Lobular Carcinoma in Situ and Synchronous Malignant Lesions
Lobular Carcinoma in Situ and Synchronous Malignant Lesions
Once a woman is diagnosed with LCIS, she faces an eightfold to 10-fold increased risk for the subsequent development of breast cancer, the pathogenesis of which is poorly understood. Emerging reports of shared molecular alterations between LCIS and adjacent invasive lesions in parallel with genomic evidence that LCIS may be one of several early identifiable lesions in the pathogenesis of low-grade cancer have re-opened the debate regarding the true significance of LCIS and its precursor potential. Using fresh frozen breast samples subject to laser-capture microdissection for isolation of pure cell populations, and using specialized statistical methods, we report evidence that LCIS is clonally related to a substantial proportion of the adjacent malignancies studied in this series, including cases of low-grade and high-grade DCIS and invasive mammary carcinoma with mixed ductal and lobular features. In addition, the immunohistochemical profiles of all cases considered clonal or equivocal were consistent with the recent theory of breast carcinogenesis whereby LCIS and ER-positive DCIS are grouped as precursors of ER-positive invasive cancer, and ER-negative DCIS is a precursor of ER-negative invasive cancer, regardless of histologic grade.
As LCIS is usually a small, incidentally detected lesion, most prior reports assessing clonality between LCIS and adjacent malignancies were based on FFPE samples. These studies also suggest clonality between LCIS and adjacent breast carcinomas, despite the fact that they differed in terms of methodology. Our re-analysis of the publically available data from the study by Hwang and colleagues using our own statistical methods provided classification results broadly consistent with our own results. The use of fresh frozen tissue samples and SNP arrays in our study allows for a substantially increased resolution for assessing clonal relatedness.
Wagner and colleagues analyzed LOH in 10 cases of co-existent ipsilateral DCIS, LCIS, and invasive carcinoma. LOH was investigated in 13 commonly informative and deleted markers on chromosome 16 (six markers), chromosome 17 (two markers), and chromosomes 1, 8, 9, 11, and 13 (one marker each). In five cases, the authors observed phenotype concordance among all three samples (LCIS, DCIS, and IDC) in at least one marker. In two additional cases, concordant LOH was found between LCIS and DCIS, but not between LCIS and the invasive lesion. This study was novel in suggesting evidence of clonality between LCIS and lesions with ductal phenotype, and our own results provide substantive support for this conclusion. We found definitive or probable clonality in three out of four LCIS-DCIS pairs and among all three lesions in two LCIS-DCIS-invasive cancer case triplets (Case #95 and Case #110). All clonal pairs displayed concordant ER-positivity between paired lesions, yet the subsequent lesions were not restricted to low-grade disease. This finding is consistent with our previous work demonstrating a prominent role for ER status over grade in breast cancer progression in a historical cohort of women with in situ and subsequent invasive lesions, and adds to the growing body of literature that suggests low-grade and high-grade ER-positive tumors are more similar to each other than to their ER-negative counterparts.
Our results also suggest that LCIS and DCIS can have the same cell of origin and therefore may be part of a morphological spectrum of the same precursor lesion. Although pathologists often use E-cadherin staining to distinguish between these two lesions, IHC has many well-described pitfalls and many pathologists prefer to differentiate these lesions based on morphology alone. If newly proposed models of breast carcinogenesis are validated, whereby both ER-positive LCIS and DCIS behave as precursor lesions to low-grade ER-positive breast cancer, this distinction may become less clinically relevant.
While results to date provide broad support for clonality among synchronous lobular lesions, the data favoring clonality among LCIS and subsequent (metachronous) invasive cancers are less clear. Aulmann and colleagues studied nine patients with LCIS who developed subsequent invasive breast carcinoma (five ILC cases and four IDC cases) between 2 and 10 years after the index biopsy. All cases (LCIS and invasive) showed an ER-positive/HER2-negative profile. This study was based on comparisons of mitochondrial DNA heteroplasmy by PCR, direct sequencing, and phylogenetic tree clustering, and used microdissected samples from FFPE tissue. They observed identical patterns of heteroplasmy in two out of five pairs of LCIS and ILC. In one case the changes were more complex in ILC than in LCIS, and in two pairs of LCIS and ILC the changes favored unrelatedness. Similarly, in all four pairs of LCIS and subsequent IDC, the changes favored unrelatedness. More work examining metachronous cancers is needed to better understand the clonal relationship between primary cancers and subsequent recurrences.
Despite the extensive data available to us from copy number profiling, the classification of cases as clonal versus independent is far from a litmus test. Ideally, the histogram plot of our likelihood ratio measure (Figure 4, red histogram) would separate clearly into two distinct groups: one group overlapping the reference (black histogram) distribution, representing the independent pairs; and one group clearly separated, representing the clonal pairs. We do see a clear separation for some pairs that can be confidently classified as clonal. However, the histogram also includes several tumor pairs in an intermediate grey zone at the upper tail of the reference distribution (P < 0.05) but within the range of values obtained by pairing tumors from different patients. This phenomenon could have various causes. Tumor evolution following clonal divergence would tend to lead to a mixed pattern of matching and nonmatching allelic changes. This concept has been hypothesized to account for the wide range of histologic and molecular diversity seen within many ductal in situ lesions and may also explain the proposed evolution from low-grade to high-grade disease among ER-positive lesions. Alternatively, contamination of the tumor samples with normal cells or technical artifacts will tend to obscure true signals, making it harder for our statistical algorithm to detect them clearly. The algorithm is especially useful in assessing the evidence for and against clonal relatedness in these difficult cases.
Others have attempted to determine clonality based on the presence of concordant mutations, yet this methodology can be limited by the fact that sporadic mutations happen recurrently at the same point in some tumors. For example, the BRAF point mutation (T1799A) occurs in 45% of papillary thyroid carcinomas and is associated with poor clinical outcome, but its high frequency limits its usefulness to address clonality. In contrast, inactivating mutations of the E-cadherin gene that occur at dozens of different locations within the gene are highly frequent in infiltrating lobular breast carcinomas and in diffuse gastric carcinomas. The specificity of the mutations makes this gene much more informative regarding clonal relatedness. These mutations can be small insertions or deletions and are frequently combined with LOH of the wild-type allele. Mutations in E-cadherin have been found at very early non-invasive stages of these diseases, leading to an association between E-cadherin mutations and loss of growth control, and to the classification of E-cadherin as a candidate tumor suppressor. Data regarding the presence of coincident mutations in E-cadherin among LCIS and adjacent invasive cancers have been mixed. In an early report, Vos and colleagues presented two LCIS-ILC pairs with matching point mutations. Rieger-Christ and colleagues, however, found no matches in a series of eight patients in which mutations were detected in LCIS-invasive pairs. Germline mutations of the E-cadherin gene have been described in families with hereditary diffuse gastric carcinomas, and family members are also at increased risk for invasive lobular cancers; however, germline mutations have not been identified among women with LCIS outside these kindreds.
Our statistical method compares all areas of gains and losses for a pair of samples, examines the concordance of the starting and ending points within chromosomes, and compares the degree of similarity of changes of an individual pair to a reference distribution created with samples paired from different patients. This provides a stronger argument for clonality than any single concordant point mutation. The use of an empirical reference distribution created using pairs of tumors from different patients is especially important since we observed some similar patterns of copy number variation between different patients. For example, the presence of 1q gain and 16q loss, consistently reported in low-grade lesions of ductal and lobular morphology, was present in 73% and 53% of our LCIS cases, respectively, and therefore these changes are not very meaningful in an analysis of clonal relatedness.
We recruited all women in a defined period presenting for risk-reducing or therapeutic mastectomy, but we only aimed to harvest fresh frozen LCIS for DNA extraction in a proportion of them; as a result, this group may not be representative of all women harboring LCIS. While this small prospective study confirms to us that LCIS is likely to be associated with ER-positive and low-grade disease, the lack of a larger group with ER-negative and more high-grade histology prevents us from drawing broad conclusions on the characteristics of clonally related lesions.
Discussion
Once a woman is diagnosed with LCIS, she faces an eightfold to 10-fold increased risk for the subsequent development of breast cancer, the pathogenesis of which is poorly understood. Emerging reports of shared molecular alterations between LCIS and adjacent invasive lesions in parallel with genomic evidence that LCIS may be one of several early identifiable lesions in the pathogenesis of low-grade cancer have re-opened the debate regarding the true significance of LCIS and its precursor potential. Using fresh frozen breast samples subject to laser-capture microdissection for isolation of pure cell populations, and using specialized statistical methods, we report evidence that LCIS is clonally related to a substantial proportion of the adjacent malignancies studied in this series, including cases of low-grade and high-grade DCIS and invasive mammary carcinoma with mixed ductal and lobular features. In addition, the immunohistochemical profiles of all cases considered clonal or equivocal were consistent with the recent theory of breast carcinogenesis whereby LCIS and ER-positive DCIS are grouped as precursors of ER-positive invasive cancer, and ER-negative DCIS is a precursor of ER-negative invasive cancer, regardless of histologic grade.
As LCIS is usually a small, incidentally detected lesion, most prior reports assessing clonality between LCIS and adjacent malignancies were based on FFPE samples. These studies also suggest clonality between LCIS and adjacent breast carcinomas, despite the fact that they differed in terms of methodology. Our re-analysis of the publically available data from the study by Hwang and colleagues using our own statistical methods provided classification results broadly consistent with our own results. The use of fresh frozen tissue samples and SNP arrays in our study allows for a substantially increased resolution for assessing clonal relatedness.
Wagner and colleagues analyzed LOH in 10 cases of co-existent ipsilateral DCIS, LCIS, and invasive carcinoma. LOH was investigated in 13 commonly informative and deleted markers on chromosome 16 (six markers), chromosome 17 (two markers), and chromosomes 1, 8, 9, 11, and 13 (one marker each). In five cases, the authors observed phenotype concordance among all three samples (LCIS, DCIS, and IDC) in at least one marker. In two additional cases, concordant LOH was found between LCIS and DCIS, but not between LCIS and the invasive lesion. This study was novel in suggesting evidence of clonality between LCIS and lesions with ductal phenotype, and our own results provide substantive support for this conclusion. We found definitive or probable clonality in three out of four LCIS-DCIS pairs and among all three lesions in two LCIS-DCIS-invasive cancer case triplets (Case #95 and Case #110). All clonal pairs displayed concordant ER-positivity between paired lesions, yet the subsequent lesions were not restricted to low-grade disease. This finding is consistent with our previous work demonstrating a prominent role for ER status over grade in breast cancer progression in a historical cohort of women with in situ and subsequent invasive lesions, and adds to the growing body of literature that suggests low-grade and high-grade ER-positive tumors are more similar to each other than to their ER-negative counterparts.
Our results also suggest that LCIS and DCIS can have the same cell of origin and therefore may be part of a morphological spectrum of the same precursor lesion. Although pathologists often use E-cadherin staining to distinguish between these two lesions, IHC has many well-described pitfalls and many pathologists prefer to differentiate these lesions based on morphology alone. If newly proposed models of breast carcinogenesis are validated, whereby both ER-positive LCIS and DCIS behave as precursor lesions to low-grade ER-positive breast cancer, this distinction may become less clinically relevant.
While results to date provide broad support for clonality among synchronous lobular lesions, the data favoring clonality among LCIS and subsequent (metachronous) invasive cancers are less clear. Aulmann and colleagues studied nine patients with LCIS who developed subsequent invasive breast carcinoma (five ILC cases and four IDC cases) between 2 and 10 years after the index biopsy. All cases (LCIS and invasive) showed an ER-positive/HER2-negative profile. This study was based on comparisons of mitochondrial DNA heteroplasmy by PCR, direct sequencing, and phylogenetic tree clustering, and used microdissected samples from FFPE tissue. They observed identical patterns of heteroplasmy in two out of five pairs of LCIS and ILC. In one case the changes were more complex in ILC than in LCIS, and in two pairs of LCIS and ILC the changes favored unrelatedness. Similarly, in all four pairs of LCIS and subsequent IDC, the changes favored unrelatedness. More work examining metachronous cancers is needed to better understand the clonal relationship between primary cancers and subsequent recurrences.
Despite the extensive data available to us from copy number profiling, the classification of cases as clonal versus independent is far from a litmus test. Ideally, the histogram plot of our likelihood ratio measure (Figure 4, red histogram) would separate clearly into two distinct groups: one group overlapping the reference (black histogram) distribution, representing the independent pairs; and one group clearly separated, representing the clonal pairs. We do see a clear separation for some pairs that can be confidently classified as clonal. However, the histogram also includes several tumor pairs in an intermediate grey zone at the upper tail of the reference distribution (P < 0.05) but within the range of values obtained by pairing tumors from different patients. This phenomenon could have various causes. Tumor evolution following clonal divergence would tend to lead to a mixed pattern of matching and nonmatching allelic changes. This concept has been hypothesized to account for the wide range of histologic and molecular diversity seen within many ductal in situ lesions and may also explain the proposed evolution from low-grade to high-grade disease among ER-positive lesions. Alternatively, contamination of the tumor samples with normal cells or technical artifacts will tend to obscure true signals, making it harder for our statistical algorithm to detect them clearly. The algorithm is especially useful in assessing the evidence for and against clonal relatedness in these difficult cases.
Others have attempted to determine clonality based on the presence of concordant mutations, yet this methodology can be limited by the fact that sporadic mutations happen recurrently at the same point in some tumors. For example, the BRAF point mutation (T1799A) occurs in 45% of papillary thyroid carcinomas and is associated with poor clinical outcome, but its high frequency limits its usefulness to address clonality. In contrast, inactivating mutations of the E-cadherin gene that occur at dozens of different locations within the gene are highly frequent in infiltrating lobular breast carcinomas and in diffuse gastric carcinomas. The specificity of the mutations makes this gene much more informative regarding clonal relatedness. These mutations can be small insertions or deletions and are frequently combined with LOH of the wild-type allele. Mutations in E-cadherin have been found at very early non-invasive stages of these diseases, leading to an association between E-cadherin mutations and loss of growth control, and to the classification of E-cadherin as a candidate tumor suppressor. Data regarding the presence of coincident mutations in E-cadherin among LCIS and adjacent invasive cancers have been mixed. In an early report, Vos and colleagues presented two LCIS-ILC pairs with matching point mutations. Rieger-Christ and colleagues, however, found no matches in a series of eight patients in which mutations were detected in LCIS-invasive pairs. Germline mutations of the E-cadherin gene have been described in families with hereditary diffuse gastric carcinomas, and family members are also at increased risk for invasive lobular cancers; however, germline mutations have not been identified among women with LCIS outside these kindreds.
Our statistical method compares all areas of gains and losses for a pair of samples, examines the concordance of the starting and ending points within chromosomes, and compares the degree of similarity of changes of an individual pair to a reference distribution created with samples paired from different patients. This provides a stronger argument for clonality than any single concordant point mutation. The use of an empirical reference distribution created using pairs of tumors from different patients is especially important since we observed some similar patterns of copy number variation between different patients. For example, the presence of 1q gain and 16q loss, consistently reported in low-grade lesions of ductal and lobular morphology, was present in 73% and 53% of our LCIS cases, respectively, and therefore these changes are not very meaningful in an analysis of clonal relatedness.
We recruited all women in a defined period presenting for risk-reducing or therapeutic mastectomy, but we only aimed to harvest fresh frozen LCIS for DNA extraction in a proportion of them; as a result, this group may not be representative of all women harboring LCIS. While this small prospective study confirms to us that LCIS is likely to be associated with ER-positive and low-grade disease, the lack of a larger group with ER-negative and more high-grade histology prevents us from drawing broad conclusions on the characteristics of clonally related lesions.
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