|Year : 2017 | Volume
| Issue : 3 | Page : 56-62
Genetic investigation of breast ductal carcinoma In situ: A literature review
Daniel Bankole Olusina1, Nnaemeka Thaddeus Onyishi2
1 Department of Morbid Anatomy, University of Teaching Hospital, Ituku Ozalla, Enugu, Nigeria
2 Department of Histopathology, Enugu State University, Teaching Hospital Parklane, Enugu, Nigeria
|Date of Web Publication||26-Dec-2017|
Dr. Nnaemeka Thaddeus Onyishi
Department of Histopathology, Enugu State University, Teaching Hospital Parklane, Enugu
Source of Support: None, Conflict of Interest: None
The incidence of ductal carcinoma in situ (DCIS) of the breast has greatly increased in countries with breast cancer early detection programs. However, uncertainty remains about its natural history and precise implication of its diagnosis with growing concern in some quarters about possible overdiagnosis and overtreatment. Various molecular techniques have been applied to the investigation of DCIS in an attempt to clarify its biology in relation to invasive breast cancer. The following is a historical tour of some of those molecular studies and the contributions they have made to our understanding of DCIS. It is preceded by a recap of the DCIS conundrum and the uncertainties surrounding its natural history. Peer-reviewed scientific publications on the subject were retrieved by search of PubMed databases. The search was conducted with the following string of keywords: “breast carcinoma in situ genetics,” “breast carcinoma in situ molecular biology,” and “ductal carcinoma in situ molecular method technique.” Reference lists of retrieved articles were scrutinized for additional, relevant publications.
Keywords: Breast carcinoma in situ, ductal carcinoma in situ genetics, molecular biology ductal carcinoma in situ
|How to cite this article:|
Olusina DB, Onyishi NT. Genetic investigation of breast ductal carcinoma In situ: A literature review. Libyan J Med Sci 2017;1:56-62
| Introduction|| |
Molecular biology holds the key to explanation of pathological features and clinical behavior of tumors. Moreover, it is anticipated that, in the study of breast cancers, understanding the genetics of ductal carcinoma in situ (DCIS) could be vital to the elucidation of evolutionary events of breast cancer given the position of DCIS phenotype as the precursor of invasive stage. DCIS has been subjected to basic molecular genetic and cytogenetic investigations in a bid to unravel its biology, explain its behavior, and identify reliable prognostic biomarkers to be used in screening and risk stratification and to uncover possible vulnerable targets of therapy. The following brief review is a historical tour of some of those molecular studies and the contributions they have made to our understanding of DCIS. It is preceded by a recap of the DCIS conundrum and the uncertainties surrounding its natural history.
| Methods|| |
Peer-reviewed scientific publications on the subject were retrieved by search of PubMed databases. The search was conducted with the following string of keywords: “breast carcinoma in situ genetics,” “breast carcinoma in situ molecular biology,” “DCIS molecular method technique,” “breast carcinoma in situ natural history,” and “breast carcinoma in situ epidemiology.” Studies were grouped in chronological order and according to molecular techniques employed in their methodology. Reference list of retrieved articles was scrutinized for additional, relevant publications.
| The Ductal Carcinoma In situ problem|| |
DCIS of the breast is a proliferation of malignant epithelial cells still confined within the mammary duct by the basement membrane and myoepithelial cells. Literature situates its full recognition and description in the early 1930s., As a disease entity, it is currently attracting tremendous research interest and attention due to its increased incidence in the clinics as a result of breast cancer screening programs. In the United States, DCIS accounted for 3.8% of breast cancers seen in the clinics before the introduction of population-wide early detection program. Following the deployment of screening mammography, incidence of DCIS increased steeply through the 1990s, continued to increase at a much slower rate up to 2011 then began to stabilize. It is reported that DCIS incidence rose from 1.87 in 1974 to 32.5 per 100,000 in 2004. It was projected that about 60,290 new cases of female breast carcinoma in situ (DCIS and lobular carcinoma in situ) would be diagnosed in the United States in 2015, and this would constitute 20% of all breast cancers. This trend is replicated in Europe as demonstrated in multiple reports.,,, Since the late 1970s, female in situ breast carcinoma incidence rates have increased by 534% in Great Britain and most of the increase happened in the late 1980s and 1990s coinciding with introduction of breast screening program. Countries with no population-based breast cancer screening program do not have that much number of DCIS diagnoses. Literature search for incidence of breast carcinoma in situ in Africa yielded very few results, probably a testament to the near absence of population-wide breast cancer screening program on the continent. One study which analyzed the outcome of mammographic investigations in a South African hospital reported 9 DCIS in 3774 screening mammograms done over 10 years. Furthermore, two different hospital-based reviews of pattern of breast cancer in Nigeria put the prevalence of DCIS at below 7%.,
| Natural History|| |
The natural history of DCIS has not been fully elucidated, and thus, the clinical significance of diagnosis of DCIS remains, to a large extent, uncertain. A good number of studies investigating possible natural history point to the fact that DCIS is a lesion with considerable propensity for local recurrence either as DCIS or invasive cancer.,,,,, In these studies, follow-up of DCIS cases treated by excision alone or excision with radiotherapy revealed rates of the second ipsilateral breast event ranging from 10.5% to 40% depending on tumor grade, status of surgical margins, and whether adjuvant radiotherapy was given or not. Just recently, Khan et al. studied the outcome for 720 patients of DCIS managed by surgical excision alone dichotomized into adequately excised group on one part and inadequately excised group on the other which was taken as surrogate for “surveillance-only” management strategy. The result of their study showed that 10-year recurrence rates for low- and high-grade diseases were 51 and 70% in the surveillance-only (inadequately excised) group or 13 and 35% in the adequately excised group. The general consensus is that DCIS is a nonobligate precursor of invasive breast cancer (IBC) meaning that not all cases of DCIS are bound to progress to invasive cancer. The proportion of DCIS which will advance, without the interference of biopsy or treatment, to invasive ductal carcinoma is not known, but it is reasonable to surmise that this will be much higher than what obtained in the partially or fully treated cases. Even though women diagnosed with DCIS often experience a second primary breast cancer event (DCIS or invasive cancer), their breast cancer-specific mortality is not overly different from that of the general population. This was revealed by Narod et al. in an observational study of 108196 women diagnosed with DCIS. They reported a 20-year breast cancer-specific mortality of 3.3%, a rate not significantly different from that of the general population and which is in agreement with the findings of earlier studies., Added to this complicated picture, is the observation in the United States that decades of early detection and treatment of DCIS in the population-based screening program have not led to a commensurate decline in late-stage breast cancer incidence or mortality. The debate about the benefits or otherwise of breast cancer screening is currently, a raging controversy.
| Molecular Investigation of Ductal Carcinoma In situ|| |
Analysis of loss of heterozygosity
The cancer phenotype is a product of multiple genetic and epigenetic aberrations in the cellular genes. Traditionally, two classes of cancer-causing genes are recognized: oncogenes and tumor suppressor genes (TSG). Loss of function of TSG by a number of mechanisms including chromosome loss, deletions, mutations, and epigenetic silencing is known to be a common event in tumor where it is thought to contribute to carcinogenesis or cancer progression. Search for TSGs in tumors involves identification of chromosomal regions with loss of heterozygosity (LOH) by the use of polymorphic DNA markers. Minimal regions of nonrandom LOH were believed to be sites of putative TSG., In breast tumors, this type of genetic investigation was first applied on IBC, and as a result, the late 80s and early 90s saw the emergence of multiple reports documenting genetic alterations and LOH frequently present in IBC.,,,,,, Subsequently, Radford et al. claimed the first report of LOH in DCIS. In this investigation and in many others following it,,,,,,,,,, microdissected DCIS cells from archival paraffin-embedded tissue were examined for LOH at chromosomal loci previously reported to exhibit high rate of allelic loss in IBC. This was done by the use of polymerase chain reaction (PCR)-amplified microsatellite markers, a technique which succeeded restriction enzyme digestion and Southern blotting in genetic linkage analysis. These studies, though thematically similar, had minor variations in methods. However, one common concern of most of the studies was to compare the allelotype and LOH profile of DCIS and IBC in other to describe shared genetic alterations and identify possible genetic changes responsible for progression from in situ to invasive stage. This approach was no doubt inspired by a successful unraveling, within this period, of the sequence and chronology of genetic changes leading to colon cancer., Loss of an allele at particular loci such as 8p, 13q, 16q, l7p, 17q, and 6q was regarded as early events because they were present in DCIS and synchronous IBC and also as evidence in support of clonal origin of invasive cancer from the adjacent DCIS.,,, Alleles lost frequently in IBC and only rarely in DCIS (11p, 18p, 18q, 22q) and were interpreted as a later genetic event and probably a feature of Invasive phenotype., Fujii et al., adapting a minor variation in method, investigated multiple foci within each individual tumor and reported intratumor allelic loss heterogeneity in DCIS but not in synchronous IBC. In another study, they described a pattern of heterogeneity suggestive of intralesional progression and reasoned that those allelic loss commonly present in all foci (16q, 17p) could be early genetic event while losses seen only in some foci (1p, 11q) were most likely, later acquisitions in tumor progression.
Knowledge generated from LOH analysis of DCIS includes identification of chromosomal sites that may harbor TSG involved in the development of breast cancer as well as evidence for genetic relatedness and precursor relationship between DCIS and IBC as shown by near similar LOH profile of the two entities. The often-stated goal of identifying the chronology of genetic changes underlying transition from DCIS to IBC was not met.
The overall utility of LOH analysis as a methodology for genetic study of tumor has been questioned by Tomlinson et al. who observed that profusion of LOH found in practically all tumor types has not often translated to successful isolation of TSG.
Comparative and array comparative genomic hybridization
These are techniques which were widely applied in the genetic interrogation of solid tumors. They enabled genome-wide identification of chromosomal copy number changes and provided means for a quick scan of the genome for regions of chromosomal aberrations. Comparative genomic hybridization (CGH) technique first reported by Kallioniemi et al. as a tool for genetic analysis involves hybridization of fluorochrome-labeled tumor and reference DNA to a normal human metaphase preparation. In array CGH, fragments of DNA oligonucleotide or bacterial artificial chromosome clones(BAC) immobilized on a solid glass substrate are used as probes. They replace the metaphase chromosome employed as probe in CGH. Just as in traditional CGH, tumor DNA and reference DNA are labeled with different fluorochromes. Following hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of tumor to reference DNA which acts as proxy for gain or loss of tumor genetic material., Theoretically, aCGH has higher resolution than CGH which is limited, in the size of alterations it can detect, to 5–20Mb., DCIS like so many solid tumors has benefited from the illuminating technique of CGH which is a very powerful tool in generating a genetic profile of tumors. Array CGH and CGH studies of DCIS revealed that: DCIS is a genetically complex lesion exhibiting multiple regions of chromosomal alteration and pattern of chromosomal abnormality in DCIS broadly resembles those implicated in IBC supporting a precursor role for DCIS.,,,,, Comparison of primary DCIS with recurrent lesion or with concurrent invasive tumor showed shared genetic alterations indicative of clonal relationship among the entities.,, Also revealed was the fact that pattern of genetic alteration differed in low- and high-grade DCIS giving the initial suggestion that low and high-grade breast tumors evolved along distinct pathways., Nuclear grade of breast cancer cells was found to be strongly associated with the number and pattern of genetic abnormalities. Thus, low-grade DCIS is characterized by frequent 16q loss and 1q gain.
CGH and LOH assay by PCR amplification of microsatellite markers were almost contemporaneous technologies. It is quite instructive that studies applying either of the two techniques in evaluation of DCIS turned out similar results, thus corroborating each other [Table 1]. CGH and aCGH are genome-wide assay and were thus able to give more global picture of chromosomal imbalance.
|Table 1: Some chromosomal alterations in ductal carcinoma in situ as detected by loss of heterozygosity assay and comparative genomic hybridization|
Click here to view
Broadly, the contribution of the technique to our understanding of DCIS includes highlighting the genetic instabilities present in DCIS and underscoring regions that may harbor candidate oncogene or TSG. The finding, in some of these studies, that recurrent DCIS or invasive cancer concurrently existing with DCIS had marginally higher numbers of chromosomal alteration or novel alteration not seen in primary DCIS hinted at genetic progression through the various stages of breast carcinoma.,,,
Gene expression microarray studies
Expression microarray is a genomic method employed to quantify nucleic acid sequences in a sample or tissue. It involves hybridization of labeled nucleic acid mixture (target) isolated from tissue of interest to oligomers (probe) arrayed on a glass substrate. Arrays have been adapted to study gene expression profile in tissues, transcription factor-binding analysis, and single nucleotide polymorphism genotyping. The result of expression microarray studies gives genes that are turned on or off in a tissue of interest. Perou et al. in a seminal work used microarray of 8102 genes to study gene expression patterns in 65 IBC specimens. Cluster analysis using a subset of the expressed genes enabled them to identify four molecular subtypes of breast cancer – normal breast-like, basal-like, Her2+, and luminal epithelial/ER+. Their work has been translated to clinical use as these subtypes were found to have prognostic implications. Following this, DCIS has similarly been subjected to expression microarray studies. The main thrust of most of the efforts in this regard was to define the gene expression profile of DCIS and identify gene set involved in progression from DCIS to IDC.,,,, This was done mainly by comparing gene expression of various morphologic entities including normal breast epithelium, ductal hyperplasia, DCIS, and IDC to identify differentially expressed genes. For instance, Adeyinka et al. compared gene expression pattern of DCIS with necrosis and DCIS without necrosis. The main assumption underpinning their study seemed to be that comedonecrosis having been identified as a morphologic indicator of DCIS recurrence and progression represented a distinct step along the evolutionary pathway of DCIS progression; thus, differential gene expression between DCIS with necrosis and DCIS without necrosis could reveal genes involved in progression. They identified 69 differentially expressed transcripts between DCIS with necrosis and DCIS without necrosis. Ma et al. studied the gene expression profile of the histologic spectrum of breast epithelium and pathological states ranging from normal terminal duct lobular unit to atypical ductal hyperplasia (ADH), DCIS, and IBC to explore the genetic changes that are associated with the various stages of breast cancer progression. Their findings indicate that the most remarkable changes in patterns of gene expression during breast tumorigenesis appear during the transition from normal tissue to ADH. Different DCIS grades were shown to have distinct transcriptional signatures. Grade I and Grade III breast tumors showed distinct gene expression patterns, whereas Grade II tumors showed a hybrid pattern of Grade I and Grade III signatures. They found the three pathologically discrete stages of breast cancer (ADH, DCIS, and IBC) to be highly similar to each other at the level of the transcriptome. However, through some robust statistical analysis of data, they were able to demonstrate that subtle quantitative differences in gene expression exist between Grade III DCIS and its corresponding IBC with 85 genes found to be overexpressed in IBC. Elias et al. performed a comprehensive analysis of DCIS cells before and after the morphologic manifestation of invasion by comparing the expression profile of epithelial cells captured from pure DCIS with that captured from DCIS coexisting with IBC, on the one hand, and DCIS and IBC components of DCIS-IBC hybrid lesion. Using the rapid subtractive hybridization approach which is a much more sensitive technique, they detected some subtle difference in gene expression between DCIS and IBC, but the expression pattern did not consistently distinguish the two entities, thus reaffirming the genetic and molecular similarity between the two lesions. The implications of this result are that molecular alterations needed for invasion are already established in earlier stages of the tumorigenic process and that genetic differences between DCIS and IBC could be quantitative rather than qualitative. Schuetz et al. analyzed matched pairs of DCIS and IBC from nine patients to understand the molecular biology of transition from DCIS to IBC. They identified a master gene set consisting of 546 significantly regulated probe sets, characterizing the transition from DCIS to IBC. Four hundred and 45 probe sets (360 genes) were upregulated and 101 probe sets (85 genes) were downregulated in IDC. Some of the expression microarray studies of DCIS sought to establish a molecular classification or grading of DCIS based on gene expression profile.,
In genetic studies of DCIS, expression microarray studies represented an important advancement over earlier methods because of its ability to identify specific genes rather than gross chromosomal alteration which are highlighted in the earlier techniques. Even though most microarray studies identified catalogs of gene sets which are differentially expressed, comparison of gene expression data obtained from different array platforms shows only marginal overlap between studies.,
The next logical step in these experiments has been to subject the gene sets identified as differentially expressed to pathway analysis to elucidate the intricate network of interactions and biological processes involved, but no specific gene has as yet been identified as the chief driver of the invasive process.
The precipitous decline in cost of next-generation sequencing coupled with some obvious advantages, it offers over microarray, has facilitated its emergence as common technique and the state of the art in genomic research prompting some predictions that it is bound to completely replace microarray in due course.,, Among other advantages, sequencing offers a direct measure of nucleic acid present in a sample and is able to detect closely related gene sequences and novel splice forms that may be missed due to cross-hybridization on DNA microarrays., The concerns of most cancer genome-sequencing studies generally have thus far included one or more of four specific aims: discovering driver mutations, identifying somatic mutational signatures, characterizing clonal evolution, and advancing personalized medicine.
Pang et al. sought to document the mutational landscape of DCIS of different grades, hormone receptor and ERBB2 status, and to identify differences between invasive and preinvasive disease by massive parallel sequencing using DNA derived from formalin-fixed, paraffin-embedded tissue. They found mutations in 19 genes including genes involved in DNA repair, cell cycle control, the Hedgehog pathway, transcription factors, receptor tyrosine kinases, serine/threonine-protein kinases, TSG, and splicing factors, implying derangement of multiple pathways in DCIS. Compared to invasive cancer, DCIS cases of all phenotypes carry mutational drivers which are similarly present in IBC.
Abba et al. performed exome sequence, transcriptome sequence, and methylome analysis of high-grade DCIS which yielded data on mutational and methylation profile of DCIS. Some conclusions from their work are in accord with another report by Kim et al. who examined DCIS and IDC for mutations and copy number variations by exome sequencing and array CGH. Both reports showed that the mutational profiles of pure DCIS and IBC are almost identical save for a moderate lower frequency of mutation in cancer driver genes in pure DCIS. Furthermore, the profile of copy number changes across the genome in high-grade DCIS was found to be similar to profiles reported in invasive breast lesions in keeping with multiple previous studies.,
Other perspectives on molecular biology of DCIS have been provided by microRNA sequencing, DNA methylation profile, and a combination of orthogonal techniques.,,,
Hernandez et al. employed a cocktail of methods and assays (aCGH, fluorescence in situ hybridization (FISH), Sequenom's massARRAY, and Sanger sequencing) to investigate the genomic and mutational profile DCIS and synchronous IBC. In agreement with numerous other aCGH studies, no statistically significant difference in copy number aberration was seen in DCIS and IDC when the two entities are subjected to grouped analysis. However, on detailed pairwise comparison of DCIS and its adjacent invasive carcinoma, differences in the presence and level of specific amplifications were detected. This pointed to the possibility that genetic difference between DCIS and IDC was quantitative rather than qualitative. In fact, they were able to show by FISH that DCIS and adjacent invasive ductal carcinoma (IDC) were composed of mosaic and clones of tumor cells. This provided circumstantial evidence for intratumor genetic heterogeneity and the prospect that progression from DCIS to IDC is by clonal selection.
Volinia et al. studied the expression of regulatory microRNA in normal breast epithelium, DCIS, and invasive cancer in a bid to unravel the regulation of transition to breast cancer. In their report, the microRNA profile regulating transition from normal breast to DCIS was found to be largely maintained in the in situ to invasive ductal carcinoma transition. The recurring thread of genetic similarity between DCIS and IBC is present in their study just as in epigenetics studies  which reports strong resemblance in gene methylation profile of DCIS and invasive ductal carcinoma. However, Volinia et al. also found that a nine-microRNA signature correlating with invasiveness and five microRNAs correlating with time to metastasis and overall survival in IDC patients were identified.
The tumor microenvironment and epithelial-mesenchymal interaction have also received attention in the search for molecular underpinnings and explanation of invasive progression of DCIS.
| Conclusion|| |
- The exponential increase in incidence of DCIS as a result of mammography screening, the ensuing clinical challenges it presented, and its unique position as the precursor of invasive stage of breast cancer have made it an object of intense research interest
- Molecular studies of DCIS have largely sought to: identify genetic alterations, copy number variations, gene expression, and mutational profiles of DCIS; compare DCIS with normal breast epithelium, ADH, and invasive cancer to identify pathways and molecular signature of breast cancer progression. Most of these studies employed, as part of their methodology, the technique of manual or laser capture microdissection which enabled harvesting of specific morphologic entities from tissue sections, thus facilitating molecular study at cellular level
- The efforts at molecular investigations of DCIS have yielded significant contributions to knowledge and exposés on what is valid or invalid about breast cancer progression
- The hypothesis of dedifferentiation which postulated that grade I breast carcinomas progressed to become grade III tumours has been substantially refuted by genetic studies. Molecular evidences show that Grade I DCIS and IDC are distinct from Grade III DCIS and IDC. In some studies, majority of Grade II tumors have Grade 1 molecular profile while few displayed Grade III or hybrid molecular profile
- The DCIS stage seemed to be as genetically advanced as invasive stage. Studies comparing the genetics of the DCIS stage and the successive invasive stage often showed no genetic difference or stage-specific genetic event on grouped analysis. Alternative pairwise analysis detected quantitative rather than qualitative genetic differences between the two entities
- DCIS is most likely composed of multifarious clones and mosaics of neoplastic cells and clonal selection of populations with specific genomic aberrations take place during progression from DCIS to IBC
- It is much more likely that no single gene or recurrent group of genes determines whether pure DCIS cells progress to IDC
- “Progression from DCIS to invasive cancer is likely to constitute a complex biological phenomenon that follows a Darwinian evolutionary model and a convergent phenotype (i.e., progression from DCIS to invasive cancer may be caused by a large constellation of genetic and/or epigenetic aberrations and/or be mediated by the microenvironment).”
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