Molecular profiling of aromatase inhibitor sensitive and resistant ER+HER2- postmenopausal breast cancers.

Aromatase inhibitors (AIs) reduce recurrences and mortality in postmenopausal patients with oestrogen receptor positive (ER+) breast cancer (BC), but >20% of patients will eventually relapse. Given the limited understanding of intrinsic resistance in these tumours, here we conduct a large-scale molecular analysis to identify features that impact on the response of ER + HER2- BC to AI. We compare the 15% of poorest responders (PRs, n = 177) as measured by proportional Ki67 changes after 2 weeks of neoadjuvant AI to good responders (GRs, n = 190) selected from the top 50% responders in the POETIC trial and matched for baseline Ki67 categories. In this work, low ESR1 levels are associated with poor response, high proliferation, high expression of growth factor pathways and non-luminal subtypes. PRs having high ESR1 expression have similar proportions of luminal subtypes to GRs but lower plasma estradiol levels, lower expression of estrogen response genes, higher levels of tumor infiltrating lymphocytes and immune markers, and more TP53 mutations.

HER2-enriched subtype and novel molecular subgroups drive aromatase inhibitor resistance and an increased risk of relapse in early ER+/HER2+ breast cancer.

BACKGROUND: Oestrogen receptor positive/ human epidermal growth factor receptor positive (ER+/HER2+) breast cancers (BCs) are less responsive to endocrine therapy than ER+/HER2- tumours. Mechanisms underpinning the differential behaviour of ER+HER2+ tumours are poorly characterised. Our aim was to identify biomarkers of response to 2 weeks' presurgical AI treatment in ER+/HER2+ BCs. METHODS: All available ER+/HER2+ BC baseline tumours (n=342) in the POETIC trial were gene expression profiled using BC360™ (NanoString) covering intrinsic subtypes and 46 key biological signatures. Early response to AI was assessed by changes in Ki67 expression and residual Ki67 at 2 weeks (Ki672wk). Time-To-Recurrence (TTR) was estimated using Kaplan-Meier methods and Cox models adjusted for standard clinicopathological variables. New molecular subgroups (MS) were identified using consensus clustering. FINDINGS: HER2-enriched (HER2-E) subtype BCs (44.7% of the total) showed poorer Ki67 response and higher Ki672wk (p<0.0001) than non-HER2-E BCs. High expression of ERBB2 expression, homologous recombination deficiency (HRD) and TP53 mutational score were associated with poor response and immune-related signatures with High Ki672wk. Five new MS that were associated with differential response to AI were identified. HER2-E had significantly poorer TTR compared to Luminal BCs (HR 2.55, 95% CI 1.14-5.69; p=0.0222). The new MS were independent predictors of TTR, adding significant value beyond intrinsic subtypes. INTERPRETATION: Our results show HER2-E as a standardised biomarker associated with poor response to AI and worse outcome in ER+/HER2+. HRD, TP53 mutational score and immune-tumour tolerance are predictive biomarkers for poor response to AI. Lastly, novel MS identify additional non-HER2-E tumours not responding to AI with an increased risk of relapse. FUNDING: Cancer Research UK (CRUK/07/015).

Human epidermal growth factor receptor-2 and endocrine resistance in hormone-dependent breast cancer.

Endocrine therapies are the main treatment strategies for the clinical management of hormone-dependent breast cancer. Despite prolonged time to recurrence in the adjuvant setting and the initial clinical responses in the metastatic setting, many patients eventually encounter tumour relapse due to acquired resistance to these agents. Other patients experience a lack of tumour regression at the beginning of treatment indicating de novo resistance that significantly limits its efficacy in the clinic. There is compelling evidence that human epidermal growth factor receptor-2 (HER2) overexpression contributes to resistance to endocrine therapies in oestrogen receptor-positive (ER+) breast cancer. ER+/HER2+ tumours comprise about 10% of all breast cancer cases and about 60% of the whole set of HER2+ tumours. Most patients with primary ER+/HER2+ disease will receive antibody-based HER2-targeted therapy, but this is generally for no more than one year while endocrine treatment is usually for at least 5 years. A number of HER2-kinase inhibitors are also now in clinical use or in clinical trials, and the interaction of these with endocrine treatment may differ from that of antibody treatment. In this review article, we aim to summarise knowledge on molecular mechanisms of breast cancer resistance to endocrine therapies attributable to the impact of HER2 signalling on endocrine sensitivity, to discuss data from clinical trials addressing the role of HER2 in the development of endocrine resistance in the metastatic, neoadjuvant and adjuvant settings and to explore rational new therapeutic strategies.

Impact of the menstrual cycle on commercial prognostic gene signatures in oestrogen receptor-positive primary breast cancer.

PURPOSE: Changes occur in the expression of oestrogen-regulated and proliferation-associated genes in oestrogen receptor (ER)-positive breast tumours during the menstrual cycle. We investigated if Oncotype® DX recurrence score (RS), Prosigna® (ROR) and EndoPredict® (EP/EPclin) prognostic tests, which include some of these genes, vary according to the time in the menstrual cycle when they are measured. METHODS: Pairs of test scores were derived from 30 ER-positive/human epidermal growth factor receptor-2-negative tumours sampled at two different points of the menstrual cycle. Menstrual cycle windows were prospectively defined as either W1 (days 1-6 and 27-35; low oestrogen and low progesterone) or W2 (days 7-26; high oestrogen and high or low progesterone). RESULTS: The invasion module score of RS was lower (- 10.9%; p = 0.098), whereas the ER (+ 16.6%; p = 0.046) and proliferation (+ 7.3%; p = 0.13) module scores were higher in W2. PGR expression was significantly increased in W2 (+ 81.4%; p = 0.0029). Despite this, mean scores were not significantly different between W1 and W2 for any of the tests and the two measurements showed high correlation (r = 0.72-0.93). However, variability between the two measurements led to tumours being assigned to different risk categories in the following proportion of cases: RS 22.7%, ROR 27.3%, EP 13.6% and EPclin 13.6%. CONCLUSION: There are significant changes during the menstrual cycle in the expression of some of the genes and gene module scores comprising the RS, ROR and EP/EPclin scores. These did not affect any of the prognostic scores in a systematic fashion, but there was substantial variability in paired measurements.

A simple digital image analysis system for automated Ki67 assessment in primary breast cancer.

AIMS: Ki67 is a well-established immunohistochemical marker associated with cell proliferation that has prognostic and predictive value in breast cancer. Quantitative evaluation of Ki67 is traditionally performed by assessing stained tissue slides with light microscopy. Automated image analysis systems have become available and, if validated, could provide greater standardisation and improved precision of Ki67 scoring. Here, we aimed to evaluate the use of the Cognition Master Professional Suite (CogM) image analysis software, which is a simple system for scoring Ki67 in primary breast cancer samples. METHODS AND RESULTS: Sections from 94 core-cut biopsies, 20 excision specimens and 29 pairs of core-cut biopsies and excision specimens were stained for Ki67 with MIB1 antibody and the Dako EnVision FLEX Detection System. Stained slides were scanned to convert them to digital data. Computer-based Ki67 scoring was performed with CogM. Manual Ki67 scoring assessment was conducted on previously stained sections from the same biopsies with a clinically validated system that had been calibrated against the risk of recurrence. A high correlation between manual and digital scores was observed [rCores  = 0.92, 95% confidence interval (CI) 0.87-0.94, P < 0.0001; rExcisions  = 0.95, 95% CI 0.86-0.98, P < 0.0001] and there was no significant bias between them (P = 0.45). There was also a high correlation of Ki67 scores between paired core-cut biopsies and excision specimens when CogM was used (r = 0.78, 95% CI 0.59-0.89, P < 0.0001). CONCLUSIONS: CogM image analysis allows for standardised automated Ki67 scoring that accurately replicates previously clinically validated and calibrated manual scores.

Interleukin-1 Receptor-Associated Kinase 4 Deficiency in a Greek Teenager.

BACKGROUND: Human interleukin- (IL-) 1 receptor-associated kinase 4 (IRAK-4) deficiency is a recently described primary immunodeficiency. It is a rare, autosomal recessive immunodeficiency that impairs toll/IL-1R immunity, except for the toll-like receptor (TLR) 3- and TLR4-interferon alpha (IFNA)/beta (IFNB) pathways. Case Report. We report the first patient in Greece with IRAK-4 deficiency. From the age of 8 months, she presented with recurrent infections of the upper and lower respiratory tract and skin abscesses. For this, she had been repeatedly hospitalized and treated empirically with intravenous antibiotics. No severe viral, mycobacterial, or fungal infections were noted. Her immunological laboratory evaluation revealed low serum IgA and restored in subsequent measurements; normal IgG, IgM, and IgE; and normal serum IgG subclasses. Peripheral blood immunophenotyping by flow cytometry and dihydrorhodamine (DHR) test revealed normal counts. She was able to make functional antibodies against vaccine antigens, including tetanus and diphtheria. She was administered with empirical IgG substitution for 5 years until the age of 12 years, and she has never experienced invasive bacterial infections so far. DNA analysis revealed a heterozygous variant in the patient: c.823delT (p.S275fs ∗ 13 at protein level) in the IRAK4 gene. CONCLUSIONS: The importance of clinical suspicion is emphasized in order to confirm the diagnosis by IRAK4 gene sequencing and provide the appropriate treatment for this rare primary immunodeficiency, as soon as possible.

The Role of TLR4 Asp299Gly and TLR4 Thr399Ile Polymorphisms in the Pathogenesis of Urinary Tract Infections: First Evaluation in Infants and Children of Greek Origin.

Urinary tract infections are one of the most common and serious bacterial infections in a pediatric population. So far, they have mainly been related to age, gender, ethnicity, socioeconomic level, and the presence of underlying anatomical or functional, congenital, or acquired abnormalities. Recently, both innate and adaptive immunities and their interaction in the pathogenesis and the development of UTIs have been studied. The aim of this study was to assess the role and the effect of the two most frequent polymorphisms of TLR4 Asp299Gly and Thr399Ile on the development of UTIs in infants and children of Greek origin. We studied 51 infants and children with at least one episode of acute urinary tract infection and 109 healthy infants and children. We found that 27.5% of patients and 8.26% of healthy children carried the heterozygote genotype for TLR4 Asp299Gly. TLR4 Thr399Ile polymorphism was found to be higher in healthy children and lower in the patient group. No homozygosity for both studied polymorphisms was detected in our patients. In the group of healthy children, a homozygote genotype for TLR4 Asp299Gly (G/G) as well as for TLR4 Thr399Ile (T/T) was showed (1.84% and 0.92 respectively). These results indicate the role of TLR4 polymorphism as a genetic risk for the development of UTIs in infants and children of Greek origin.

Altered Expression of TLR2 and TLR4 on Peripheral CD14+ Blood Monocytes in Children with Urinary Tract Infection.

Urinary tract infection (UTI) is the second most common bacterial infection, after otitis media, in infants and children. The mechanisms of disease susceptibility and the role of immunity in the pathogenesis of UTI in children have been evaluated. In recent years, Toll-Like Receptors (TLRs) have been recognized as specific components of the innate immune system constituting important mediators in host immune recognition. The aim of the present study was to determine ΤLR2 and TLR4 expression during the acute phase of UTI in infants and children by measuring the CD14/TLR2 and CD14/TLR4 expression on monocytes. We also attempted to compare the TLRs expression with the immunological status of the patients to healthy children. The study group consisted of 60 children (36 females and 24 males) and the control group included 60 age-matched pediatric subjects (27 females and 33 males). In our study, no antibody deficiency was found either in the children with UTI or in healthy subjects. There might be a connection between low IgA, IgG, and IgG subclasses serum levels and UTI as there was a statistically significant difference between patients and healthy children. A higher expression of CD14/TLR2 was revealed in patients (90,07%) compared to controls (85,48%) as well as CD14/TLR4 in patients (90,53%) compared to controls (87,25%) (statistically significant difference, p < 0,05). The results of this study could provide new understanding of UTIs' pathogenesis in children.