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The major changes in hormone levels that occur through the menstrual cycle have been postulated to affect the expression of hormone-regulated and proliferation-associated genes (PAGs) in premenopausal ER+ breast cancer. Whilst previous studies have demonstrated differences in gene expression, here, we investigated if there are within patient changes in the expression of oestrogen- and progesterone-regulated genes (ERGs and PRGs) and PAGs in ER+ breast cancer during the menstrual cycle. Samples from 96 patients in two independent prospective studies of the effect of menstrual cycle on ER+ breast cancer were used. Plasma hormone measurements were used to assign tumours to one of three pre-defined menstrual cycle windows: W1 (days 27–35 and 1–6; low oestradiol and low progesterone), W2 (days 7–16; high oestradiol and low progesterone) and W3 (days 17–26; intermediate oestradiol and high progesterone). RNA expression of 50 genes, including 27 ERGs, 11 putative PRGs and seven PAGs was measured. The AvERG (geomean of PGR, GREB1, TFF1 and PDZK1) was used as a composite measure of ERG expression and showed significant changes between the three windows of the menstrual cycle increasing over 2.2-fold between W1 and W2 and decreasing between W2 and W3 and between W3 and W1. Proliferation gene expression also varied significantly, following the same pattern of changes as ERG expression, but the changes were of lower magnitude (1.4-fold increase between W1 and W2). Significant changes in the expression of eight individual ERGs, including GREB1, PGR and TFF1, and two PAGs were observed between W1 and either W2 or W3 with all genes showing higher levels in W2 or W3 (1.3–2.4-fold; FDR 0.016–0.05). The AvProg, a composite measure of PRG expression, increased significantly (1.5-fold) in W3 compared to W1 or W2 but no significant changes were observed for individual PRGs. In conclusion, we observed significant changes in ERG, PRG and PAG expression in ER+ breast tumours during the menstrual cycle that may affect the assessment and interpretation of prominent biomarkers (e.g. PgR) and commonly used multigene prognostic signatures in premenopausal ER+ 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.

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TO THE EDITOR: Cui et al 1 discussed the biology and etiology of estrogen receptor (ER) -positive/progesterone receptor (PR) -negative breast cancers highlighting recent data on molecular crosstalk between ER and growth factor signaling pathways also demonstrating how PR might be a useful marker of these activities.In their review, Cui et al missed our report on the negative association between PR and HER-2 in 1,104 women with an ERpositive breast cancer using semiquantitative immunohistochemistry with monoclonal antibodies NCL-ER-6F11/2 for ER, NCL-PgR-312 for PR, and CB11 for HER-2 (Novocastra Laboratories, Newcastleupon-Tyne, United Kingdom). The negative association between PR and HER-2 is only seen after age 45; in younger women, HER-2 positive ER-positive breast tumors were as likely PR-positive with similar median PR levels as HER-2 negative tumors; patients with triple-positive cancer (that is, ER-, PR-, and HER-2-positive cancer) were younger than the others. 2 This suggests cross talk is age related and may explain why such breast cancers remain sensitive to antiestrogens in young women. 3 In ER-positive breast cancers, PR-negative tumors are more aggressive than PR-positive cancers. Cui et al stated that the reason for the poor clinical course of PR-negative tumors is unclear. We previously reported that a negative PR in women with an ER-positive breast cancer predicts lymph node invasion independent of other predictors of lymph node invasion especially in younger women. 4 Consequently, we repeated our analysis for a negative PR as a predictor for a positive lymph node status in ER-positive breast cancers taking tumor size and tumor grade into account in an updated cohort of 1,472 previously untreated and consecutive women with a unilateral invasive breast cancer that was surgically treated between 2000 and 2004 in one center. With multivariate logistic regression analysis using stepwise selection in the LOGISTIC procedure from the SAS software package version 9.1 (SAS Institute, Cary, NC), the following variables were first considered for inclusion in the model predicting the nodal status: PR (PR-positive v PR-negative), tumor grade (grade 3 v grade 1 and 2) and maximal tumor size (Ͼ 20 mm v Յ 20 mm). After variable selection, the final model retained only variables having a coefficient significantly different from zero (P Ͻ .05; Wald 2 statistic). 5 As described in Table 1 and in agreement with previously reported findings from a recent paper 6 of Cui et al's group, this approach did not retain PR as a predictor for a positive lymph node status when the model was derived from all ER-positive breast cancer patients. In our analyses, considering women 50 years or younger at the time of breast cancer diagnosis, PR, tumor grade, and tumor size were all independent predictors for a positive lymph node status. In women older than age 50, only tumor size and tumor grade predicted a positive lymph node status.In analyses of the prognostic effect of a negative PR in women with an ER-positive breast cancer...