Modelling the early growth of ductal carcinoma in situ of the breast

The growth of a tumour in a rigid walled cylindrical duct is examined in order to model the initial stages of tumour cell expansion in ductal carcinoma in situ (DCIS) of the breast. A nutrient-limited growth model is formulated, in which cell movement is described by a Stokes flow constitutive relation. The effects on the shape of the tumour boundary of the material properties (i.e. the viscosity) and the extent to which the cells adhere to the duct wall are studied using numerical and asymptotic methods. It is shown how stable, non-planar, interface configurations result and that, during these initial stages, before the duct wall has been breached, few cells die and a nutrient-rich model is usually sufficient to capture the behaviour. Finally, we discuss the relevance of this approach to DCIS and suggest possible avenues for further work. Send offprint requests to:S.J. Franks at Centre of Mathematical Medicine.     

A quantitative theoretical model for the development of malignancy in ductal carcinoma in situ

Mathematical models and clinical observations have demonstrated that microenvironmental hypoxia and acidosis are important selection factors during the later stages of the somatic evolution of breast cancer. The consequent promotion of constitutive upregulation of glycolysis and resistance to acid-induced cellular toxicity is hypothesized to be critical for the ability of cancer cells to invade host tissue. In this work we developed a 3D fixed lattice cellular automata model to study the role of these two phenotypes in determining morphology and the potential for invasion of ductal carcinoma in situ (DCIS), which in this work is defined as the erosion of a healthy epithelial cell layer and direct contact with the basement membrane. The model was conceived as a 40-cell wide epithelial duct surrounded by blood vessels and composed of a basement membrane and one internal layer of epithelial cells. Our results show that an increment in the order of 8-fold in glucose metabolism and an increase in acid resistance corresponding to pH thresholds of approximately 6.8 and 6.45 for quiescence and death, respectively, are required for the tumor to breach through the layer of healthy epithelial cells and reach the basement membrane as a first step for invasion. Our model also suggests correlations between classic morphologies and different values of hyperglycolytic and acid-resistant phenotypes, indicating that immunohistochemistry studies targeting these genes may improve the predictive power of morphological analyses of biopsies.     

Microarray and RNA in situ hybridization assay for recurrence risk markers of breast carcinoma and ductal carcinoma in situ: Evidence supporting the use of diverse pathways panels

Breast tumor stratification by recurrence-risk is critical for deciding patient treatment. Here an approach combining cancer pathways microarray data complemented by RNA in situ hybridization (ISH) was investigated as a means for recurrence marker discovery and visualization in pathology specimens. LncRNA and mRNA expressions in breast carcinomas with low (n = 8) vs intermediate/high (n = 10) recurrence-scores as estimated by 21-gene assay and pathology review were compared by microarray assay. Tissue microarrays were prepared from breast carcinomas (n = 20) and ductal carcinoma in situ (DCIS) specimens (n = 84 patients) with known outcomes. Thirteen RNA ISH assays were performed: lncRNAs (BBC3-1, FER3, RAD21-AS1, ZEB1-2) and mRNAs (GLO1, GLTSCR2, TGFB1, TLR2) (implicated by the microarray data); MKI67; a pooled panel of recurrence-associated proliferation markers (BIRC5, Cyclin B1, MKI67, MYBL2, STK15); a pooled panel of non-proliferation recurrence-associated markers (CEACAM5, HTF9C, NDRG1, TP53, SLC7A5); and lncRNAs H19 and HOTAIR. Seven lncRNAs and 10 mRNAs showed significantly (P < .05) altered upregulation or downregulation by microarray assay: carcinoma RNA ISH staining did not mirror these patterns. HOTAIR staining was associated with a higher breast cancer recurrence score (P = .0152); qualitatively, H19 was massively expressed in a metaplastic triple negative breast carcinoma. Among the DCIS cohort, significant associations with multiple outcome variables were noted for TGFB1 and the non-proliferation panel (P-value range: .0001 to .047); proliferation panel staining showed an association with increasing DCIS grade (P = .0269) but not with outcomes. The findings support recurrence-risk estimation by the use of multi-marker panels that are representative of diverse cellular pathways rather than over-reliance on proliferation targets. H19, HOTAIR, and TGFB1 RNA ISH show potential for selective diagnostics.     

Loss of caveolin-1 and gain of MCT4 expression in the tumor stroma: Key events in the progression from an in situ to an invasive breast carcinoma

The progression from in situ to invasive breast carcinoma is still an event poorly understood. However, it has been suggested that interactions between the neoplastic cells and the tumor microenvironment may play an important role in this process. Thus, the determination of differential tumor-stromal metabolic interactions could be an important step in invasiveness.

The expression of stromal Caveolin-1 (Cav-1) has already been implicated in the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Additionally, stromal Cav-1 expression has been associated with the expression of stromal monocarboxylate transporter 4 (MCT4) in invasive breast cancer. However, the role of stromal MCT4 in invasiveness has never been explored, neither the association between Cav-1 and MCT4 in the transition from breast DCIS to IDC.

Therefore, our aim was to investigate in a series of breast cancer samples including matched in situ and invasive components, if there was a relationship between stromal Cav-1 and MCT4 in the progression from in situ to invasive carcinoma. We found loss of stromal Cav-1 in the progression to IDC in 75% of the cases. In contrast, MCT4 stromal expression was acquired in 87% of the IDCs. Interestingly, a concomitant loss of Cav-1 and gain of MCT4 was observed in the stroma of 75% of the cases, when matched in situ and invasive carcinomas were compared. These results suggest that alterations in Cav-1 and MCT4 may thus mark a critical point in the progression from in situ to invasive breast cancer.     

Ductal carcinoma in situ of the breast: morphological and molecular features implicated in progression

The spread of mammographic screening programmes around the world, including in developing countries, has substantially contributed to the diagnosis of small non-palpable lesions, which has increased the detection rate of DCIS (ductal carcinoma in situ). DCIS is heterogeneous in several ways, such as its clinical presentation, morphology and genomic profile. Excellent outcomes have been reported; however, many questions remain unanswered. For example, which patients groups are overtreated and could instead benefit from minimal intervention and which patient groups require a more traditional multidisciplinary approach. The development of a comprehensive integrated analysis that includes the radiological, morphological and genetic aspects of DCIS is necessary to answer these questions. This review focuses on discussing the significant findings about the morphological and molecular features of DCIS and its progression that have helped to uncover the biological and genetic heterogeneity of this disease. The knowledge gained in recent years might allow the development of tailored clinical management for women with DCIS in the future.     

Cellular automaton simulation examining progenitor hierarchy structure effects on mammary ductal carcinoma in situ

A computer simulation is used to model ductal carcinoma in situ, a form of non-invasive breast cancer. The simulation uses known histological morphology, cell types, and stochastic cell proliferation to evolve tumorous growth within a duct. The ductal simulation is based on a hybrid cellular automaton design using genetic rules to determine each cell's behavior. The genetic rules are a mutable abstraction that demonstrate genetic heterogeneity in a population. Our goal was to examine the role (if any) that recently discovered mammary stem cell hierarchies play in genetic heterogeneity, DCIS initiation and aggressiveness. Results show that simpler progenitor hierarchies result in greater genetic heterogeneity and evolve DCIS significantly faster. However, the more complex progenitor hierarchy structure was able to sustain the rapid reproduction of a cancer cell population for longer periods of time.     

Lapatinib inhibits stem/progenitor proliferation in preclinical in vitro models of ductal carcinoma in situ (DCIS)

Breast-conserving surgery for ductal carcinoma in situ (DCIS) is often combined with irradiation, reducing recurrence rates to 20% within 10 years; however, there is no change in overall survival. Evidence in the invasive breast indicates that breast cancer stem cells (CSCs) are radiotherapy-resistant and are capable of re-initiating a tumor recurrence; hence, targeting CSCs in high risk DCIS patient may improve survival. HER2 is overexpressed in 20% of DCIS and is known to be highly active in breast CSCs; we therefore investigated the effect of Lapatinib on DCIS CSC activity using 2 in vitro culture systems. Two DCIS cell lines DCIS.com (HER2 normal) and SUM225 (HER2 overexpressed) as well as DCIS cells from patient samples (n = 18) were cultured as mammospheres to assess CSC activity and in differentiated 3D-matrigel culture to determine effects within the non-CSCs. Mammosphere formation was reduced regardless of HER2 status, although this was more marked within the HER2-positive samples. When grown as differentiated DCIS acini in 3D-matrigel culture, Lapatinib only reduced acini size in the HER2-positive samples via decreased proliferation. Further investigation revealed lapatinib did not reduce self-renewal activity in the CSC population, but their proliferation was decreased regardless of HER2 status. In conclusion we show Lapatinib can reduce DCIS CSC activity, suggesting that the use of Lapatinib in high-risk DCIS patients has the potential to reduce recurrence and the progression of DCIS to invasive disease.     

Molecular subtypes of ductal carcinoma in situ in African American and Caucasian American Women: Distribution and correlation with pathological features and outcome

Background: Molecular subtypes of breast cancer have been extensively studied in invasive carcinoma. They were shown to have a different distribution within the various ethnic populations. Few studies have applied the same classification to Ductal Carcinoma in Situ (DCIS). We report the distribution of the molecular breast cancer subtypes in DCIS between African American (AA) and Caucasian American (CA) women, their association with pathological features and outcome. Materials and methods: Tissue microarrays were constructed from paraffin blocks of 94 DCIS cases (67 AA and 27 CA) selected from a cohort of AA and CA patients diagnosed with DCIS between 1996 and 2000; mean age at diagnosis was 61 ± 12 for the AA and 58 ± 11 years for the CA group. TMA blocks were labeled with antibodies for ER, PR, HER2, Ki-67, and CK5/6. The cases were subtyped as Luminal A (ER+ and/or PR+; HER2?), Luminal B (ER+ and/or PR+; HER2+), HER2+ (ER?, PR?; HER2+), basal-like (BL) (ER?, PR?, HER2?; CK5/6+) or unclassified triple negative (UTN) (ER?, PR?, HER2?, CK5/6?). Information on grade, size and follow-up were obtained. Results: (1) Most DCIS cases were Luminal A, comprising 80% of the DCIS cases in AA and 92.6% in CA patients. (2) HER2+, BL and UTN DCIS subtypes were not seen in the CA population, and formed 9% of the DCIS cases in the AA population; these cases were all high grade. (3) In the cases with recurrence (8 AA and 1 CA patients), DCIS was Luminal A in 6 AA and 1 CA and Luminal B in 2 AA patients. Conclusion: The distribution of the molecular subtypes of DCIS did not show a significant difference between the two ethnic groups in our study. In addition, the risk of recurrence might not be higher in the non-luminal subtypes than in Luminal A and B.     

EZH2 and ALDH1 expression in ductal carcinoma in situ: Complex association with recurrence and progression to invasive breast cancer

The diagnosis of ductal carcinoma in situ (DCIS) is an increasingly common event due to widespread use of screening mammography. However, appropriate clinical management of DCIS is a major challenge in the absence of prognostic markers. Tumor-initiating cells may be particularly relevant for disease pathogenesis; therefore, two markers associated with such cells, EZH2 and ALDH1, were evaluated. A cohort of 248 DCIS patients was used to determine the association of EZH2 and ALDH1 with ipsilateral breast event, DCIS recurrence and progression to invasive breast cancer (IBC). In this cohort, high EZH2 expression was associated with the risk of an ipsilateral breast event and DCIS recurrence but not invasive progression. ALDH1 expression was observed in both the tumor and stromal compartment; however, in neither compartment were ALDH1 levels independently associated with evaluated study endpoints. Interestingly, the combination of high EZH2 with high epithelial ALDH1 was associated with disease progression. Therefore, ALDH1 within the DCIS lesion can add to the prognostic significance of EZH2, particularly in the context of risk of development of invasive disease.     

A novel, patient-specific mathematical pathology approach for assessment of surgical volume: application to ductal carcinoma in situ of the breast

We introduce a novel "mathematical pathology" approach, founded on a biophysical model, to identify robust patient-specific predictors of tumor growth useful in clinical practice to improve the accuracy of diagnosis/prognosis and intervention. In accordance with biological observations, our model simulates the diffusion-limited in situ tumors with a relatively short phase of fast initial growth, followed by a prolonged slow-growth phase where tumor size is constrained primarily by the relative weight of cell mitosis and death. The former phase may only last for a few months, so that at the time of diagnosis, we may assume that most tumors will have entered the phase where their size is changing slowly. Based on this prediction, we hypothesize that the volume of breast with ducts affected by in situ tumors at the time of diagnosis will be closely approximated by a model-derived mathematical function based on the ratio of tumor cell proliferation-to-apoptosis indices and on the extent of diffusion of cell nutrients (diffusion penetration length), which can be measured from immunohistochemical and morphometric analysis of patient histopathology specimens without the need for multiple-time measurements. We tested this idea in a retrospective study of 17 patients by staining breast tumor specimens containing ductal carcinoma in situ for mitosis with Ki-67 and for apoptosis with cleaved caspase-3 and counting cells positive for each marker. We also determined diffusion penetration by measuring the thickness of viable rims of tumor cells within ducts. Using the ensuing ratios, we applied the model to determine a predicted surgical volume or tumor size. We then corroborated our hypothesis by comparing the predicted size of each tumor based on our model with the actual size of the pathological specimen after tumor excision (R2 = 0.74-0.88). In addition, for the 17 cases studied, both histological grade and mammography were not found to correlate with tumor size (R2 = 0.08-0.47). We conclude that our mathematical pathology approach yields a high degree of accuracy in predicting the size of tumors based on the mitotic/apoptotic index and on diffusion penetration. By obtaining these ratios at the time of initial biopsy, pathologists can employ our model to predict the size of the tumor and thereby inform surgeons how much tissue to remove (surgical volume). We discuss how results from the model have implications concerning the current debate on recommendations for screening mammography, while the model itself may contribute to better planning of breast conservation surgery.     

Neuroendocrine ductal carcinoma in situ of the breast: cytological features in 32 cases

T. Kawasaki, S. Nakamura, G. Sakamoto, T. Kondo, H. Tsunoda‐Shimizu, Y. Ishii, T. Nakazawa, K. Mochizuki, T. Yamane, M. Inoue, S. Inoue and R. Katoh
Neuroendocrine ductal carcinoma in situ of the breast: cytological features in 32 cases Objective: The purpose of this study was to clarify the cytological features of neuroendocrine ductal carcinoma in situ (NE‐DCIS) of the breast. Methods: We analysed the cytopathological findings in 22 fine needle aspiration (FNA) smears and 17 nipple discharge smears obtained from 32 Japanese patients with NE‐DCIS. Results: The background of the FNA smears was clear (59%), mucoid (23%), haemorrhagic (14%) or necrotic (5%). Most of the FNA smears (95%) showed high cellularity. Characteristically, NE‐DCIS cells were loosely arranged in three‐dimensional solid clusters or singly dispersed. Well‐developed vascular cores with or without malignant cells were occasionally recognized. The tumour cells were polygonal or spindle‐shaped with a fine granular, abundant cytoplasm. Nuclei with finely granular chromatin were round or oval and often eccentrically located (plasmacytoid appearance). Mitotic figures were infrequent. Nuclear grade was estimated to be low in 86%. Most nipple discharge smears had fairly low cellularity with poorly preserved cell clusters in a markedly haemorrhagic background, although two (12%) were extremely cellular with cytological characteristics similar to those of the FNA smears. Pre‐operative cytological malignant diagnoses were made in 42% of FNA smears and 0% of nipple discharge smears. Immunohistochemistry for neuroendocrine markers (chromogranin A and synaptophysin) confirmed the neuroendocrine nature of this tumour in adequate cytological specimens. Conclusions: NE‐DCIS has distinctive cytological features and can therefore be diagnosed as a neuroendocrine tumour in most FNAs and some nipple discharge smears by cytological examination employing immunohistochemical techniques. We emphasize that a breast lesion with these features may be in situ and not invasive, and also that there is a risk of under‐diagnosis.     

Nuclear chromatin characteristics of breast solid pattern ductal carcinoma in situ.

OBJECTIVE: To characterize nuclei from breast solid pattern ductal carcinoma in situ (DCIS) by their karyometric features and to search for the presence of statistically significantly different subsets of nuclei. STUDY DESIGN: One hundred nuclei from each of 6 normal, 13 solid DCIS, (9 low and intermediate grade and 4 high grade DCIS) histopathologic samples of breast tissue were digitally recorded. Karyometric features were computed and subjected to a nonsupervised learning algorithm (P-index) to identify significantly different subgroups. RESULTS: Nuclei in low grade lesions displayed a diploid/near diploid pattern, while the majority of intermediate grade lesions fell into a range beyond 5N. The high grade lesions showed substantial genomic instability and represented three statistically different subsets or phenotypes. CONCLUSION: There is a progression of nuclear abnormality from low grade to high grade DCIS. The nuclei from high grade DCIS form a heterogeneous set that represents three phenotypes. One of these phenotypes shows a nuclear chromatin pattern that more closely resembles poorly differentiated, infiltrating disease. The observation of such a phenotype may have prognostic implications.     

Computerized image analysis of Ki-67 in ductal breast carcinoma in situ.

OBJECTIVE: To develop and determine the staining protocols and computerized image analysis methods that are the most effective combination for performing quantitative analysis of Ki-67. STUDY DESIGN: We compared conventional bright-field light microscopy and refractive optical enhancement methods in combination with various immunodetection and filter enhancement methods, including immunogold in combination with epipolarization refractive optics and enzymatic conversion of chromogenic substrates in combination with optical filter enhancement. Initial Ki-67 tests were performed on lymph node tissues and cultured human breast cells and then applied to 200 ductal carcinoma in situ (DCIS) samples. DCIS acini were digitally acquired, and a region of interest was manually outlined in each one with a digital stylus to include only the cellular component; then the Ki-67 staining index was quantified by segmentation analysis. RESULTS: Although combining epipolarization analysis with immunohistogold staining was the most sensitive detection method, nonspecific binding was too high. The streptavidin-horseradish-peroxidase enzymatic conversion of 3,3'-diaminobenzidine (DAB) in combination with optical enhancement filters was the most effective method tested. Ki-67 stain was associated with dense fibrillar structures of the nucleoli in the less intensely staining nuclei and was most intense in paired nuclei. CONCLUSION: The method of measuring Ki-67 expression by DAB staining combined with optical enhancement filters and quantification via computer-assisted image analysis techniques produced objective and reproducible results. As such, this method can offer (1) less intraobserver and interobserver variability, (2) a digital archival record, and (3) a baseline for digital exchange of information between studies.     

Aromatase and in situ estrogen production in DCIS (ductal carcinoma in situ) of human breast

Ductal carcinoma in situ or DCIS belongs to intraductal proliferative lesions, which are a group of cytologically and architecturally diverse ductal proliferations, typically originating from the terminal duct–lobular units. In these intraductal proliferative diseases, estrogens are considered to be involved in the progression of the disease especially from ductal non-neoplastic hyperplasia to DCIS and possibly development of invasive carcinoma from DCIS. Estrogen receptor (ER) alpha is abundantly expressed in atypical ductal hyperplasia and low grade DCIS. Suppression of estrogenic actions using tamoxifen resulted in inhibition of recurrence of DCIS and/or of progression into invasive carcinoma. Intratumoral estrogen concentration in DCIS determined by liquid chromatography/electrospray tandem mass spectrometry is significantly higher than that in non-neoplastic breast tissues with statistically not lower than that in invasive carcinoma. Aromatase mRNA expression in both stromal and parenchymal cells of DCIS determined by quantitative RT-PCR following laser capture microdissection was also much higher than that in non-neoplastic breast, although lower than that in invasive carcinoma. Immunohistochemistry of aromatase also revealed the similar patterns of immunolocalization as in invasive carcinoma. Aromatase is overexpressed in noninvasive breast malignancies including DCIS and results in elevated concentrations of intratumoral estradiol. These findings could provide the scientific rationale as to employing aromatase inhibitors in the management of ER positive DCIS patients.     

Cytologic characteristics of endocrine ductal carcinoma in situ of the breast. A case report

BACKGROUND: Endocrine ductal carcinoma in situ is a rare form of ductal carcinoma in situ. It is regarded as a distinct subgroup of mammary carcinoma. However, the cytologic features of endocrine ductal carcinoma in situ have not been previously reported. CASE: A case of endocrine ductal carcinoma in situ exhibited characteristic cytologic findings on a specimen obtained by the scrape method (stained with hematoxylin and eosin and Diff-Quik). CONCLUSION: The cytologic criteria for endocrine ductal carcinoma in situ are sufficiently distinct and are useful for making the diagnosis on fine needle aspiration.     

Molecular differences between ductal carcinoma in situ and adjacent invasive breast carcinoma: a multiplex ligation-dependent probe amplification study

Ductal carcinoma in situ (DCIS) accounts for approximately 20% of mammographically detected breast cancers. Although DCIS is generally highly curable, some women with DCIS will develop life-threatening invasive breast cancer, but the determinants of progression to infiltrating ductal cancer (IDC) are largely unknown. In the current study, we used multiplex ligation-dependent probe amplification (MLPA), a multiplex PCR-based test, to compare copy numbers of 21 breast cancer related genes between laser-microdissected DCIS and adjacent IDC lesions in 39 patients. Genes included in this study were ESR1, EGFR, FGFR1, ADAM9, IKBKB, PRDM14, MTDH, MYC, CCND1, EMSY, CDH1, TRAF4, CPD, MED1, HER2, CDC6, TOP2A, MAPT, BIRC5, CCNE1 and AURKA.There were no significant differences in copy number for the 21 genes between DCIS and adjacent IDC. Low/intermediate-grade DCIS showed on average 6 gains/amplifications versus 8 in high-grade DCIS (p=0.158). Furthermore, alterations of AURKA and CCNE1 were exclusively found in high-grade DCIS, and HER2, PRDM14 and EMSY amplification was more frequent in high-grade DCIS than in low/intermediate-grade DCIS. In contrast, the average number of alterations in low/intermediate and high-grade IDC was similar, and although EGFR alterations were exclusively found in high-grade IDC compared to low/intermediate-grade IDC, there were generally fewer differences between low/intermediate-grade and high-grade IDC than between low/intermediate-grade and high-grade DCIS.In conclusion, there were no significant differences in copy number for 21 breast cancer related genes between DCIS and adjacent IDC, indicating that DCIS is genetically as advanced as its invasive counterpart. However, high-grade DCIS showed more copy number changes than low/intermediate-grade DCIS with specifically involved genes, supporting a model in which different histological grades of DCIS are associated with distinct genomic changes that progress to IDC in different routes. These high-grade DCIS specific genes may be potential targets for treatment and/or predict progression.     

Molecular characterization of the transition to malignancy in a genetically engineered mouse-based model of ductal carcinoma in situ

A transplantable model of human ductal carcinoma in situ that progresses to invasive carcinoma was developed from a genetically engineered mouse (GEM). Additional lines were established using early mammary premalignant lesions from transgenic MMTV-PyV-mT mice. These lines were verified to be premalignant and transplanted repeatedly to establish stable and predictable properties. Here, we report the first in-depth molecular analysis of neoplastic progression occurring in one premalignant transplantable GEM-derived line. Oligonucleotide microarrays showed that many genes are differentially expressed between the quiescent and prelactating mammary gland and the premalignant GEM outgrowth. In contrast, a small but consistent group of genes was associated with the transformation from premalignancy to tumor. This suggests that the majority of gene expression changes occur during the premalignant transition from normal to premalignancy, whereas many fewer changes occur during the malignant transition from premalignancy to invasive carcinoma. The premalignant transition is associated with several cell cycle-related genes and the up-regulation of oncogenes is associated with various cancers (Ccnd11, Cdk4, Myb, and Ect2). The changes identified in the malignant transition included genes previously associated with human breast cancer progression. Misregulation of the insulin-like growth factor and transforming growth factor-beta signaling pathways and the stromal-epithelial interaction were implicated. Our results suggest that this transplantable GEM-based model recapitulates human ductal carcinoma in situ at both histologic and molecular levels. With consistent tumor latency and molecular profiles, this model provides an experimental platform that can be used to assess functional genomics and molecular pharmacology and to test promising chemoprevention strategies.