Invasive Ductal Mammary Carcinoma Research Articles

Analysis of TP53 gene and particular infrastructural alterations in invasive ductal mammary carcinoma.

This study was conducted in order to determine the mutational status of TP53 gene and to determine some particular aspects from ultrastructural level in invasive mammary ductal carcinoma. The cellular signaling pathway involving the TP53 gene acts in biological deoxyribonucleic acid (DNA) repair processes and cell cycle arrest following a signal transmitted to the p53 protein when posttranslational changes occur in the cell due to stress induced in the cell by both intrinsic and extrinsic factors. Cellular stress activates the transcription factor function of the protein that initiates, as the case may be, either DNA repair or programmed cell death (apoptosis). The TP53 gene is commonly mutated in many human cancers and also has a highly polymorphic grade. To determine the mutational status of the exons 4–9 of the TP53 gene, we used extracted DNA from fresh breast tissue, and we analyzed it through direct sequencing. In mammary carcinoma, the mutation frequency of TP53 is running between 20–40% and, in regards the polymorphism, at least 14 different forms were identified, that are associated with cancer risk. The mutation type distribution showed a predominance of deletions and a reduced frequency of substitutions comparing with International Agency for Research on Cancer (IARC) database. Taken in consideration the importance of the tumor associated stroma in tumor development, we have also investigated some particular aspects at the infrastructural level of invasive mammary ductal carcinoma, notably concerning telocytes as tumor stroma interstitial cells by transmission electron microscopy analysis.

Abstract A209: Targeting immune cell-specific sphingosine-1-phosphate receptor 4 to restore antitumor immunity resulting in improved therapy response

Abstract The inflammatory tumor microenvironment is a critical element of tumor development and progression, as well as a parameter predicting the response to cancer treatment. An increased abundance of suppressive immune cells such as tumor-associated macrophages (TAMs) and regulatory T-cells (Tregs) appears thereby as a limiting factor for a successful treatment due to the inhibition of tumoricidal lymphocytes such as cytotoxic CD8+ T-cells. The bioactive lipid sphingosine-1-phosphate (S1P), which signals mainly through its five G-protein-coupled receptors (S1PR1-5), emerges as an important regulator of carcinogenesis that promotes tumor growth, angiogenesis and metastasis, among others, by affecting tumor immunity. We investigated S1PR4, which is exclusively expressed by immune cells, as a potential regulator of antitumor immunity. Using a transgenic mouse model of spontaneous invasive ductal mammary carcinoma (PyMT mice) and the AOM/DSS model of colitis-associated cancer, we show that S1PR4 deficiency (S1PR4-/-) leads to a significant decrease in lung metastasis and a delay of tumor growth in case of the PyMT breast cancer model and reduced colon tumor incidence and size in S1PR4-/- mice subjected to the AOM/DSS model. S1PR4-/- PyMT tumors contained macrophages with an altered activation profile, characterized by reduced IL-10, IL-1β and IL-6 production. Likely as a consequence, S1PR4-/- mice showed a less immunosuppressive lymphocyte infiltrate composed of reduced Tregs and Th17 cells, but increased cytotoxic CD8+ T-cell numbers. Interestingly, AOM/DSS-treated colons of S1PR4-/- mice also displayed a marked increase in CD8+ intestinal epithelial lymphocytes, underlining the negative impact of S1PR4 signaling on CD8+ T-cell abundance during tumor development. Next, we asked whether increased CD8+ T-cell numbers in S1PR4-/- tumors would increase the efficacy of chemotherapy or immunotherapy. Indeed, S1PR4-/- PyMT mice exhibited a significantly improved response to treatment with doxorubicin (DXR), characterized by the absence of relapse, whereas WT PyMT tumors relapsed after DXR treatment. This enhanced tumor control was strictly CD8+ T-cell dependent, as shown by experiments utilizing CD8 antibody depletion in combination with DXR treatment. Finally, using a MC38 colon adenocarcinoma model, where MC38 cells were engrafted into the flanks of WT and S1PR4-/- mice, we demonstrate a synergistic effect of S1PR4 depletion and anti-PD-1 immunotherapy reflected by reduced tumor growth and an increased survival rate of tumor-bearing S1PR4-/- compared to WT mice. A similar effect of additional tumor growth reduction by combinatorial anti-PD-1 treatment was seen in S1PR4-/- PyMT mice compared to their WT counterpart, although less pronounced compared to the MC38 allograft model. Taken together, our data indicate that S1PR4 depletion restores antitumor immunity by, among other mechanisms, increasing CD8+ T-cell abundance, translating into enhanced response to chemotherapy and anti-PD-1 immunotherapy. Thus, we reveal the potential of S1PR4 as a target to improve cancer therapy. Citation Format: Catherine Olesch, Evelyn Sirait-Fischer, Bernhard Brüne, Andreas Weigert. Targeting immune cell-specific sphingosine-1-phosphate receptor 4 to restore antitumor immunity resulting in improved therapy response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A209.

302. Quantitative analysis and SVM-based classification of 18F-FDG PET data in mammary carcinoma

Purpose Invasive ductal carcinoma is the most common type of breast cancer. The identification of the tumors with worst prognoses is important to achieve better clinical management of pathology. Aims of this study were the analysis of 18F-FDG PET radiometric features with Support Vector Machines (SVM) and correlation with prognostic clinical-pathological parameters. Methods The PET-CT scans of 51 women (age ⩾ 18 years) with invasive ductal mammary carcinoma selected for chirurgical resection were considered. Images were corrected voxel-by-voxel for Partial Volume Effect using an iterative post-reconstruction method based on measured system Point Spread Function. Mean Standardized Uptake Value (SUVmean) was extracted for segmented Regions Of Interest (ROIs) representing tumor lesions (L) and background mammary healthy tissue (BK). Pearson correlations of tumor proliferative index with SUVmean(L) and SUVmean(L) normalized to SUVmean(BK) (SUVmean(L)-norm) were computed. The relevance of SUVmean(L)-norm and SUVmean(BK) to discriminate tumor luminal subtypes (A, B, B2, HER2, TN) [1] was investigated implementing linear-kernel SVM classification [2] . Performance was evaluated computing the Area (AUC) Under the Receiver Operating Characteristic (ROC) curve [3] , estimated in leave-one-out cross-validation to ensure an unbiased estimate of the classifier performance. Results Significant positive correlations of SUVmean(L) (p = 0.007) and SUVmean(L)-norm (p = 0.003) with proliferative index were found. Considering the features SUVmean(L)-norm and SUVmean(BK), multiclass classification was not able to discriminate between all luminal categories with performances significantly different from the chance level. By contrast, considering two-class classification, AUC was enhanced to 81% when considering luminal TN vs others and 89% for luminal A vs others. Conclusions Quantitative features extracted from PET images were correlated with prognostic clinical and histopathological factors in mammary carcinoma. Moreover, the SVM-based classification demonstrated a predictive value of PET images.

Invasive Ductal Mammary Carcinoma in a Cow

Background: Mammary gland carcinoma is a malignant epithelial neoplasm that can be classified as solid, papillary, ductal, invasive micropapillary, or invasive ductal. They are rarely diagnosed in livestock animals. Reports describing the breast as primary site of tumor in cows are scarce. In this case report, pathological and clinical findings of primary carcinoma ofthe mammary gland in a cow are reported.Case: A 7-year-old crossbred pregnant cow exhibited an increase in volume in the posterior region of the udder. Palpation raised suspicion of mastitis. The prefemoral lymph nodes (LNs) were enlarged. After calving, the clinical condition worsened, and edema was observed in the ventral region, from the udder to the neck. Poisoning by Amaranthus sp. was suspected. The cow died fifteen days after calving. Necropsy was performed. The affected mammary gland and its LNs, fragments of abdominal organs, brain, pelvic limbs, and udder skin were routinely prepared and stained with hematoxylin and eosin. Gross examination revealed significant alterations in the mammary gland, and in the mammary and prefemoral LNs. The mammary gland was firm and swollen; with loss of normal architecture, and released purulent, pinkish, fetid contents. The mammary LNs were enlarged, with diffuse coalescentmultifocal lesions, loss of corticomedullary delimitation, and the content was similar to that observed in the udder. The internal prefemoral LNs were also enlarged and contained the same fetid contents. Histopathology revealed proliferation of neoplastic ducts coated by numerous neoplastic cells; some areas were solid. There was moderate to severe pleomorphism, with moderate anisocytosis and anisokariosis. Most of the cells had vesicular nuclei, with up to two evident nucleoli. Other cells had dense chromatin, and moderately eosinophilic cytoplasm. There was marked cell dissociation, and cell detachment. The central portionsof the ducts were necrotic, with a predominantly neutrophilic inflammation. The mitotic index was low, but some areas had up to four mitotic figures per high power field. Numerous areas exhibited multiple foci of deposition of organized granular basophilic material, interpreted as mineralized areas, and stained positively for Von Kossa stain. Some areas of the mammary gland exhibited proliferative acini and ducts, composed of a single layer of cuboidal or columnar cells with moderately eosinophilic, vacuolated cytoplasm, and a basal rounded nucleus of generally aggregated chromatin (mammary hyperplasia). Many acini were dilated, and some contained circumscribed basophilic material (corpora amylacea). The structural architecture of the LNs was altered by malignant neoplastic infiltration, similar to that found in the mammary gland. No metastatic lesions were detected in the other organs. Immunohistochemistry (IHC) was performed using the streptavidin-biotin-peroxidase technique. Anti-pan-cytokeratin and antivimentin antibodies were used. Neoplastic cells exhibited strong and uniform cytoplasmic immunoreactivity for pan-cytokeratin. Expression of vimentin was absent in the neoplastic cells, but was present in the connective tissue associated with the neoplasm.Discussion: Invasive ductal mammary carcinoma was confirmed by histopathological and IHC analyses. Edema can be explained by the neoplasm in the mammary gland and in the mammary LNs, with accumulation of fluid in the interstitium, caused by growth of the tumor and compression of large blood and lymphatic vessels. IHC confirmed the epithelial origin of the tumor and demonstrated the presence of epithelial tumor cells in the LNs, proving to be metastatic. Similar findingswere reported in a cow with a highly aggressive secretory mammary carcinoma, in which neoplastic cells exhibited immunoreactivity to cytokeratin, indicative of their epithelial origin.Keywords: histology, immunohistochemistry, neoplasms, ruminant.

Severe cognitive decline following chemotherapy of breast cancer

Chemotherapy for different types of cancer is often life-saving. On the other hand, the spectrum of side effects may involve the central nervous system (CNS) and lead to severe emotional and cognitive disturbances, a syndrome often referred to as “The Chemo Brain”. Here we report on a then 43-year-old female patient who first presented to our clinic in 2013 and in whom breast cancer of the right breast had been diagnosed four years earlier. At that time she became a patient of the Comprehensive Cancer Center of the University of Ulm (CCCU) and underwent breast-conserving therapy starting with systemic chemotherapy according to the rules of the GeparQuinto Study. This was followed by breast-conserving operation including axillary lymphonodectomy. Histological examination of the excised material revealed an invasive ductal mammary carcinoma the exact staging of which will be reported in detail. Convalescence of the patient was protracted, and she continued complaining of difficulties to concentrate and to remember what she had been told or read shortly before. In addition, she complained of frequent mood swings. Therefore, she was referred to our department. Exploring her case history, we learned that she had worked as a manager of a car rental company which she had no longer been able to do after her cancer treatment because of her cognitive deficits. Beside clinical and psychiatric examination, our diagnostic procedures included electroencephalography (EEG), magnetic resonance tomography (MRT) of the brain using gadolinium enhancement, and extensive neuropsychological testing. In detail, components of the Wechsler Adult Intelligence Scale (WAIS), the Ray Auditory Verbal Learning Test (RAVLT), the Multiple Choice Vocabulary (“Mehrfachwahl-Wortschatztest”), the “Regensburger Wortflussigkeitstest” (Regensburg Word Fluency Test, RWT), and the “Testbatterie zur Aufmerksamkeitsprufung” (Test Battery for the Assessment of Attention, TAP) were applied. Whereas the EEG was unremarkable, the MRT performed in 2013 showed brain atrophy with frontotemporal accentuation. As an accessory finding, an arachnoid cyst was detected at the left temporal pole. No metastases were found. Interestingly, we had the opportunity to compare these findings with those of an earlier MRT performed in 2011. In the latter one, the arachnoid cyst was also visible, but there was no pronounced brain atrophy. These observations raise the question if progressive brain atrophy might be associated with the syndrome of “Chemo Brain”. Neuropsychological examination revealed severe cognitive deficits in this patient – details of which will be reported – apparently due to the chemotherapy she had undergone. Possible mechanisms of action, treatment options, and ethical implications will be discussed.

Correlation of sonographic features of invasive ductal mammary carcinoma with age, tumor grade, and hormone‐receptor status

To determine whether presenting sonographic features of invasive ductal carcinomas (IDC) are associated with patient age, tumor histologic grade, and hormonal receptor status. Sonographic features of 101 consecutive cases of IDC seen at ultrasound were retrospectively assessed based on the BI-RADS criteria of posterior acoustic appearance, tumor margins, and echogenicity. Associations between sonographic features and tumor characteristics were statistically evaluated with attention to patient age. IDC with shadowing compared with unchanged posterior acoustic appearance were significantly more likely to be of low histologic grade (Odds Ratio [OR] = 5.00; p < 0.05) and estrogen receptor (ER) -positive (OR = 10.00; p < 0.05). Conversely, posterior enhancement was associated with ER-negative status (OR = 4.45; p < 0.01), particularly among patients younger than 60 years of age (OR = 5.36, p < 0.05). Circumscribed tumors were more often high grade, particularly among older women (p < 0.01), and hormone receptor--negative regardless of age group. Among older women, tumors with mixed echogenicity tended to be high grade and progesterone receptor--negative (p values < 0.05). Noncircumscribed borders were observed for all tumors with posterior shadowing, and 97% of such tumors were also ER positive. Sonographic features were significantly associated with tumor grade and hormone receptor status, with some differences based on patient age. Specifically, the presence of posterior shadowing was associated with lower histologic grade and ER-positive status, especially in older patients. In contrast, we found that posterior acoustic enhancement was more commonly associated with ER-negative status, especially in younger patients.

Abstract P5-03-06: Progesterone Activates MAPK Pathway in Estrogen Induced Mammary Carcinogenesis

Abstract In women, breast cancer is the most prevailing endocrine-related malignancy. Epidemiological and experimental studies argue that estrogens are central to its etiology. Estrogen-induced carcinogenesis in ACI rats is highly relevant as a model for human breast cancer. These estrogen-induced cancers exhibit aneuploidy, as do invasive ductal mammary carcinomas in women. It has been demonstrated that ovary intact ACI rats given estrogen induced high incidence of mammary cancers while ovariectomized rats given the same dose of estrogen for the same length of time did not induce mammary cancers. Recently, we demonstrated that ovariectomized ACI rats given estrogen plus progesterone had a high incidence of mammary cancer. In this context, it is essential to understand the possible role of progesterone, if any, as the other main ovarian hormone in mammary carcinogenesis. Our goal was to determine the role of progesterone in mammary cancer development by using carcinogenesis induced solely by hormones in ACI rats. To achieve our goal, we designed the following experiment. Ovary intact and ovariectomized rats were used in this study. The groups (n=30) tested were as follows: 1) ovary intact control (no hormone treatment); 2) ovary intact rats given 30 mg estradiol; 3) ovary intact rats given 30 mg estradiol plus 30 mg mifepristone; 4) ovary intact rats given 30 mg progesterone; 5) ovariectomized control (no hormone treatment); 6) ovariectomized rats given 30 mg estrogen; and 7) ovariectomized rats given 30 mg estradiol plus 30 mg progesterone. Mammary glands were surgically excised and snap frozen in liquid nitrogen and stored at -80°C from a subset (n=4) of intact animals after four weeks of treatment and ovariectomized rats after twelve weeks of treatment. In rest of the animals mammary carcinogenesis was followed for nine months. As expected, estradiol induced mammary cancers in ovary intact rats and not in ovariectomized rats. Addition of progesterone to ovariectomized rats treated with estradiol induced high incidence of mammary cancer. Mifepristone treatment inhibited mammary carcinogenesis in ovary intact rats receiving estradiol. Pathway focused microarray analysis of mammary glands from the rats of different groups and treatment indicated alterations in apoptosis, MAPK and PI3K pathways. Estrogen plus progesterone treatment down regulated apoptotic genes such as Apaf1, Bad, Bax, Bid, Casp9 and up regulated Mapk3, Grb10, Itgb1, PrKca, Prkcz and Map4k1, Map2k1ip1, Mapk8ip2, Mapkapk5 in PI3K and MAPK pathways respectively when compared with estrogen alone treatment. We have observed differences both at transcriptional and translational levels. Our results clearly demonstrate the significance of progesterone in mammary carcinogenesis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-03-06.

Statistical analysis of labelling patterns of mammary carcinoma cell nuclei on histological sections

It is of central interest for tumour biology to explore the mechanisms of tumour cell proliferation. In this study, methods of spatial statistics were used to study the spatial distribution of proliferating cells within tumour tissue quantitatively and objectively. Mammary cancer tissue was studied as an example. It was attempted to clarify whether cell division occurs entirely at random (random labelling), i.e. the process of division occurs at random, independently from the state of the neighbouring nuclei, or whether the spatial distribution of proliferation is more complex, e.g. in the form of actively proliferating clusters alternating with relatively silent zones. In the case of random labelling, the reduced second moment functions K(r) of the labelled and the unlabelled nuclei would be identical. The same would hold for the pair correlation functions g(r). The alternative hypothesis is that the second-order properties of the processes of the labelled and the unlabelled nuclei are different. Twenty cases of invasive ductal mammary carcinomas were studied. The nuclei of proliferating cells were stained immunohistochemically with the monoclonal antibody MIB-1, which detects specifically the proliferation-associated nuclear antigen Ki 67. The planar coordinates of the tumor cell nucleus profiles from two rectangular visual fields per case were recorded. For each visual field, the following investigations were performed: estimation of the explorative summary characteristics K(r) and g(r), fitting of the parameters of a stationary Strauss hard-core model to the observed point patterns, estimation of two distance-dependent Simpson indices and Monte Carlo tests of all individual patterns on the null hypothesis of random labelling. Significant differences between the mean K-functions and the mean g-functions of the labelled and the unlabelled nuclei were found. Moreover, the mean interaction parameter gamma of the stationary Strauss hard-core model was significantly higher for the labelled nuclei than for the unlabelled nuclei. The estimates of the two distance-dependent Simpson indices showed a tendency of points with the same label towards a positive spatial correlation. In the Monte Carlo tests, the null hypothesis of random labelling was rejected for the majority of the visual fields. These four lines of investigation led to the concordant conclusion that the labelling of mammary carcinoma nuclei by MIB-1 is not simply random. The data suggest that the second-order properties of the point process of the labelled nuclei are significantly different from those of the unlabelled nuclei. In particular, the process of the labelled nuclei shows a higher degree of clustering (increased strength of interaction) than the process of the unlabelled points.

Pit-1 is expressed in normal and tumorous human breast and regulates GH secretion and cell proliferation

The transcription factor pituitary-1 (Pit-1) is mainly expressed in the pituitary gland, where it has critical roles in cell differentiation and as a transcriptional factor for GH and prolactin (PRL). It is also expressed in human extrapituitary tissues (placenta, lymphoid and haematopoietic tissues) and cell lines (human breast adenocarcinoma cells, MCF-7). Despite the widely suggested roles of GH and PRL in the progression of proliferative mammary disorders, Pit-1 expression in human mammary gland has not yet been reported. To evaluate the expression of Pit-1 in human breast and, using the MCF-7 cell line, to investigate whether Pit-1 overexpression regulates GH expression and increases cell proliferation. Using real-time RT-PCR, western blotting and immunohistochemistry, we evaluated the expression of Pit-1 mRNA and protein in seven normal human breasts and 14 invasive ductal mammary carcinomas. GH regulation by Pit-1 in MCF-7 cells was evaluated using RT-PCR, western blotting, ELISA and transfection assays. Cell proliferation was evaluated using bromodeoxyuridine. We found expression of Pit-1 mRNA and protein in both normal and tumorous human breast. We also found that Pit-1 mRNA levels were significantly increased in breast carcinoma compared with normal breast. In MCF-7 cells, Pit-1 overexpression increased GH mRNA and protein concentrations and significantly increased cell proliferation. These findings indicate that Pit-1 is expressed in human breast, that it regulates endogenous human mammary GH secretion, and that it increases cell proliferation. This suggests that, depending on its level of expression, Pit-1 may be involved in normal mammary development, breast disorders, or both.

Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1.

Almost any growth of tumors is to some extent associated with an inflammatory reaction which may be anti-tumorigenic by acting directly on tumor cells or protumorigenic cells presumably by inducing tumor-associated angiogenesis. In this study, we have analyzed the angiogenesis-inducing capacity of monocyte chemoattractant protein-1 (MCP-1), a key regulatory molecule of monocyte trafficking to sites of inflammation. MCP-1 was found to be potently angiogenic when implanted into rabbit cornea, exerting potency similar to the specific angiogenic vascular endothelial growth factor (VEGF)-A(121). MCP-1-induced angiogenesis in the cornea is associated with prominent recruitment of macrophages, whereas VEGF-A(121)-induced corneal angiogenesis is devoid of inflammatory cell recruitment. Based on these findings, we studied MCP-1 expression and macrophage recruitment in human invasive ductal mammary carcinomas in comparison with the physiological angiogenic processes in bovine ovarian corpus luteum. Macrophage recruitment was always associated with MCP-1 expression. High macrophage counts in mammary tumors corresponded with poor prognosis. In contrast, physiological ovarian angiogenesis was associated with only minimal inflammatory recruitment of macrophages. Our data show that MCP-1 is an indirect inflammation-associated inducer of angiogenesis and demonstrate distinct qualitative differences between tumor angiogenesis in human mammary tumors and physiological angiogenesis in the ovary.

Relationship Between p53 Expression and Other Prognostic Factors in Human Breast Carcinoma:An Immunohistochemical Study

Among 843 cases of breast cancer, p53 oncoprotein was detected by the monoclonal antibody (MoAb) Pab-1801 in only 13%. Low-grade carcinomas (tubular, mucinous, papillary, and invasive cribriform types) did not express p53 protein, but it was observed in 4.2% of infiltrating lobular carcinomas (6 of 140 cases) and 50% of pure medullary carcinomas (5 of 10 cases). In intermediate-grade neoplasms, no correlation was seen between p53 status and other putative determinants of a poor prognosis. The latter included high tumor stage, lymph nodal involvement, high growth fraction (as determined by labeling with the MoAb Ki-67), negative results for estrogen receptor (ER) and progesterone receptor (PR) proteins, and amplification of the c-erbB-2 oncogene product in the neoplastic cells. Ninety-nine of 640 (15.5%) cases of high-grade, invasive, ductal breast carcinoma, however, showed an inverse relationship between expression of p53 protein and positive results for ER/PR proteins and a direct correlation with large tumor size, Ki-67-determined growth fraction, and amplification of c-erbB-2 oncopeptide. All of the latter associations were highly significant statistically. The authors conclude that mutant p53 protein may serve a prognostic role in a subset of cases of invasive ductal mammary carcinoma.

The Impact of Nucleolar Organizer Regions for the Lymph Node Spread and Prognosis of Invasive Ductal Mammary Carcinoma

In primary tumours of 40 patients with invasive ductal carcinomas the significance of nucleolar organizer regions (NORs) for metastatic spread to the axillary lymph nodes and for the prognosis was assessed. Silver-stained tissue sections were investigated by means of semiautomated image analysis. The nucleolar organizer regions of 100 tumour cell nuclei per specimen were measured. The number as well as the area of the NORs were evaluated together with morphometrical and DNA features, histopathological and clinical data. By means of multivariate discriminant analysis, significant differences between tumours of 20 node-negative and 20 node-positive patients could be found. The mean number of NORs was significantly higher in patients with lymph node metastases (p = 0.0059), whereas the mean area was significantly lower in node-positive patients. By using the NOR number as the only parameter both groups were classified with an overall efficiency of 95%. There was also a significant difference between long-term and short-term survivors by considering the mean number of NORs, but the Auer-type, the 2 cDl value, and the DNA-grade of malignancy were of better predictive value. Within the group of node-negative patients the NOR number was most suitable for distinguishing between good and poor prognosis, whereas within the group of node-positive patients once more the DNA parameters played the most decisive role for predicting prognosis. With regard to the small number of patients the results have to be considered as preliminary. Further investigations in a more extensive population are necessary.

Endogenous Lectins with Specificity to β-Galactosides and α- or β-N-Acetyl-Galactosaminides in Human Breast Cancer: Their Glycohistochemical Detection in Tissue Sections by Synthetically Different Types of Neoglycoproteins, Their Quantitation on Cultured Cells by Neoglycoenzymes and Their Usefulness as Targets in Lectin-Mediated Phototherapy in vitro

Endogenous lectins may augment the panel of tumor markers. Specific protein-carbohydrate interactions especially involve carbohydrate moieties that are located at sequence termini, e.g. D-galactose and N-acetyl-D-galactosamine. Respective endogenous lectins can be detected by suitably constructed neoglycoproteins. In order to evaluate the influence of sugar and label density as well as coupling mode of the carbohydrate moiety to the carrier protein for lectin localization in histopathology, four different types of neoglycoproteins, carrying beta-galactosides or alpha- and beta-anomers of N-acetyl-D-galactosamine were employed to reveal the presence of specific receptors in invasive ductal mammary carcinomas with propensity for metastasis formation. Staining of tumor cells was more intense than staining of normal cell types. Coupling of the diazo derivatives of p-aminophenyl glycosides led in most cases to the relatively highest extent of staining in terms of number of stained cells and staining intensity. Classified next according to these categories attachment of sugars via p-isothiocyanato derivatives or via an aliphatic linker after his reaction with the C6-hydroxyl group of the sugar moiety was rather equally well effective, whereas reductive amination with concomitant ring opening at the reducing end of the disaccharide lactose resulted in neoglycoproteins, yielding the lowest extent of staining. The alpha-anomer is preferred as a ligand to endogenous lectins of tumor cells to the beta-anomer of N-acetyl-D-galactosamine. To reduce the number of steps in glycohistochemical processing, glycosylated enzymes were successfully employed. They also allowed to measure the lectin density on breast carcinoma cells, leading to rational selection for demonstrated lectin-mediated targeting of neoglycoprotein-hematoporphyrin conjugates. Immobilization of ligands as an approach to prepare histochemically valuable reagents to localize respective receptors is not confined to tumor lectinology, as emphasized by additional application of hormone-protein conjugates, termed neohormoproteins.

Grading and Prognosis of Invasive Ductal Mammary Carcinoma by Nuclear Image Analysis in Tissue Sections

In primary tumours of 84 patients with invasive ductal carcinomas the prognostic significance of the nuclear image was assessed. Feulgen stained tissue sections were investigated by image cytometry. At least 150 tumour cell nuclei as well as 50 lymphocytes or trout erythrocytes for diploid reference were measured in each specimen. 35 morphological features, including those of the chromatin pattern, were derived from the digitized nuclear images. By means of multivariate discriminant analysis, significant differences could be found between tumours of 41 node-negative and 43 node-positive patients. The differences were sufficiently large to distinguish subgroups with low and high risk of lymph node involvement. Irrespective of lymph node status, 66 patients with an adequate 5 year follow-up were divided into two groups according to their survival time. The classification results revealed a clear relationship between characteristics of the nuclear image and prognosis. By image cytometry it is thus possible to separate both node-positive and node-negative patients into subgroups with increasing risk of recurrence. The results indicate that nuclear image analysis provides prognostic information in addition to established prognostic factors, such as histological grade and tumour stage.