Genomic Instability Within Tumor Stroma and Clinicopathological Characteristics of Sporadic Primary Invasive Breast Carcinoma FREE

A high degree of variability is observed in both biological be havior and clinical outcome in sporadic breast cancer, and this interpatient diversity in breast cancer biology and behavior may confound clinical management based on averages. Breast conserving surgery has become the standard of care for early stage breast cancer. In a recently published study, 2929 patients with early stage breast cancer were examined for the relative impact of the patient, the surgeon, hospital factors, or all 3 on surgical treatment outcome variation in patients with breast cancer. The study by Gort et al¹ showed that 91.2% of the total variance was attributable to the patient level (there was large interpatient variability). These data suggested that interpatient variation accounts for the high degree of clinical variability.¹

Indeed, the demand for personalized medicine illustrates the medical community's and public's recognition of interpatient variability. It has been recognized for decades that identical chemotherapeutic regimens for similar stage and grade in patients with, for example, breast cancer (or virtually any malignancy) respond differently.^1- 2 The complexities of genetic alterations in breast cancer may provide a primary basis for these consequent (secondary) clinicopathological features, an idea supported by prior positive correlations between certain breast cancer genotype and phenotype.³ For example, well-differentiated (grade 1) breast cancers show a low number of genetic alterations with highly recurrent losses of 16q, although poorly differentiated (grade 3) cancers show complex genetic changes containing DNA losses as well as DNA amplifications.³

However, many previous studies focused only on restricted regions of the genome-harboring known tumor-associated genes, such as TP53, or were limited to a small sample of patients. High throughput genome-wide scanning for genetic alterations can now be performed on larger series of clinical samples to discover genotypic-phenotypic correlations unbiased by prior work. Moreover, virtually all previous studies exploring these somatic genotype-phenotype correlations fail to separately analyze malignant epithelium and reactive host elements. Tumor microenvironment, incorporating both invasive epithelium and reactive host elements, dynamically determines cancer behavior.^4- 5 The contribution of cancer-associated stromal cell genetic changes to this interaction have been variously ascribed to epigenetic changes (DNA methylation^6- 7), or mutation, which has been shown by tumor-associated stroma from breast, colon, bladder, and ovarian cancers.^8- 13 Our previous work with breast cancer revealed that tumor-associated stroma may contain a higher density of genetic alterations than the malignant epithelium itself.¹²

In our study of sporadic breast carcinomas, we test the hypothesis that stromal cell genomic alterations significantly alter tumor behavior, as reflected in clinicopathological features at the time of diagnosis.

A total of 220 unrelated samples of primary sporadic invasive carcinomas of the female breast annotated by basic clinicopathological features were obtained under the approval of the respective institutional review boards (Brigham and Women's Hospital, Boston, Mass; and Cleveland Clinic, Cleveland, Ohio). Samples from men with breast cancer, women with a personal history of ovarian cancer, and women with 1 or more first-degree relatives with breast or ovarian cancer were excluded. Widely metastatic disease (TX NX M1) was also an exclusion criterion. Anonymized sections from archived blocks were linked only to their respective clinicopathological features (Table 1). No personal identifiers or linking files were maintained.

Laser capture microdissection was performed using the Arcturus PixCell II microscope (Arcturus Engineering Inc, Mountain View, Calif) to isolate neoplastic epithelium and tumor stroma separately.^{9,11- 12,18- 19} Tumor-associated stromal fibroblasts were collected from locations proximate to epithelial tumor cells, being within 5 mm of an epithelial tumor nodule. Corresponding germline reference DNA for each case was procured from normal tissue, either within the breast, but at least 1 cm distant from malignant epithelial cells, or from histologically normal tissues outside the breast. The different origins of the corresponding germline DNA had no effect on the frequency or pattern of loss of heterozygosity/allelic imbalance (LOH/AI).

Genomic DNA was extracted as previously described,^9,11 with incubation in proteinase K at 65°C for 2 days.¹² The primer sets for multiplex polymerase chain reaction (PCR) defined 386 microsatellite markers in 72 multiplex panels (ResGen, Invitrogen, Carlsbad, Calif). Genotyping was performed with the ABI 3730 DNA analyzer (Applied Biosystems, Foster City, Calif). The genotyping results were analyzed by automated fluorescence detection using the ABI Genemapper version 3.5 (Applied Biosystems). Scoring of LOH/AI and retention of heterozygosity was performed by inspection of the Genemapper outputs (Figure 1). A ratio of peak heights of alleles between germline and epithelial carcinoma or surrounding stromal DNA of at least 1.5 was used to define LOH/AI.^19- 22 The methodological accuracy of LOH/AI using multiplex PCR on archived templates was validated as published.¹² Three samples were excluded from statistical analyses because none of the tested loci were informative (all loci homozygous in germline). Data were collected from October 2003 through June 2006.

Statistical analyses were performed on the remaining 217 samples, each of which was informative for at least 79 chromosomal loci. The total number of 386 microsatellite markers were used for total genome LOH/AI scan, and each chromosome contained from 7 (chromosome 21) to 31 (chromosome 1) markers. Standard quality control measures for both laser capture microdissection procurement and replicability of compartment-specific LOH/AI calls are detailed in our previous publications, including the comparisons between the results of PCR on the DNA extracted from laser capture microdissection−captured tissues and those on the DNA from the corresponding frozen tissues, and between the results from multiplex PCR genotyping and those of quantitative PCR and the lack of cross contamination between compartments.^{9,12,18- 19,23- 24}

Mutation analysis was performed on the 112 breast cancer samples, which had informative LOH/AI data at D17S796. Genomic DNA from the epithelium and stroma from each breast carcinoma was subjected to mutation analysis for TP53. The classic mutation cluster region of this gene, exons 4 through 9, exon-intron boundaries, and flanking intronic regions of TP53 were analyzed by PCR-based direct sequence analysis using the ABI3730xl, as previously described.¹² When a mutation was found in the epithelium, stroma, or both, the corresponding germline was examined. No germline TP53 mutations were found; therefore, all mutations found in the breast cancer samples were somatic.

Presenting demographic and clinicopathological features included age, tumor grade (modified Scarff-Bloom-Richardson grades 1-3),^14- 16 tumor size, estrogen-receptor and progesterone-receptor expression status, and human epidermal growth factor receptor 2 (HER2/neu) expression status, as well as primary tumor status, regional lymph node metastasis status (pN), and clinical stage grouping, which was based on the 6th edition of the American Joint Committee on Cancer Cancer Staging Manual.¹⁷ For hormone receptor analysis, the percentage of immunoreactive nuclei was assessed visually and the results were categorized as (+) equals more than 10% of nuclei, (±) equals more than 0% but less than 10% of nuclei, and (−) equals 0% of nuclei immunoreactive. In HER2/neu analysis, the results were scored as 0 indicates no immunoreactivity or immunoreactivity in less than 10% of tumor cells, 1+ indicates faint weak immunoreactivity in more than 10% of tumor cells but only a portion of the membrane is positive, 2+ indicates weak to moderate complete membrane immunoreactivity in more than 10% of tumor cells, 3+ indicates moderate to strong complete membrane immunoreactivity in more than 10% of tumor cells. Scores of 0 and 1+ were regarded as negative (−), and scores of 2+ and 3+ as positive (+), respectively. Cytoplasmic immunoreactivity alone was scored as a negative result.

McNemar tests were performed to compare the LOH/AI between each compartment-pair (epithelium and stroma) from each of the tumors with the pooled samples to test whether LOH/AI is more frequent in one compartment vs the other compartment. Dissimilarities between each compartment-pair (epithelium and stroma) from each of the tumors can be measured by the percentage of discordant pairs of LOH/AI (the proportion of markers showing loss of heterozygosity in one compartment and retention of heterozygosity in the other compartment among all the markers, which were informative in both compartments). Multidimensional scaling using principal coordinate analysis measures the distance between a pair of samples and approximates the dissimilarity between the 2 as measured by the percentage of discordant LOH/AI. Based on the results for multidimensional scaling, 2 of 217 tumors appeared to have different LOH/AI patterns from those of the rest of the samples. This was most likely due to the small number of informative markers for these 2 tumors (39 and 46 informative markers in the epithelium and stroma combined). Therefore, these 2 samples were excluded from multidimensional scaling and hierarchical clustering analyses.

We first performed hierarchical clustering with average linkage and multidimensional scaling for 430 samples, epithelium and stroma separately, derived from the 215 tumors. The clustering was performed by using a function in the statistical package R (which was used for all statistical analyses and detailed by Venables and Ripley^25- 26), and the standard option of average linkage was used. As an unsupervised (unbiased) method, we correlated genotype with the presenting clinicopathological features by repeating the same analysis using 1 clinicopathological variable at a time. The same analysis was then performed by combining the epithelium and stroma samples from the same tumor to study the overall LOH/AI profile of the tumor.

We applied statistical models to study the relationships between compartment-specific LOH/AI and clinicopathological data. Logistic regression models were used for clinicopathological features with binary features, and proportional odds regression models were used for clinicopathological features with more than 2 ordered classes. The covariates in these models are chromosome-wise LOH/AI frequencies for either compartment (stroma or epithelium) from each tumor. From these analyses, we obtained a P value across each chromosome in each compartment and each clinicopathological feature, representing the strength of evidence for the correlation between LOH/AI on that particular chromosome in that compartment and the clinicopathological feature. For the group of tests for a specific compartment and clinicopathological feature, Bonferroni adjustment was applied to correct for multiple testing, resulting in a significance level of P<.05 divided by 23 (P<.0022). For any association that was statistically significant, 2-tailed Fisher exact tests were used to associate the clinicopathological feature with LOH/AI at individual markers on that chromosome in that compartment.

Overall, LOH/AI was more frequent in the epithelium than in the stroma. In the epithelium across all tumors, 43 598 PCR reactions were informative for evaluation of LOH/AI, and 22 288 PCR reactions (51.1%) showed LOH/AI compared with an overall 18 644 (47.6%) of 39 192 PCR reactions in the stroma (χ² test, P = 2.2×10⁻¹⁶). At the chromosomal level, model-based estimates for the LOH/AI frequency¹² were significantly higher in the epithelium than in the stroma for 5 chromosomes (chromosomes 7, 8, 13, 16, and 17) at the P<.05 level (Table 2), and remained so for 3 of the 5 chromosomes (chromosomes 8, 13, and 17) after Bonferroni adjustment for multiple testing (P<.0022).

As proof of concept that regions with significantly high LOH/AI often have relevant genes, we looked at the p13 region of chromosome 17, which contains the TP53 tumor suppressor gene. One of the major regions of loss of heterozygosity is within 17p13, where loss of heterozygosity at D17S796 (17p13.2) in the epithelium occurs in 72 (64%) of 112 informative (germline heterozygous at this marker) breast cancer samples from our series and, in the stroma, occurs in 56 (53%) of 106 informative breast cancer samples from our series (7 stromal samples failed to amplify). D17S796 is a proximal marker for the TP53 tumor suppressor gene.

We therefore performed direct mutation analysis by sequencing of the classic mutation cluster region, exons 4 through 9, and flanking intronic sequences of TP53 of all epithelial and stromal samples from the 113 breast cancer samples with informative loss of heterozygosity data at this locus. We found that 29 (27%) of 112 tumors had somatic intragenic TP53 mutations in the epithelium and 28 (26%) of 106 had somatic TP53 mutations in the stroma. Only 8 tumors had somatic TP53 mutations in both the epithelium and stroma, but for each of these 8 samples, the mutation found in the epithelium was different from that in the stroma. Thus, 21 tumors had TP53 mutations only in the epithelium and another 20 tumors had somatic mutations only in the stroma. Of the 30 tumors with TP53 mutations in the epithelium, 80% had loss of heterozygosity at D17S796. Among the 28 tumors with TP53 mutations in the stroma, 65% had loss of heterozygosity at this marker.

The results of the McNemar test comparing the LOH/AI between the epithelium and stroma samples derived from the 217 participants revealed that for a larger number of participants, LOH/AI was observed more frequently in the epithelium, represented by the positive P values. This result is consistent with the overall test, which indicated strong evidence for more frequent LOH/AI in the epithelium (P<.001). Neither multidimensional scaling nor hierarchical clustering revealed any strong similarity between LOH/AI profiles for the epithelial or the stromal samples from the same participant, providing a good control for noncontamination between compartments (data not shown). The hierarchical clustering did result in the samples clustering progressively, with the most similar samples clustered together first.

We took a 2-stage approach to look for associations between compartment-specific LOH/AI and clinicopathological features. First, these associations were screened at the chromosome level. The chromosomes that yielded significant correlations were then subjected to analysis at the individual marker level to determine associations between LOH/AI at the specific markers or loci and the clinicopathological features. For the first stage, we applied formal model-based methods to examine the correlations between LOH/AI and the presenting clinicopathological features. We used compartment-specific LOH/AI data to classify the clinicopathological features using logistic and ordinal regression models, with chromosome-wise LOH/AI as the independent variable, for each chromosome in turn, and obtained P values for each presenting clinicopathological feature. The obtained P values (Table 3) represent the strength of evidence for the correlation between LOH/AI on a particular chromosome and the particular presenting clinicopathological feature.

More statistically significant associations (after Bonferroni adjustment) with clinicopathological features were found for LOH/AI in the stroma (7 associations) than in the epithelium (1 association). Specifically, we found significant associations between tumor grade and LOH/AI on chromosome 11 in the stroma (P = .0013), between tumor grade and LOH/AI on chromosome 14 in the epithelium and progesterone receptor (P = .002), and between tumor grade and LOH/AI on chromosomes 1 (P = .0006), 2 (P = .0016), 5 (P = .0009), 18 (P = .0009), 20 (P = .001), and 22 (P = .0002) in the stroma and pathological pN status (Table 3 and Figure 2).

Once promising chromosomes were identified, we proceeded with the second stage to associate LOH/AI at specific loci and the clinicopathological features. To determine if specific markers were responsible for the LOH/AI along the chromosomes noted above that were significantly associated with tumor grade, pN, and progesterone-receptor status, 2-tailed Fisher exact tests were used to test the association of the corresponding clinicopathological feature with LOH/AI at each marker in the corresponding compartment (Table 4). Markers along chromosome 14 in the epithelium associated with progesterone-receptor status were D14S588 (P = .029) and D14S1426 (P = .027). Specific markers contributing to the LOH/AI on chromosome 11 in the stroma associated with tumor grade were D11S1999 (P = .00055) and D11S1986 (P = .042). Importantly, LOH/AI at various markers in the stroma was significantly associated with pN: ATA42G12 (chromosome 1, P = .00095), D5S1457 (P = .00095), D5S1501 (P = .0011), D5S816 (P = .0008), D18S858 (P = .0026), D20S103 (P = .0027), D20S851 (P = .0045), D22S683 (P = .00033), and D22S1045 (P = .0013).

Eight significant associations were found between compartment-specific, chromosome-specific LOH/AI, and clinicopathological features. Although only 2 markers on chromosome 14 in the epithelium were significantly associated with any clinicopathological feature at all (in this case, progesterone receptor), genomic instability within 7 chromosomes in the stroma of primary invasive breast carcinomas were significantly associated with tumor grade (chromosome 11) and the presence of regional lymph node metastases (chromosomes 1, 2, 5, 18, 20, and 22). Our previous observational studies of total genome LOH/AI in breast cancer compartments have also revealed specific regions of chromosome 11 as an important target of genomic alteration.^9,12 Furthermore, because this 11q region is also a hot spot (a significantly higher frequency of LOH at a marker or markers compared with other markers along the same chromosome) in the stroma of head and neck cancers, the role of this region in the stroma might be more universal.²⁷

The ataxia-telangiectasia (ATM) gene is mapped to chromosomal region 11q23.1, the locus associated with tumor grade. Because the ATM gene is responsible for maintaining genomic integrity,²⁸ it may be postulated that LOH/AI at the ATM locus in the stroma might initiate general genomic instability in that compartment. Lack of the ATM gene has also been shown to be associated with increased neoangiogenesis and with increased tumor grade and poor clinical outcome in non-Hodgkin lymphoma.²⁹ At least 1 previous study has shown that LOH/AI at the ATM locus correlated with increased tumor grade in whole (without compartment-specific analysis) primary breast carcinomas.³⁰ However, sometimes genotype–clinicopathological feature associations may not be as straightforward as merely reflecting a gene or genes within an identified hot spot. For example, we used our model-based statistic to look for potential associations between compartment-specific presence or absence of somatic TP53 mutations and specific hot spot LOH/AI. Interestingly, the presence of somatic TP53 mutations in the stroma, but not in the epithelium, were associated with the presence of LOH/AI at our 2 stroma-specific hot spot markers on chromosome 11 associated with tumor grade (A.P. and C.E., unpublished data, 2007). Thus, it is possible that somatic mutation of TP53 in the stroma results in genomic instability leading to LOH/AI, including LOH/AI at 11q23, and affecting the ATM locus that sets up a perpetuating cycle of increasing genomic instability and, therefore, high-grade tumors.

The association of LOH/AI at 9 specific loci residing on 6 chromosomes in the stroma of primary breast carcinomas with pathological pN status is worth mentioning. In the process of lymph node metastases, there would be at least 2 rate-limiting steps: gaining access to the lymphatics at the site of the primary lesion and tumor formation at the regional lymph node.³¹ For successful metastasis, it would seem straightforward that the primary tumor stroma should have many important roles, providing a permissive microenvironment that permits invasion. Our observation that genetic alterations at the 9 loci (on 6 chromosomes) in the stroma associating with pN should reflect the genetically altered microenvironment favorable to metastasis. There are 2 broad categories of metastasis-associated genes (metastasis activators and metastasis suppressors).³² As an example, at least 4 of these are located at those chromosomal loci significantly associated with pN in this study, such as Maspin³¹ at 18q21.3 (D18S858), EP300³³ at 22q13 (D22S1045), and PLCB1³⁴ at D20S851, as well as MYH9³⁵ on D22S683, which are known to be associated with metastasis, invasion, or both. In particular, EP300 encodes p300, which is a transcriptional cofactor and prototype histone acetyltransferase that plays a role in multiple cellular processes. In vitro, p300-deficient cells appeared to have an aggressive phenotype with loss of cell-cell adhesion and defects in cell-matrix adhesion.³³ In vivo, embryos lacking p300 were shown to arrest development and die between E8.5 and E11, suggesting that p300 would be necessary for healthy organ development.³⁶ Our observation might also explain why epigenetic phenomena are more prominent in tumor stroma.^6- 7 However, our current data suggest that structural loss of such genes as EP300 occur first, with consequent epigenetic alterations important in tumor stroma occurring thereafter. Within or close to 7 of the 9 pN-associated markers lie genes or loci associated with immune modulation (eg, IL2RB, IBD5³⁷), and several quantitative trait loci for rheumatoid arthritis.^38- 39 Overall, our observations lend evidence that genetic alterations in the tumor stroma activates or promotes genomic instability and neovascularization (ATM locus LOH/AI and tumor grade) followed by further dysfunction in such genes as EP300 and Maspin whose consequences interact with inflammation and immune suppressive responses (IL2RB, IBD5, and quantitative trait loci for rheumatoid arthritis), which promote cell migration and invasion.⁴⁰

These results support a model in which genetic changes in both stromal and epithelial compartments occur during tumorigenesis, and progression is codetermined by local interaction between these cell populations within the primary tumor.¹² We previously found that the stroma had a greater multiplicity of genetic alterations than the epithelium and the targets of genetic alterations in the stroma were more numerous and widely distributed than those in the epithelium. This suggests that epithelium only requires a small number of LOH/AI events to undergo malignant transformation, but local behavior of the resultant epithelial neoplasm is substantially modified by a broader repertoire of genetic changes in adjacent stroma.

Our data suggest that clinical tumor progression, reflected in the measured clinicopathological features, may be more influenced by locally acquired changes in the stromal environment than carcinoma cell genotype per se.¹² Stromal genetic changes that contribute to clinically relevant outcomes can be mapped to particular chromosomal loci, including 2 markers on chromosome 11 that correlate with tumor grade and 9 markers on 6 chromosomes associated with regional lymph node metastasis. Genetic changes acquired in the stroma adjacent to transformed epithelial cells contribute an additional dimension of progression modulation beyond that contributed by the carcinoma cells themselves. The combination of stromal and epithelial genetic changes produces a greater range of outcome scenarios than can otherwise be explained by carcinoma cell genotype alone.

The genetics and genomics of tumor stroma from human patients is a relatively new field of exploration compared with the cell biology of epithelial-stromal interactions in in vitro and nonhuman solid tumor models, which may date back to 20 or more years ago. Given the technology of the day, albeit modern, there are always caveats to studies such as this. For example, despite our best intention to avoid cross contamination between compartments, there might have been a few stray cells from each compartment that crossed over. When this occurs, very low level LOH/AI (eg, in subpopulations) will be missed, and so subtle clinicopathological feature−associations may be missed. Furthermore, we used a 385-marker total genome coverage (10-Mb mean intermarker distance); therefore, it is possible that a few important regions or genes that are relatively distant from each marker were missed. For example, the 17q markers closest to the HER2/neu gene showed a relatively low allelic imbalance (genomic amplification) frequency compared with HER2/neu protein expression by immunohistochemistry. It is possible that the 386-marker whole genome coverage still did not have enough resolution to capture the 1-Mb HER2/neu amplicon, as this marker set did not include a marker within this gene. Added confidence is provided by similar findings of reproducible genomic, epigenomic, and expressional changes found by different technologies, such as chromosome genomic hybridization and expression profiling in breast and other carcinomas published by several other articles.^6- 7,10 Nonetheless, as with any patient-oriented study, our data should be validated, perhaps with emerging novel technologies, in larger series especially those with event-free survival data and therapeutic trials with longer follow-up.