Image analysis of immune cell patterns in the human mammary gland during the menstrual cycle refines lymphocytic lobulitis

Purpose

To improve microscopic evaluation of immune cells relevant in breast cancer oncoimmunology, we aim at distinguishing normal infiltration patterns from lymphocytic lobulitis by advanced image analysis. We consider potential immune cell variations due to the menstrual cycle and oral contraceptives in non-neoplastic mammary gland tissue.

Methods

Lymphocyte and macrophage distributions were analyzed in the anatomical context of the resting mammary gland in immunohistochemically stained digital whole slide images obtained from 53 reduction mammoplasty specimens. Our image analysis workflow included automated regions of interest detection, immune cell recognition, and co-registration of regions of interest.

Results

In normal lobular epithelium, seven CD8\(^{+}\) lymphocytes per 100 epithelial cells were present on average and about 70% of this T-lymphocyte population was lined up along the basal cell layer in close proximity to the epithelium. The density of CD8\(^{+}\) T-cell was 1.6 fold higher in the luteal than in the follicular phase in spontaneous menstrual cycles and 1.4 fold increased under the influence of oral contraceptives, and not co-localized with epithelial proliferation. CD4\(^{+}\) T-cells were infrequent. Abundant CD163\(^{+}\) macrophages were widely spread, including the interstitial compartment, with minor variation during the menstrual cycle.

Conclusions

Spatial patterns of different immune cell subtypes determine the range of normal, as opposed to inflammatory conditions of the breast tissue microenvironment. Advanced image analysis enables quantification of hormonal effects, refines lymphocytic lobulitis, and shows potential for comprehensive biopsy evaluation in oncoimmunology.

     

The effect of age and menstrual cycle upon proliferative activity of the normal human breast

The aim of this study was to determine the proliferative activity within the epithelial cells of the normal human breast in 122 patients (6 reduction mammoplasties and 116 fibroadenoma excisions) in relation to age and the phase of the menstrual cycle. Thirty three of the patients were on oral contraceptives and 33 were parous. Thin tissue slices were incubated with tritiated thymidine and processed for autoradiography. Other samples were fixed directly and prepared for histology. The labelling, mitotic and apoptotic indices (LI, MI and AI) were determined and all illustrated considerable variability. The labelling indices are significantly (P less than 0.05) influenced by both patient age and stage during the menstrual cycle and ranged from 0-11.5%. Maximum LI values were obtained on the 20.8th day of the cycle. A square root transformation of the data was used to reduce the skewness of the data to a more normal distribution. The square root of the LI declined by 0.22 per decade. The mitotic data showed similar significant (P less than 0.05) correlations against age and day of cycle with a peak on the 21.5th day of the cycle, a decline by 0.072 per decade and a range from 0-0.6%. The data for apoptotic cells were less clearly influenced by the stage of the menstrual cycle but showed a significant (P less than 0.5) decline with age. The AI in parous patients was significantly higher than that in non-parous patients. There was no significant effect of oral contraceptives on any of the parameters measured when age and stage of cycle were taken into account. The considerable variability in the data could not be fully accounted for by either technical factors, the age of the patients, or the day of the cycle. We conclude that proliferation is negatively related to age and is influenced by the menstrual cycle but that additional as yet unknown factors must account for a large part of the variability seen in the data.     

Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium

Introduction

Although estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ER–, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects will bring insight into the cellular origin of breast cancer subtypes.

Methods

We used fluorescence-activated cell sorting of primary human mammary epithelial cells along with in vitro and in vivo functional assays to examine the hierarchic relation between cells with aldehyde dehydrogenase enzymatic activity (ALDH+ cells) and ER+ cells in the normal human breast epithelium. We assessed the proliferation and lineage differentiation potential of these cells in vitro and in vivo. A gene reporter assay was used to separate live ER+ and ER– mammary epithelial cells. With shRNA-mediated knockdown, we investigated the role of ALDH isoforms in the functionality of mammary epithelial progenitor cells.

Results

We describe a cellular hierarchy in the normal human mammary gland in which ER–/ALDH+ cells with functional properties of stem/progenitor cells generate ER+ progenitor cells, which in turn give rise to cells of luminal lineage. We show that the ALDH1A1 isoform, through its function in the retinoic acid metabolism, affects the proliferation and/or early differentiation of stem/progenitor cells and is important for branching morphogenesis.

Conclusions

This study presents direct evidence that ER+ cells are generated by ER–/ALDH+ stem/progenitor cells. We also show that ER+ cells are able to generate cell progeny of luminal lineage in vitro and in vivo. Loss of ALDH1A1 function impairs this process, as well as branching morphogenesis and clonogenicity in suspension culture. This latter effect is reversed by treatment with retinoic acid.     

Proliferation of the breast epithelium in relation to menstrual cycle phase,hormonal use,and reproductive factors

Summary The proliferative rate in normal breast epithelium from 58 women undergoing reduction mammoplastics was studied using the formalin resistant antibody Ki-S5, and related to age at operation, menstrual cycle phase, family history of breast cancer, height and weight, parity, and hormonal use.The breast tissue from women operated on in the luteal menstrual cycle phase (day 15–28 among oral contraceptive (OC) users) had significantly higher proliferative rate than breast tissue removed from women in the follicular phase (day 1–14) (p = 0.01). Among women presently exposed to hormones, those with a positive family history of breast cancer among first and second degree relatives had significantly higher values than cases without such history (p = 0.02). Weight was not significantly related to proliferation rate, while a short height was associated with a significantly higher proliferation rate (p = 0.04). Women who used OCs before the first full-term pregnancy (FFTP) had a significantly higher proliferation rate compared with never users or late users (p = 0.04). No significant difference was seen between parous versus nulliparous women.The results from the univariate analysis persisted in multivariate models. An especially high proliferation rate was seen in young women with both a positive family history and present hormonal use (p = 0.001). Overall, it was found that young women had a non-significantly higher proliferation rate than older women (p = 0.10). Due to small sample size, these results must be regarded as preliminary, especially in the subgroup analyses.     

Effect of dietary calcium and cholic acid on the proliferative indices of murine coionic epithelium

The effect of dietary calcium on the cholic acid-induced coloncell proliferative response was investigated with C57BL/6J mice.The control diets were formulated on the basis of the AIN-76diet with the levels of calcium set at 0.1, 0.5 or 1.0% by weightand the ratio of Ca: P at approximately 1: 1. Cholic add wasadded at 0.1% and 0.25% by weight. Hie cytokinetk parametersof the colons were evaluated after 2 weeks of feeding by themetaphase arrest technique and by autoradiogra-phy. Mitoticactivity was found to monotonically decrease with increasingcalcium level from 0.1 to 1.0% for both the cholic acid andcontrol groups, cholic acid-fed groups exhibiting significantlyhigher mitotic activity than the control groups. The effectof calcium (0.1 versus 1.0%) was evident throughout the 24-hperiod with both the control and cholic acid-supplemented diets.Autoradiographk analyses of the colons yielded similar results.Among the controls, the 0.1% Ca group exhibited significantlyhigher (P0.05) crypt height, higher whole crypt labelling indexand higher labelling indices at various positions along thecrypt height than the 1.0% Ca group. Cholic acid-fed groupshad higher values for crypt height, whole crypt labelling indicesand the position of the uppermost labelled cells than the controlgroups. The level of calcium affected the bile acid-inducedcell proliferation in the colonic epithelium; the groups fed0.1% Ca + cholic add had a markedly higher whole crypt labellingindex, a higher position of the uppermost labelled cells andhigher labelling indices at various cell positions along thecrypt column compared with the 1.0% Ca + cholic add group. Thepresent study thus demonstrated that increased dietary cakiumalleviates the mitogenic effect of a cholic add supplementeddiet on the colonic epithelium.     

Ovarian tumor marker HE4 is differently expressed during the phases of the menstrual cycle in healthy young women

The objective of the present study was to investigate in healthy young women the fluctuations in serum concentration of human epididymal secretory protein human epididymis-specific protein 4 (HE4) and CA125 during the phases of the menstrual cycle and the correlation between HE4 and CA125 values and age. Forty women with regular menstrual cycles were included in the study. Pelvic and transvaginal ultrasound were performed in order to exclude ovarian pathologies. Blood samples were collected at follicular (FP), ovulatory (OP), and luteal (LP) phases of the hormonal cycle. The values of HE4 (expressed as picomoles per liter) observed were (mean ± SEM) 39.1 ± 1.1 (FP), 45.3 ± 1.19 (OP), and 42.0 ± 1.3 (LP). The difference between FP and OP was statistically significant (p = 0.0002). By contrast, serum CA125 levels (expressed as units per milliliter) were 14.35 ± 0.66 (FP), 13.15 ± 0.54 (OP), and 13.70 ± 0.54 (LP), respectively. The levels of HE4 observed in serum samples of women below 35 years were 37.5 ± 1.28 in the FP, 46.6 ± 1.4 in the OP, and 42.8 ± 1.49 in the LP. In this group, a statistically significant difference was observed in the FP compared with the OP (p < 0.0001), whereas no statistically significant difference was observed during the different hormonal phases in the group of women over 35. In conclusion, the correct interpretation of laboratory data is essential to define a threshold of normality, and for what concerns HE4 levels, the menstrual cycle phase-dependent variability appears indicated in the interpretation of the results.     

Effect of a high fat diet on the proliferative indices of murine colonic epithelium

The present study investigated the effect of low (5%) and high (23.5%) corn oil diets on the cytokinetic parameters of colonic crypts. C57BL/6J mice were fed the designated diets for 10 or 15 weeks. In comparison to the low corn oil group, the high corn oil group had an increased number of labelled cells per crypt, with increased labelling indices and crypt heights and larger proliferative compartments in their colons at both time periods. The findings of the present study suggest that a high corn oil diet may exert an enhanced tumorigenic effect by altering the cell diversion parameters of the colonic crypts.     

Cell proliferation,apoptosis, and expression of Bcl-2 and Bax in non-lactating human breast epithelium in relation to the menstrual cycle and reproductive history

Cell proliferation, apoptosis, and the expression of Bcl-2 and Bax were investigated in breast tissue of healthy premenopausal women in order to study the effect of the menstrual cycle and reproductive history on the cell turnover in the non-lactating mammary gland epithelium. Immunohistochemistry was used to detect the proliferation-associated antigen Ki-67, as well as Bcl-2 and Bax. Apoptotic cells were identified by enzymatic labelling of fragmentized DNA (TUNEL-technique) and morphologic analysis. Consistent with published data, the proliferative activity and the frequency of apoptotic events as detected by morphologic analysis was higher in the luteal than in the follicular phase of the menstrual cycle. Parity, lactation, and age correlated with lower proliferative activity, whereas the frequency of apoptosis was not significantly influenced by the reproductive history. Staining patterns for Bax and Bcl-2 showed characteristic changes due to the menstrual cycle with a maximum of immunoreactivity for Bcl-2 in the follicular phase and for Bax in the luteal phase. However, there was no statistically significant association between Bcl-2/Bax immunoreactivity and menstrual cycle or reproductive parameters. We conclude that other molecular pathways than the Bax/Bcl-2 antagonism may additionally be involved in the regulation of apoptotic cell death in the breast epithelium. Knowledge of the entire complexity of apoptosis regulation is necessary to understand the observed effects of parity and lactation on mammary epithelial biology, and possibly to be able to influence pathological processes caused by an imbalance between cell renewal and elimination.     

Mammalian target of rapamycin is the key effector of phosphatidylinositol-3-OH-initiated proliferative signals in the thyroid follicular epithelium

Activation of the phosphatidylinositol-3-OH kinase (PI3K) signaling cascade is becoming increasingly recognized as a common feature of thyroid follicular neoplasms. We have recently shown that conditional loss of Pten in the mouse thyroid follicular cells is sufficient to stimulate continuous autonomous growth, leading to a homogeneously hyperplastic gland and to the development of follicular adenomas. Because the PI3K/AKT cascade can activate a plethora of different signaling pathways, it is still unclear which of these may represent the key mitogenic output of PI3K-initiated signaling. Here, we show that the in vivo proliferative response to chronic PI3K activation profoundly relies on the activation of the mammalian target of rapamycin (mTOR)/S6K1 axis, and that mTOR inhibition in Pten mutant mice and cells restores virtually normal proliferation rates, despite the presence of still elevated Akt activity, at least in part by down-regulating cyclins D1 and D3, and without affecting cell survival.     

Performance of cytology and human papillomavirus testing in relation to the menstrual cycle

Cervical smears prepared around the time of menses have been linked to unsatisfactory specimens and false negative results; however, it is unclear whether liquid-based cytology is similarly affected and data relating date of last menstrual period (LMP) to human papillomavirus (HPV) DNA testing are conflicting. Accordingly, we evaluated liquid-based cytology and HPV test results using Hybrid Capture 2 and PCR by LMP (days 0-10; 11-21; 22-28). We studied 5060 participants in ALTS, the Atypical Squamous Cells of Undetermined Significance (ASCUS) Low Grade Squamous Intraepithelial Lesion (LSIL) Triage Study. On average, women had 3.4 examinations (median 4, range 1-5) during a 2-year period of observation permitting an examination of intra-individual variation in cytology and HPV by LMP. Although uncommon, unsatisfactory cytology specimens were most likely on days 0-10. For satisfactory specimens, the frequency with which cytologic categories were reported varied by time since LMP, although differences were modest and did not affect the chance of abnormal cytology or its severity among women diagnosed with CIN2+. The frequency of positive HC2 tests did not vary with date of LMP. Among HPV infected women, independent of eventual diagnosis and the number of viral genotypes present, mid-cycle specimens yielded the highest frequency of LSIL cytologic interpretations and the highest HPV load; however, the magnitude of these effects were small. Intraindividual correlations of cytology or HPV by LMP were generally weak. We conclude that mid-cycle specimens yield slightly higher HPV DNA loads and slightly increased LSIL interpretations, but the clinical impact is marginal. Standardizing collection times would slightly improve interpretation of trends in HPV load. Finally, these data are consistent with the view that the biological properties of the HPV-infected cervix vary with the date of the LMP.     

Fluctuations in prevalence of cervical human papillomavirus in women frequently sampled during a single menstrual cycle

In the last few years much attention has been focused on the implementation of human papillomavirus detection in population based screening programmes to identify women at risk for cervical cancer. Short-term fluctuations in prevalence of human papillomavirus were investigated within a single menstrual cycle. The highest prevalence was found at the follicular phase (55%), whereas the cumulative prevalence was 75%.     

A prospective study of variability in mammographic density during the menstrual cycle

Mammographic breast density has been proposed as a surrogate endpoint in breast cancer prevention studies, but little is known about its variability over time, particularly in relation to menstrual cycle phase. The purpose of this study was to assess variation in breast density on digital mammograms using quantitative and qualitative density measures. Menstrual cycle phase was determined by salivary estradiol and progesterone assays. 73 healthy subjects with regular menses had 1–3 mammograms with paired saliva collection during a 12-month period. The mean difference in density as a percentage of the mean density was calculated for follicular–luteal (n = 50), luteal–luteal (n = 26) and follicular–follicular (n = 23) pairs in the same woman using the same breast. Two density measures (measurement of dense area and BIRADS) were used. The mean luteal density exceeded the mean follicular density by 7.1–9.2%, but density differences between luteal pairs and follicular pairs did not exceed 5%. The intraclass correlation for measurement of dense area was greater than 85% in all phases of the menstrual cycle, but was below 50% for BIRADS for luteal–follicular and follicular–follicular pairs. Our study provides estimates of the amount of variation in mammographic density during the menstrual cycle, and that inherent in repeated density measurement in premenopausal women, and suggests that menstrual phase of mammographic evaluation should be controlled for in intervention studies where density is being used as a surrogate measure.     

beta-Ionone suppresses mammary carcinogenesis, proliferative activity and induces apoptosis in the mammary gland of the Sprague-Dawley rat

beta-Ionone demonstrates potent anticancer activity both in vitro and in vivo. We determined tumor incidence and the number of rats bearing tumors as well as cell proliferation and apoptosis in a rat mammary cancer model induced by 7, 12-dimethylbenz[a]anthracene (DMBA). Rats were fed an AIN-76A diet containing beta-ionone (0, 9, 18 or 36 mmol/kg), starting 2 weeks before DMBA administration and continuing for 24 weeks. A dose-dependent inhibition of mammary carcinogenesis by dietary beta-ionone was observed. Corresponding tumor incidence values were 82.1, 53.3, 25.9 and 10.0% (p < 0.01 or 0.05). Time to tumor appearance increased and tumor multiplicity decreased with increasing dietary beta-ionone. Histopathological and immunohistochemical evaluations of tumors were performed on the 64, 31, 15 and 3 tumors, respectively, identified in rats from the respective groups of 30. The proportions of adenocarcinomas, adenomas and benign masses were equally distributed in the latter group. In proportions within the other groups, the proportions of adenocarcinomas and benign masses decreased and increased with increasing dietary beta-ionone. Proliferating cell nuclear antigen (PCNA), cyclin D1 and Bcl-2 expression decreased, and Bax expression and nuclear fragmentation increased with increasing dietary beta-ionone. These results demonstrate the potent capacity of dietary beta-ionone to suppress DMBA-initiated mammary cancer in rats.     

Altered expression of cell cycle and apoptotic proteins in human liver pathologies

Τhe expression of cell cycle (P53, Ki-67, P21, and P27) and apoptotic proteins (BCL-2 and BAX) was investigated by immunohistochemistry in paraffin-embedded formalin-fixed tissues of normal and pathologic liver. An increased frequency of expression of P21 in cirrhosis and hepatocellular carcinoma (HCC) (p=0.003 and p=0.001 respectively) was found; P27 protein expression was more frequent in hepatitis (p=0.001) and HCC (p=0.003) when compared with normal tissue. BCL-2 protein was markedly more frequent in steatohepatitis (p<0.05) as compared to normal liver, in hepatitis cases (p=0.002) and in metastases (p<0.033). The expression of BAX was more frequent in hepatitis (p=0.001) and cirrhosis (p<0.001). We demonstrated in our study the expression of these proteins at different levels in liver pathologies. These findings have implications for understanding the evolution from liver inflammation to cirrhosis and associated carcinogenesis.     

The phase of the menstrual cycle has no influence on the disease-free survival of patients with mammary carcinoma.

We evaluated the outcome of treatment for mammary carcinoma in 89 premenopausal women in relation to the phase of the menstrual cycle. The phase of the cycle was determined on the basis of serum concentrations of 17 beta-oestradiol and progesterone. The serum samples were collected 1 day prior to or on the day of operation. After a median follow-up of 4.1 years no significant differences in disease-free survival were found between the preovulatory (proliferative), periovulatory and post-ovulatory (luteal) groups. No differences in survival were found in these subgroups between the N0 and N1 subgroup. On the basis of this study we cannot confirm that the phase of the menstrual cycle during surgery has any effect on the eventual outcome in mammary carcinoma patients. However larger studies of this type are required before definitive conclusions can be reached.