recruited. The incidence of cardiac events during neoadjuvant treatment was 2.4%. No type A cardiac event was observed. The overall pCR rate was 56.6% (95% confidence interval (CI) 45.3–67.5%). The HER2-enriched subtype, which represented 52.0% of all baseline samples, was associated with a higher pCR rate compared to non-HER2-enriched tumors (83.3% vs. 46.3%; odds ratio 5.76, 95% CI 1.71–19.42). The association of subtype with pCR was independent of known clinicopathological variables, including hormone receptor status. Compared to baseline samples, surgical specimens showed a significant downregulation of proliferation-related genes (MKI67 and CCNB1) and ERBB2 levels, and a significant upregulation of luminal-related (ESR1 and PGR) and immune (CD8A) genes. Conclusions The combination of dual HER2 blockade with trastuzumab and pertuzumab with paclitaxel and non-pegylated liposomal doxorubicin is associated with a low rate of cardiac events. The HER2-enriched subtype is associated with a high rate of pCR. Trial registration clinicaltrials.gov, NCT01669239, Registered 20 August 2012.

Introducción: la curación de las metástasis a distancia, una vez que ocurren, es improbable, aunque pueden lograrse remisiones a largo plazo y paliación. La identificación durante el diagnóstico anatomopatológico de los factores pronósticos determinantes en su probable aparición es imprescindible pues de ello dependerá la supervivencia. Objetivo: identificar los factores pronósticos clínicos, inmunohistoquímicos, clasificadores y eventos adversos con capacidad predictora de metástasis a distancia. Métodos: se realizó una investigación observacional longitudinal prospectiva en una muestra de 333 pacientes con diagnóstico de carcinomas mamarios infiltrantes en un período de 16 años. Las variables fueron agrupadas en clínicas, inmunohistoquímicas, clasificado-res y eventos adversos (recaída local). Se realizó un análisis de la supervivencia por el método de Kaplan-Meier en relación con las metástasis a distancia y con la técnica de Log-Rank se hicieron las comparaciones en los subgrupos de cada variable para identificar las mejores predictoras. Resultados: las variables muy significativas (p5,4 del índice pronóstico de Nottingham, los estadios IIIC y IV, la clasificación N3 y M1 del TNMp y la recaída local y las significativas (0,005≤p

Introducción: las metástasis a distancia disminuyen la supervivencia de las pacientes con carcinomas mamarios infiltrantes, es necesaria la detección de factores pronósticos determinantes en su aparición. Objetivo: identificar factores pronósticos morfológicos con capacidad predictora de metástasis a distancia. Métodos: se realizó una investigación observacional longitudinal prospectiva en una muestra de 333 pacientes con carcinomas mamarios infiltrantes en un período de 16 años. Las variables fueron la talla y el volumen tumoral, la multicentricidad, la multifocalidad, el estado ganglionar, la infiltración extracapsular o periganglionar, los niveles ganglionares de Berg, el subtipo histológico, el grado histológico, el borde y la necrosis tumoral, la respuesta inflamatoria, la invasión vascular linfática, el componente intraductal y los márgenes de sección quirúrgicos. Se hizo un análisis de la supervivencia para todas las variables por el método de Kaplan-Meier en relación con las metástasis a distancia y con la técnica de Log-Rank se establecieron las comparaciones en los subgrupos de cada variable para identificar posibles predictores. Resultados: de las 333 pacientes 59 presentaron metástasis a distancia, con una media de presentación de 14,49 años. Fueron muy significativos (p125cm3, los subtipos histológicos ductal-lobular infiltrante, metaplásico epidermoide y apocrino, el grado histológico 3 y de sus componentes el conteo mitótico >22 mitosis en 10 campos de mayor aumento y el pleomorfismo nuclear 3 y fueron significativas (0,005≤p

Objective: Pheochromocytomas (PHEOs) and paragangliomas (PGLs) arise from adrenal extra-adrenal paraganglia. They often secrete catecholamine and approximately 1/3 are hereditary. Almost 50% of metastatic PGLs are caused by mutations in the succinate dehydrogenase gene, particularly in subunits B and D (SDHB/D). These tumors remain a diagnostic and therapeutic challenge due to a limited number of effective treatment options. To our best knowledge, we present the first report that uses induction therapy to elicit a significant response in both primary and metastatic lesions, followed by complete surgical resection of a primary SDHB-related PGL. Case description: A 45-year-old female presented with an 8.5 cm unresectable primary SDHB-related PGL with multiple bone metastases. She had an initial blood pressure of 210/130 mmHg and a heart rate of 150 bpm. Initially, she was treated with cisplatin, vinblastine, and dacarbazine (CVD) chemotherapy and lanreotide (120 mg/sc/28d), which resulted in tumor shrinkage. The patient later progressed, and monotherapy with a shortened interval of lanreotide (120 mg/sc/14d) was initiated. Following 2.5 months at this interval, the patient nearly achieved complete control of her clinical symptoms, and experienced a 30% reduction in 18F-flurodeoxyglucose uptake in her primary and metastatic lesions. Following the surgical resection of her primary PGL, all prior antihypertensive medications were stopped. Conclusions: CVD, together with a 14-day regimen of high dose lanreotide, may be an effective treatment option for SDHB-related metastatic PGLs. Therefore, further evaluation of somatostatin analogues, preferably lanreotide, in the treatment of metastatic SDHB-related PGLs is warranted. We believe that ours is the first report detailing the successful use of lanreotide treatment prior to a surgical rescue after systemic treatment. Keywords: Paraganglioma, Lanreotide, SDHB, Metastatic

Downregulation of the T cell system has been proposed as a mechanism to block immunity in colonic cancer (CC). However, little has been studied about circulating αβ and γδ T cells and their immunological status in newly diagnosed patients. The aim of this study was to characterize the αβ and γδ T cell subsets in peripheral blood of patients with CC matched with healthy volunteers. In this prospective case-control study, blood samples were obtained from 96 patients with newly diagnosed treatment-naïve infiltrating colonic adenocarcinoma and 48 healthy volunteers. Pathological report at surgery was obtained from all CC patients. A significant decrease in CD3+ γδ T cells and CD3+CD8+ γδ T cells (p

Lung metastasis is the major cause of death in osteosarcoma patients. However, molecular mechanisms underlying this metastasis remain poorly understood. To identify key molecules related with pulmonary metastasis of pediatric osteosarcomas, we analyzed high‐throughput miRNA expression in a cohort of 11 primary tumors and 15 lung metastases. Results were further validated with an independent cohort of 10 primary tumors and 6 metastases. In parallel, we performed immunohistochemical analysis of activated signaling pathways in 36 primary osteosarcomas. Only phospho‐AKT associated with lower overall survival in primary tumors, supporting its role in osteosarcoma progression. CTNNB1 expression also associated with lower overall survival but was not strong enough to be considered an independent variable. Interestingly, miR‐200c was overexpressed in lung metastases, implicating an inhibitory feed‐back loop to PI3K‐AKT. Moreover, transfection of miR200c‐mimic in U2‐OS cells reduced phospho‐AKT levels but increased cellular migration and proliferation. Notably, miR‐200c expression strongly correlated with miR‐141 and with the osteogenic inhibitor miR‐375, all implicated in epithelial to mesenchymal transition. These findings contrast epithelial tumors where reduced miR‐200c expression promotes metastasis. Indeed, we noted that osteosarcoma cells in the lung also expressed the epithelial marker CDH1, revealing a change in their mesenchymal phenotype. We propose that miR‐200c upregulation occurs late in osteosarcoma progression to provide cells with an epithelial phenotype that facilitates their integration in the metastatic lung niche. Thus, our findings identify phospho‐AKT in the primary tumor and miR‐200c later during tumor progression as prognostic molecules and potential therapeutic targets to prevent progression and metastasis of pediatric osteosarcomas.

After the publication of this work [1] the authors have reported that in Table 3 The letter “T” in columns 5 and 7 should not be there.

Metastatic pheochromocytoma and paraganglioma (mPHEO/PGL) are frequently associated with succinate dehydrogenase B ( SDHB ) mutations. Cyclophosphamide-dacarbazine-vincristine (CVD) regimen is recommended as standard chemotherapy for advanced mPHEO/PGL. There is limited evidence to support the role of metronomic schemes (MS) of chemotherapy in mPHEO/PGL treatment. We report 2 patients with SDHB -related mPGL who received a regimen consisting of MS temozolomide (TMZ) and high-dose lanreotide after progression on both CVD chemotherapy and high-dose lanreotide. Molecular profiling of the tumor tissue from both patients revealed hypermethylation of the O6-methylguanine-DNA-methyltransferase ( MGMT ) promoter. In one patient, progression-free survival was 13 months and the second patient remained under treatment after 27 months of stabilization of metabolic response of his disease. Treatment was well tolerated, and adverse effects were virtually absent. A modification in the scheme of TMZ from standard schemes to MS is safe and feasible and can be considered in patients with progressive mPHEO/PGL refractory to dacarbazine in standard doses.

The clinical and morphological prognostic factors were evaluated regarding their predictive capacity on the evolutionary appearance of death in patients with invasive breast carcinoma. Material and method: a nonrandom sample of 373: it was defined a women with invasive breast carcinomas by a period of 15 years (1992-2007). Results: the death event occurred more frequently before the first five years of evolution. 87 patients died with an overall survival of 76.7% and an average of 13.53 years. From the evaluated factors the most contributed to overall survival were: tumor position in the lower inner quadrant, with 21patients -10 of them died, 52.4% overall survival and an average of 4.35 years; bilateralism, with 12 patients –seven of them died, 41.7% overall survival and an average of 7.27 years; tumor volume of more than 125cm3, with six patients – three died, 50.0% survival and an average of 8.87 years; nodal status with more than three metastatic nodes, with 77 patients -33 died, 57.1% overall survival and an average of 8.54 years; the presence of periganglionar infiltration tumor, with 72 patients -31 dead, 56.9% of overall survival and an average of 8.63 years; involvement of level III, with 53 patients died -20 died, 62.3% survival and an average of 9.47 years; the modified histological grade poorly differentiated, with 116 patients -41 died, 64.7% overall survival and an average of 11.84 years and the mitotic count with more than 22 mitoses per field further increase with 65 cases -26 dead, 60.0% overall survival and an average of 11.24 years. Conclusion: tumor volume can be considered a morphological prognosis factor determinant in overall survival in infiltrating breast cancer.

Immunohistochemical prognosis factors or moleculars and qualifying constructs were evaluated for the contribution to overall survival, and adverse events. It was defined a nonrandom sample of 373 women with invasive breast carcinomas operated on a 15-year period (1992-2007). Results: From the evaluated factors the most contributed to overall survival were the p53 with seven patients (three deaths and 57.1% survival to the value 2 +) and 20 cases (five deaths and 75.0% overall survival for the value 3 +); the Bcl2 gene with 47 patients -13 died and 72.3% survival rate for the zero value and 40 cases (five deaths and 87.5% survival rate for the value 1 +); Top Nottingham prognostic index of 5.4 with 91 patients Nottingham greater forecasting 5.4 with 91 patients -38 died, 69.7% overall survival and an average of 9.33 years, stage IV according to the classification TNMp with a female patient -who died, with an average of 1.49 years, followe by the stage IIIC with 31 patients -21 of them dead, 32.3% survival and an average of 4.68 years, the N3 component with 32 patients -22 died, 31.3% overall survival and an average of 4.58 years; the M1component with a female patient -who died, with an average of 1.49 years, local recurrence with 17 patients –nine of them died, 47.1% survival and an average of 9.57 years, and distant metastases with 51 patients - 59 dead, with an overall survival of 13.6% and an average of 5.16 years. Conclusion: p53 positive values and negative ​​of Bcl-2, the largest group prognosis of 5.4, stages IV and III C, N3 and M1 components, local recurrence and distant metastases were most influenced in overall survival.

To determine the frequency of breast cancer (BC) patients with hereditary risk features in a wide retrospective cohort of patients in Spain.a retrospective analysis was conducted from 10,638 BC patients diagnosed between 1998 and 2001 in the GEICAM registry "El Álamo III", dividing them into four groups according to modified ESMO and SEOM hereditary cancer risk criteria: Sporadic breast cancer group (R0); Individual risk group (IR); Familial risk group (FR); Individual and familial risk group (IFR) with both individual and familial risk criteria.7,641 patients were evaluable. Of them, 2,252 patients (29.5%) had at least one hereditary risk criteria, being subclassified in: FR 1.105 (14.5%), IR 970 (12.7%), IFR 177 (2.3%). There was a higher frequency of newly diagnosed metastatic patients in the IR group (5.1% vs 3.2%, p = 0.02). In contrast, in RO were lower proportion of big tumors (> T2) (43.8% vs 47.4%, p = 0.023), nodal involvement (43.4% vs 48.1%, p = 0.004) and lower histological grades (20.9% G3 for the R0 vs 29.8%) when compared to patients with any risk criteria.Almost three out of ten BC patients have at least one hereditary risk cancer feature that would warrant further genetic counseling. Patients with hereditary cancer risk seems to be diagnosed with worse prognosis factors.

В останні роки в науковій пресі з'явилисm численні дані про молекулярну біологію раку молочної залози. У зв'язку з цим, гістологічна класифікація ВООЗ (2004 ) виявилась застарілою. Сьогодні більшість відомостей про прогноз, лікування та клінічний результат захворювання грунтується на результатах молекулярних і біологічних досліджень останніх років. Розглянуто сучасні підходи до питань гістопатологічної діагностики раку молочної залози на основі нових технологій. Обговорено рекомендації з імуногістохімічного тестування рецепторів естрогенів і прогестерону , а також рецепторів до епідермального фактора росту 2 (HERB2) у зразках пухлинної тканини. Запропоновано алгоритм оцінки отриманих даних, включаючи позитивні, спірні й негативні результати, що стосуються експресії білків HERB2 і ампліфікації гена. На основі характеру експресії генів, вивченого за технологією мікрочіпів з подальшою ієрархічної кластеризацією РНК нативної тканини, виділено нові генетичні профілі пухлини. Показано можливість застосування методів імуногістохімії в тканинах, залитих у парафін. Рекомендовано імуногістохімічну панель для діагностики наступних типів раку молочної залози: люмінальної А (Luminal А) , люмінальної В (Luminal В), базальноподібний і HER2/neu.Заключна частина роботи присвячена варіантам раку молочної залози з генетичною сприйнятливістю до BRCA1 і 2. Такі пухлини вкрай агресивні, характеризуються високим ступенем злоякісності і мають несприятливий прогноз. Проаналізовано нові підходи до терапії захворювання.

Objetivo: Caracecterizar morfológica e inmunohitoquimicamente el carcinoma apocrino mamamario. Materiales y métodos: Este estudio toma 24 casos diagnosticados como carcinomas apocrinos o con rasgos apocrinos, clasificándolos como puros y mixtos, reconociendo la presencia de componentes in situ asociados, para evaluar la expresión inmunohistoquímica del receptor de andrógenos (AR), receptor de estrógenos (ER), receptor de progesterona (PR), gross cistyc disease proteína (GCDFP-15), BCL2,KI67 y ERB-2 tanto en las áreas infiltrantes como intraepiteliales, estudiando adicionalmente la amplificación génica del ERB-2 en el cromosoma 17 por métodos de FISH. Resultados: Los tumores clasificados como puros correspondieron a 11 casos que expresan la siguiente positividad inmunohistoquímica en las áreas infiltrativas: AR (100%), ER (18%), PR (18%), GCDFP (63%), BCL2 (54%), KI67 (28%) y ERB-2(28%) con una positividad de GCDFP-15(100%) en la áreas in situ. Los tumores mixtos correspondientes a 13 casos mostraron la siguiente positividad inmunohistoquímica: AR (58%), ER (46%), PR (46%), GCDFP-15 (50%), BCL2 (33%), KI67 (58%) y ERB-2(16%), con una positividad de GCDFP-15 (100%) en las áreas in situ. Las áreas in situ expresan GCDFP-15 en todos los casos con una reducción de la expresión en las zonas de infiltración del mismo al 63 y 50% en los tumores puros y mixtos, respectivamente. Conclusiones: Los carcinomas mamarios apocrinos puros deben ser distinguidos de los mixtos mediante un examen morfológico detallado y por su perfil inmunohistoquímico (AR+, ER-, PR-/GCDFP+, BCL2+/-, KI67-, ERB2/-).

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 104 features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.

OBJECTIVE:Growing evidences indicate that the histone methyltransferase EZH2 (enhancer of zeste homolog 2) may be an appropriate therapeutic target in some tumors. Indeed, a high expression of EZH2 is correlated with poor prognosis and metastasis in many cancers. In addition, 3-Deazaneplanocin A (DZNep), an S-adenosyl-L homocysteine hydrolase inhibitor which induces EZH2 protein depletion, leads to cell death in several cancers and tumors. The aim of this study was to determine whether an epigenetic therapy targeting EZH2 with DZNep may be also efficient to treat chondrosarcomas. METHODS:EZH2 expression was determined by immunohistochemistry and western-blot. Chondrosarcoma cell line CH2879 was cultured in the presence of DZNep, and its growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using western-blot. Cell migration was assessed by wound healing assay. RESULTS:Chondrosarcomas (at least with high grade) highly express EZH2, at contrary to enchondromas or chondrocytes. In vitro, DZNep inhibits EZH2 protein expression, and subsequently reduces the trimethylation of lysine 27 on histone H3 (H3K27me3). Interestingly, DZNep induces cell death of chondrosarcoma cell lines by apoptosis, while it slightly reduces growth of normal chondrocytes. In addition, DZNep reduces cell migration. CONCLUSION:These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treat chondrosarcomas.