Adjacent Mammary Glands Research Articles

Abstract 5104: Prolactin-induced mammary tumors: A preclinical model of luminal B breast cancer which exhibits mutations in the RAS pathway

Abstract Metastatic therapy-resistant breast cancers expressing estrogen receptor alpha (ER+) account for the majority of breast cancer deaths. Understanding of the underlying biology of aggressive luminal B cancers would improve preventive/treatment strategies. However, this has been limited by the paucity of preclinical immunocompetent models. Strong epidemiologic data link prolactin (PRL) exposure to development of ER+ metastatic breast cancer. We have capitalized on this relationship to generate a murine model which overexpresses PRL in mammary epithelial cells (NRL-PRL). Transgenic mammary PRL expression leads to the development of spontaneous metastatic ER+ carcinomas with characteristics of luminal B human cancers. In order to understand the mechanisms that underlie the ability of PRL to drive the development of cancers in this model, we used functional genomic analyses to identify genomic alterations and patterns of gene expression with cancer progression. Following development of spontaneous tumors, ER+ tumors and matched adjacent mammary glands and tail samples (N=5) were collected and examined by whole genome sequencing and RNA-seq analysis. A set of mammary cell preparations from the caudal glands of 12-week-old NRL-PRL females (N=5) were also analyzed. Unexpectedly, we found that all of the tumors contained somatic alterations of Kras, including four with canonical hotspot mutations (G12 and Q61) and one with an amplification of the Kras locus. In contrast to the tumors, no Kras mutations were detected in the matched mammary glands and tails, or the cell preparations. Our findings were further validated by targeted sequencing of an extension set consisting of frozen archived spontaneous ER+ tumors from NRL-PRL females (N=15), additional samples from mice with matched tumor, adjacent mammary gland and tails (N=7) and ER+ cell lines generated from PRL-induced spontaneous tumors (N=4). The Kras hotspot mutations were detected in 14 of 22 of the extension tumors and all four cell lines. One additional tumor contained a hotspot activating mutation in Nras. Immunohistochemistry demonstrated that RAS downstream effectors, pAKT and pERK, were upregulated, whereas nuclear STAT5A was decreased, in the tumors compared to the adjacent normal gland. Moreover, RNA-Seq analyses showed that acquisition of RAS mutations by the tumors was associated with alterations in multiple pathways compared to non-tumor glands, including distinct immune cell subpopulations. Although few clinical breast cancers display RAS protein mutations, studies have shown that the RAS pathway is aberrantly activated in more than 50% of all breast lesions. This enhanced RAS activity confers many benefits to these tumors resulting in a poor prognosis. The NRL-PRL model provides a valuable tool to dissect mechanisms that drive metastatic luminal B breast cancer and identify therapeutic opportunities. Citation Format: Kathleen A. O'Leary, Katie M. Campbell, Debra E. Rugowski, Kilannin Krysiak, William A. Mulligan, Malachi Griffith, Obi L. Griffith, Linda A. Schuler. Prolactin-induced mammary tumors: A preclinical model of luminal B breast cancer which exhibits mutations in the RAS pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5104.

Obesity-Induced Inflammation Is Associated with Alterations in Subcellular Zinc Pools and Premature Mammary Gland Involution in Lactating Mice1–3

Background: Lactation failure is common in overweight and obese women; however, the precise mechanism remains unknown.Objective: We tested the hypothesis that obesity-induced inflammation in the mammary gland (MG) redistributes subcellular zinc pools to promote cell death of mammary epithelial cells (MECs) and premature involution.Methods: Female DBA/2J mice were fed a high-fat (obese; 45% kcal from fat, n = 60) or control diet (lean; 10% kcal from fat, n = 50) for 5 wk and bred. MG cytokines and macrophage infiltration were determined by reverse transcriptase-polymerase chain reaction and F4/80 staining, respectively. Zinc concentration was analyzed by atomic absorption spectroscopy, and zinc transporters and markers of endoplasmic reticulum (ER) stress, autophagy, and involution were measured by immunoblot. To confirm effects of inflammation, tumor necrosis factor-α (TNF) or vehicle was injected into adjacent MGs of lean lactating C57BL/6 mice (n = 5) and cultured MECs (HC11 cells) were treated with TNF in vitro.Results: Seventy-seven percent of obese mice failed to lactate (lean: 39%; P < 0.001). Obese mice capable of lactating had greater macrophage infiltration (obese: 135 ± 40.4 macrophages/mm2; lean: 63.8 ± 8.9 macrophages/mm2; P < 0.001) and elevated TNF expression (P < 0.05), concurrent with lower zrt- irt-like protein 7 abundance (P < 0.05) and higher ER zinc concentration (obese: 0.36 ± 0.004 μg Zn/mg protein; lean: 0.30 ± 0.02 μg Zn/mg protein; P < 0.05) compared with lean mice. Heat shock protein 5 (HSPA5) expression (P < 0.05) was suppressed in the MG of obese mice, which was consistent with HSPA5 suppression in TNF-injected MGs (P < 0.01) and MECs treated with TNF in vitro (P < 0.01). Moreover, obesity increased lysosomal activity (P < 0.05) and autophagy in the MG, which corresponded to increased zinc transporter 2 abundance and lysosomal zinc concentration compared with lean mice (obese: 0.20 ± 0.02 μg Zn/mg protein; lean: 0.14 ± 0.01 μg Zn/mg protein; P < 0.05). Importantly, MGs of obese mice exhibited markers of apoptosis (P = 0.05) and involution (P < 0.01), which were not observed in lean mice.Conclusions: Diet-induced obesity created a proinflammatory MG microenvironment in mice, which was associated with zinc-mediated ER stress and autophagy and the activation of premature involution.

Cyclooxygenase-2 Expression in Feline Mammary Carcinomas and Adjacent Mammary Gland

Cyclooxygenase-2 Expression in Feline Mammary Carcinomas and Adjacent Mammary Gland

Significance and expression of p53, p21(Cip1/WAF1) and Gadd45α in breast neoplasm tissues

To explore the expression and significance of p53, p21(Cip1/WAF1) and Gadd45α protein in breast cancer and their correlations with clinicopathologic features and prognosis in breast cancer. The expressions of p53, p21(Cip1/WAF1) and Gadd45α proteins were determined by immunohistochemical staining. The relationship between these three proteins and clinicopathologic features in breast cancer was analyzed by χ(2) test and Spearman's rank correlation analysis. And the survival analyses were performed with the Kaplan-Meier method and Cox regression. The differences between the curves were examined with the two-tailed Log-rank test. In 133 cases of invasive breast cancer, the positive rates of p53, p21(Cip1/WAF1) and Gadd45α protein were 58.6%, 47.4% and 41.4% respectively. The expressions of p21(Cip1/WAF1) and p53 in cancer were significantly higher than those in the adjacent mammary gland tissue (P < 0.05) while the expression of Gadd45α was lower than that in the control mammary gland tissue (P < 0.05). The positive rate of p21(Cip1/WAF1) was correlated with the histological stage, local recurrence and positive C-erbB-2. And the positive rate of Gadd45α was correlated with the histological stage, lymph node metastasis, metastasis and positive estrogen receptor/progesterone receptor (ER/PR). The positive rate of p53 was correlated with the lymph node metastasis and TNM stage. Spearman's rank correlation analysis showed that p21(Cip1/WAF1) was correlated positively with p53, p53 negatively with Gadd45α while p21(Cip1/WAF1) had no correlation with Gadd45α. With the follow-up data, Kaplan-Meier analysis showed that p21(Cip1/WAF1), Gadd45α, p53, lymph node metastasis, C-erbB-2 positive and TMN stage were associated with prognosis. Furthermore, Cox stepwise hazard analysis shows that p21(Cip1/WAF1), Gadd45α, C-erbB-2 and TMN stage were correlated with prognosis of breast cancer. Also the Kaplan-Meier analysis showed that p53(+)Gadd45α(-) and p53(+)p21(Cip1/WAF1)(+) were correlated with a poor prognosis of breast cancer. The expressions of p21(Cip1/WAF1), Gadd45α and p53 are associated with the clinicopathologic features and prognosis in breast cancer. Indicating a poor prognosis of breast cancer, p53(+)Gadd45α(-) and p53(+)p21(Cip1/WAF1)(+) may become independent indices for prognostic evaluations.

Detection of mutations within exons 4 to 8 of the p53 tumor suppressor gene in canine mammary glands

Fifteen female canines with mammary tumors and 6 normal females were used to study mutations in exons 4 to 8 of the p53 gene. DNA samples from the tumors, respective adjacent normal mammary tissue and mammary glands from healthy animals were sequenced and analyzed for the presence of mutations. Mutations were found in 71.8% of the samples and the most frequent were missense mutations. The most attacked exons in the mammary tumor were 5, 7 and 8, with 23.4, 31.6 and 23.4% mutations, respectively. Canine mammary tumors are related to mutations in gene p53 and mutations mostly occur in the region of the protein that is linked to the DNA in the cell nucleus, which can change the functionality of the cell and propitiate tumor growth. Despite being macroscopically normal, the mammary tissue adjacent to the tumors has mutations that can lead to recurrence if not removed together with the tumor.

Abstract 525: Functional screen of paracrine signals in breast carcinoma fibroblasts

Abstract Stromal fibroblasts actively participate in normal mammary gland homeostasis and in breast carcinoma growth and progression by secreting paracrine factors. However, our knowledge about the identity of paracrine mediators operating in individual patients remains incomplete. The purpose of this study was to characterize paracrine signaling pathways between breast carcinoma cells and breast carcinoma-associated fibroblasts (CAF) or normal mammary fibroblasts (NF), respectively. CAF and NF were isolated from breast carcinoma tissue samples and adjacent normal mammary gland tissue of 28 patients. The fibroblasts were grown in 3D collagen gel co-culture with T47D human breast carcinoma cells and T47D cell growth was measured. CAF stimulated T47D cell growth to a significantly greater degree than NF. Collectively, neutralizing antibodies to fibroblast growth factor 2, heparin-binding epidermal growth factor, SDF1 and TGF- β1 and the enzymes heparanase-1 and MT1-MMP significantly abrogated CAF-stimulated T47D cell growth, although, a considerable degree of inter-individual heterogeneity was observed. CAF from low-grade but not high-grade carcinomas required IGF1 and TGF- β1 to stimulate carcinoma growth. CAF from estrogen or progesterone receptor negative carcinomas depended on HB-EGF activity for carcinoma growth. Paradoxically, blocking of MT1-MMP stimulated T47D cell growth in co-culture with NF. The results were largely replicated by treating the fibroblasts with siRNA oligonucleotides prior to co-culture, implicating the fibroblasts as principal production site for the paracrine mediators examined in this study. In summary, we characterize the paracrine signaling network of primary breast cancer fibroblasts with inhibitor screens. These findings have significant implications for the design of stroma-targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 525. doi:10.1158/1538-7445.AM2011-525

The Tammar Wallaby and Fur Seal: Models to Examine Local Control of Lactation

Mammary development and function are regulated by systemic endocrine factors and by autocrine mechanisms intrinsic to the mammary gland, both of which act concurrently. The composition of milk includes nutritional and developmental factors that are crucial to the development of the suckled young, but it is becoming increasingly apparent that milk also has a role in regulating mammary function. This review examines the option of exploiting the comparative biology of species with extreme adaptation to lactation to examine regulatory mechanisms that are present but not readily apparent in other laboratory and livestock species. The tammar wallaby has adopted a reproductive strategy that includes a short gestation (26 d), birth of an immature young, and a relatively long lactation (300 d). The composition of milk changes progressively during the lactation cycle, and this is controlled by the mother and not the sucking pattern of the young. Furthermore, the tammar can practice concurrent asynchronous lactation; the mother provides a concentrated milk high in protein and fat for an older animal that is out of the pouch and a dilute milk low in fat and protein but high in carbohydrates from an adjacent mammary gland for a newborn pouch young. This phenomenon suggests that the mammary gland is controlled locally. The second study species, the Cape fur seal, has a lactation characterized by a repeated cycle of long at-sea foraging trips (up to 28 d) alternating with short suckling periods of 2 to 3 d ashore. Lactation almost ceases while the seal is off shore, but the mammary gland does not progress to apoptosis and involution, most likely because of local control of the mammary gland. Our studies have exploited the comparative biology of these models to investigate how mammary function is regulated by endocrine factors, and particularly by milk. This review reports 3 major findings using these model animals. First, the mammary epithelial cell has an extraordinary intrinsic capacity for survival in our culture model, and the path to either function or death by apoptosis is actively driven. The second outcome is that the route to apoptosis is most likely regulated by specific milk factors. Finally, whey acidic protein, a major milk protein in some species, may play a role in normal mammary development, but that role in vivo may be limited to marsupials. Evolutionary pressure has led to changes in the structure of the protein with an accompanying change in function. Therefore, we propose that a loss of function of this protein in eutherians may relate to a reproductive strategy that is less dependent on lactation.

Experimental staphylococcal mastitis in bitches: Clinical, bacteriological, cytological, haematological and pathological features

The objectives of the work were to study the features of experimentally induced canine mastitis and to present hypotheses regarding the pathogenesis of the disease. The right caudal abdominal mammary gland of six bitches was inoculated on day 8 after whelping with Staphylococcus intermedius to induce mastitis; adjacent mammary glands were used as controls. Clinical examination, bacteriological and cytological (whiteside test, Giemsa) examination of mammary secretion, as well as haematological tests were performed from 5 days before until 34 days after challenge. Mastectomy was sequentially performed 1, 2, 4, 18, 26 and 34 days after challenge in each of the bitches, in order to carry out a pathological examination of mammary glands. All animals developed clinical mastitis: challenged glands became painful, hot, enlarged and oedematous; secretion was brownish, purulent, with flakes or clots, subsequently becoming yellowish and thick. Staphylococci were isolated from all inoculated glands (up to 22 days). WST was positive in 41/46 samples from inoculated glands and 66/138 samples from control glands; neutrophils predominated during the acute stage. Blood leukocyte counts increased, whilst platelet counts decreased. Gross pathological findings initially included congestion, purulent discharge and subcutaneous oedema; then abscesses, brownish areas and size decrease were seen. Salient histopathological features were initially neutrophilic infiltration, haemorrhages, destruction of mammary epithelial cells and alveoli, and then infiltration by lymphocytes, shrunken alveoli, loss of glandular architecture and fibrous tissue proliferation. We conclude that in bitches, intrammamary inoculation of Staphylococcus intermedius can induce clinical mastitis, followed by subclinical disease. The disorder is characterized by bacterial isolation and leukocyte influx in challenged glands, by leukocyte presence in adjacent mammary glands, by increased blood leukocyte counts and by destruction of mammary parenchyma.

Significance and correlation of MAPK/ERK2 and PI3-K in human breast tumorigenesis

ObjectiveMAPK ((Mitogen-actived Protein Kinase) and PI3-K (Phosphatidylinositol 3-kinase) pathways have been implicated in the mitogenic pathways regulating cell growth, proliferation, differentiation and transformation and thus involved in tumorigenesis. This study was designed to examined the protein expression, activity and mRNA levels of both ERK and PI3-K in a series of breast tumors and adjacent mammary glands, and to figure out the changes of ERK2 and PI3-K during the dynamic process of breast tumorigenesis.MethodsA series of breast tumors and adjacent mammary glands were collected at surgery, including 37 cases of breast cancer, 6 cases of atypical hyperplasia-breast carcinoma in situ and 15 cases of benign conditions. Western blot, kinase activity assay and RT-PCR were used to detect the protein expression, kinase activity and mRNA level, respectively.ResultsThe reveals of protein, activity and mRNA of ERK2 were elevated during the stages of both initiation and progression. The increasing tendency in breast cancer was equal to atypical hyperplasia-in situ carcinoma, but higher than in benign lesion and adjacent normal mammary gland. PI3-K was activated during the stage of progression of breast cancer. An inverse correlation between the activity of PI3-K and ERK2 in breast cancer was found.ConclusionOur findings indicate that ERK2 may perform its function during both the stages of breast cancer initiation and breast cancer progression, while PI3-K may exert its effect during the stage of breast cancer progression. Both PI3-k and ERK2 are involved in the tumorigenesis of breast cancer.

Phosphorylated Akt1 in human breast cancer measured by direct sandwich enzyme-linked immunosorbent assay: Correlation with clinicopathological features and tumor VEGF-signaling system component levels

Protein kinase B (Akt) plays a major role in the regulation of breast cancer growth, survival, hormone, drug and radiosensitivity, but the clinical value of its expression and activation in human tumors is unclear. Activated Akt1 (pAkt1) expression was quantified in a series of 46 breast cancer and adjacent mammary gland samples by a direct Path-Scan PhosphoAkt1 (Ser473) sandwich ELISA kit. VEGF, sVEGFR1 and sVEGFR2 levels were measured simultaneously by standard ELISA kits. Forty-nine percent of the tumors had an increased pAkt1 level as compared to adjacent tissue. pAkt1 levels were significantly higher in stage IIb than in stage I-IIa tumors. The frequency of pAkt1 elevation was positively associated with tumor size and malignancy grade. pAkt1 was also twice as frequently increased in PgR-negative as in PgR-positive tumors, while its mean level was significantly higher in ER-positive than in ER-negative tumors. VEGF, sVEGFR1 and sVEGFR2 were increased in 73-85% of the tumors, but no associations with most clinicobiological factors and pAkt1 level were found. In conclusion, activation of Akt1 is not associated with VEGF signaling protein expression in breast cancer but is related to tumor size, grade of malignancy, and steroid receptor status.

Phosphorylated Akt1 in Human Breast Cancer Measured by Direct Sandwich Enzyme-Linked Immunosorbent Assay: Correlation with Clinicopathological Features and Tumor VEGF-Signaling System Component Levels

Protein kinase B (Akt) plays a major role in the regulation of breast cancer growth, survival, hormone, drug and radiosensitivity, but the clinical value of its expression and activation in human tumors is unclear. Activated Akt1 (pAkt1) expression was quantified in a series of 46 breast cancer and adjacent mammary gland samples by a direct Path-Scan™ PhosphoAkt1 (Ser473) sandwich ELISA kit. VEGF, sVEGFR1 and sVEGFR2 levels were measured simultaneously by standard ELISA kits. Forty-nine percent of the tumors had an increased pAkt1 level as compared to adjacent tissue. pAkt1 levels were significantly higher in stage IIb than in stage I-IIa tumors. The frequency of pAkt1 elevation was positively associated with tumor size and malignancy grade. pAkt1 was also twice as frequently increased in PgR-negative as in PgR-positive tumors, while its mean level was significantly higher in ER-positive than in ER-negative tumors. VEGF, sVEGFR1 and sVEGFR2 were increased in 73–85% of the tumors, but no associations with most clinicobiological factors and pAkt1 level were found. In conclusion, activation of Akt1 is not associated with VEGF signaling protein expression in breast cancer but is related to tumor size, grade of malignancy, and steroid receptor status.

Preferential Occurrence of Breast Carcinomas with Loss of Chromosome 16q and der(16)t(1;16)/der(1;16) in Middle-aged Patients with Hyperplasia of Mammary Glands

Structural and numerical alterations of chromosome 16 are considered to be commonly involved in the genesis of breast cancer. To reveal etiological factors that predispose cells to these alterations, we examined the frequencies of chromosome 16 aneusomy, 16q loss and 1;16 fusion indicating der(16)t(1;16)/der(1;16) by multi‐color fluorescence in situ hybridization in 46 tumors resected mostly from young (≤34 years old) or elderly (≥75 years old) women, and compared the results with those in a patient group representing a common age distribution of Japanese patients in whom chromosome 16 status in the tumor had already been studied. The correlation of these chromosome 16 alterations with age, hyperplasia in adjacent mammary glands, cancer history, and obesity indices was investigated in a total of 244 patients. In the present 46 tumors, the frequency of 16q loss and der(16)t(1;16) did not differ between 20 younger patients (30% and 15%) and 23 elderly patients (43% and 13%). However, the incidences of 16q loss and der(16)t(1;16) were low in comparison with the values of 64% and 38% in the 198 Japanese patients representing the common age distribution (P< 0.001). In addition, 16q loss and der(16)t(1;16) were more frequent in tumors arising in hyperplastic mammary glands (68%, 44%) than in those without (52%, 24%) (P< 0.01). Such correlations were not evident for 16cen aneusomy. Other etiological factors examined were not correlated with these chromosome 16 alterations. The 16q loss and der(16)t(1;16) formation were more frequently involved in the development of breast cancer in middle‐aged patients than in young and elderly patients. High‐level estrogens and/or sensitivity of mammary glandular cells to estrogens might induce breast cancers with structural changes of chromosome 16.

Phospatidylinositol 3-kinase expression in human breast cancer

Phospatidylinositol 3-kinase (PI 3-kinase) expression was analysed by Western blotting with monoclonal antibodies to the p85 subunit in a series of tumour and adjacent mammary gland samples collected at surgery from 33 breast cancer patients. Seventy-nine percent of the investigated pairs of the samples were characterised by an increased level of PI 3-kinase in the tumour in comparison with the adjacent mammary gland. PI 3-kinase activation was not associated with tumour steroid receptor status, histologic grade and other clinico-morphological characteristics. Furthermore, immunoblotting of epidermal growth factor receptor (EGFR) in the tumours with increased PI 3-kinase and corresponding adjacent tissues revealed no association between EGFR and PI 3-kinase activation. Thus, increased PI 3-kinase expression appears to be a widespread feature of breast cancer not associated with the main biological markers of its prognosis and hormone sensitivity.

Etoposide enhances the lethal effect of radiation on breast cancer cells with less damage to mammary gland cells.

In the present study, the cytotoxic effects of anticancer drug treatment plus radiation on breast cancer and normal mammary gland cells were investigated in vitro. Breast cancer and adjacent mammary gland tissue was obtained from surgically resected specimens from 13 premenopausal female patients, and was cultured. Cultured cells were treated with radiation and etoposide, and sensitivities were evaluated using an MTT assay. In breast cancer cells, radiation alone had a cytotoxic effect. When etoposide was added, synergistic effects were achieved which were independent of the radiation dose. The cytotoxicity of etoposide against normal gland cells, however, was much the same as that against cancer cells, and no synergistic effects were observed in normal cells even when radiation was added. Radiation combined with etoposide showed lethal effects against breast cancer cells with less damage to normal mammary gland cells.

Autocrine inhibition of milk secretion in the lactating tammar wallaby (Macropus eugenii).

The lactating tammar wallaby progressively alters the rate of secretion and composition of its milk to provide appropriate nutrition for the developing offspring, whose needs are signalled by changes in the pattern and efficiency of its sucking. Tammars are also capable of asynchronous concurrent lactation, when the mother provides a dilute milk for a newborn young permanently attached to the teat (phase 2A of lactation), and a concentrated milk from an adjacent mammary gland for a young-at-heel (phase 3). The relationship between suckling behaviour and milk secretion, and the ability of adjacent glands to function independently, suggests that milk secretion is controlled locally, within each mammary gland, by a mechanism sensitive to frequency and completeness of milk removal. To determine if tammar milk contains a factor able to control milk secretion, milk fractions have been screened in tissue and cell culture bioassays. A 6-30 kDa fraction of phase 3 whey was found to inhibit milk constituent synthesis and secretion in vitro, and inhibitory activity was associated with two discrete fractions obtained by anion exchange chromatography, which contained protein bands migrating anomalously at 66 kDa and 63 kDa in SDS-PAGE. These bands were recognised in Western blotting by antiserum raised against a bovine autocrine inhibitor of milk secretion. By the same criteria, milk secreted in phase 2B of tammar lactation, when milk secretion is low and suckling intermittent but less vigorous than phase 3, also contained a feedback inhibitor of milk secretion. The results indicate that, as in dairy animals, marsupial milk secretion is under local control through feedback inhibition by a milk protein, and raise the possibility that autocrine feedback may influence the transition from phases of low milk secretion (phase 2A, 2B) to a high rate in the final third phase of lactation.

A Case of Apocrine Carcinoma of the Breast Presenting as Two Cysts.

A case of apocrine carcinoma of the breast presenting as two cysts is reported. A 60-year-old woman had two soft, mobile, well-defined masses measuring 4 x 2.5 cm and 1.5 x2.5 cm in diameter in the lower-inner quadrant of the right breast. Tumor markers such as CEA, CA15-3, BCA225 and NCC-ST-439 were within normal limits. Mammography showed two oval radioopaque masses with microcalcificatin in the smaller one. Ultrasonography disclosed two cysts and papillary projection in the larger one. Fine needle aspiration cytology revealed apocrine carcinoma of the breast. Excisional biopsy confirmed the diagnosis of apocrine carcinoma with focal invasion. Modified radical mastectomy was performed. Pathological study showed that the tumor mainly proliferated in the cysts and the two cysts communicated with each other through an involved duct. There was no apocrine metaplasia in adjacent mammary gland. There were no metastatic lymph nodes. This case may be regarded as intraductal apocrine carcinoma developing two cysts with focal invasion.

Progressive changes in milk protein gene expression and prolactin binding during lactation in the tammar wallaby (Macropus eugenii).

Changes in milk protein gene expression and specific prolactin binding were quantified in mammary tissue from the tammar wallaby (Macropus eugenii) at different stages of lactation. The transition from early (phase 2) lactation to late (phase 3) lactation was characterized by the induction of the gene for late lactation protein, a novel whey protein. During the same period, the levels of beta-lactoglobulin and beta-casein gene expression increased, whereas there was no change in the levels of expression of alpha-lactalbumin and alpha-casein genes. Prolactin binding in the mammary gland doubled during the latter half of phase 2 of lactation but declined significantly during the transition to phase 3 of lactation. These changes in prolactin binding resulted from changes in the number of receptors and not from a change in the affinity of the receptor for prolactin. Treatment of membranes with concanavalin A increased the number of prolactin-binding sites by 40% in membranes from phase 2 mammary tissue but decreased binding by 40% in membranes from phase 3 tissue, indicating that significant changes had occurred in the membranes of cells during this period. The tammar wallaby can secrete phase 2 and phase 3 milk from adjacent mammary glands (asynchronous concurrent lactation) and the developmental changes in milk protein gene expression and prolactin binding observed during lactation were reflected in these individual glands. Taken collectively, these findings suggest that mammary development and milk secretion in the tammar wallaby are regulated by both endocrine and local (intramammary) mechanisms.

Afferent projections from the mammary glands to the spinal cord in the lactating rat—I. A neuroanatomical study using the transganglionic transport of horseradish peroxidase-wheatgerm agglutinin

Horseradish peroxidase-wheatgerm agglutinin was injected subcutaneously into one or more nipples of lactating rats to determine the spinal organization of sensory afferents emanating from the mammary glands. After survival periods of 45–96 h, dorsal root ganglia and segments of the spinal cord and/or medulla oblongata were sectioned and reacted histochemically with tetramethylbenzidine to reveal the transganglionically transported tracer. For each nipple injected, the peroxidase reaction product was found in somata, ranging in diameter from 15 to 60 μm and fibres in 5–11 contiguous dorsal root ganglia. The number of labelled profiles was highest in the 2–4 central-most ganglia of the series and generally decreased progressively rostrally and caudally. After separate injections into each of the six ipsilateral nipples, labelling occurred in all ipsilateral dorsal root ganglia between the 5th cervical and 6th lumbar spinal segments. Substantial overlap of the spinal projections from adjacent mammary glands was seen, a given dorsal root ganglion innervating 2–3 different glands. Label in the spinal cord was restricted to the medial portion of the superficial dorsal horn. It occurred in what appeared to be terminal fields and fibres essentially in the substantia gelatinosa, but was also seen to extend into the marginal zone and sometimes into deeper regions of the dorsal horn. Label was found in both the gracile and cuneate nuclei of the medulla oblongata, though only occasionally and then only very sparsely. The substantial spread and segmental overlap of labelled mammary afferents and the fact that most labelled afferents terminated in the dorsal horn, suggest that this spinal region may be an important site for the integration of sensory input from the mammary glands that may play a role in the sensory induction of reflex milk ejection.

Intratumoral BCG and Corynebacterium parvum therapy of canine mammary tumours before radical mastectomy.

In two parallel studies, bitches with mammary tumour received single intralesional injections of BCG (1 mg: 10(7) living bacteria) and Corybacterium parvum (10(9) killed bacteria) (53 bitches) or C. parvum alone (129 bitches) at the same dosage. Control groups received injections, following the same protocol, of 1 ml BCG suspension medium diluted in saline in the first study (51 bitches) or no injections at all (120 bitches in the second study). A block dissection, including mammary tumours, adjacent mammary glands, and regional lymph nodes, was performed 2 weeks later in all animals. On the basis of histologically confirmed malignant tumours, 48 bitches (25 treated by-immunotherapy and 23 controls) in the first study and 67 bitches (30 treated by immunotherapy and 37 controls) in the second study remained for postsurgical follow-up. The clinical tolerance of the treatment was generally good. No significant differences were found in cumulative survival rates between treated and control group in either studies.