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Int J Cancer. Author manuscript; available in PMC 2019 January 15.
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Published in final edited form as:

Int J Cancer. 2018 January 15; 142(2): 347–356. doi:10.1002/ijc.31051.

Contralateral Breast Cancers: Independent Cancers or

Metastases?
Colin B Begg1, Irina Ostrovnaya1, Felipe C Geyer2, Anastasios D Papanastasiou2,3,
Charlotte KY Ng2,4, Rita Sakr5, Jonine L Bernstein1, Kathleen A Burke2,6, Tari A King7,
Salvatore Piscuoglio2,4, Audrey Mauguen1, Irene Orlow1, Britta Weigelt2, Venkatraman E
Seshan1, Monica Morrow5, and Jorge S Reis-Filho2
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1Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New
York, NY, USA 2Department of Pathology, Memorial Sloan Kettering Cancer Center, New York,
NY, USA 3Metaxa Cancer Hospital/University of Patras, Patras, Greece 4Institute of Pathology,
University Hospital Basel, Switzerland 5Breast Service, Department of Surgery, Memorial Sloan
Kettering Cancer Center, New York, NY, USA 6IBM Watson Health, Cambridge, MA USA 7Dana-
Farber Cancer Institute/Brigham and Women’s Hospital, Boston, MA USA

Abstract
A cancer in the contralateral breast in a woman with a previous or synchronous breast cancer is
typically considered to be an independent primary tumor. Emerging evidence suggests that in a
small subset of these cases the second tumor represents a metastasis. We sought to investigate the
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issue using massively parallel sequencing targeting 254 genes recurrently mutated in breast cancer.
We examined the tumor archives at Memorial Sloan Kettering Cancer Center for the period 1995–
2006 to identify cases of contralateral breast cancer where surgery for both tumors was performed
at the Center. We report results from 49 patients successfully analyzed by a targeted massively
parallel sequencing assay. Somatic mutations and copy number alterations were defined by state-
of-the-art algorithms. Clonal relatedness was evaluated by statistical tests specifically designed for
this purpose. We found evidence that the tumors in contralateral breasts were clonally related in 3
cases (6%) on the basis of matching mutations at codons where somatic mutations are rare.
Clinical data and the presence of similar patterns of gene copy number alterations were consistent
with metastasis for all 3 cases. In 3 additional cases there was a solitary matching mutation at a
common PIK3CA locus. The results suggest that a subset of contralateral breast cancers represent
metastases rather than independent primary tumors. Massively parallel sequencing analysis can
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provide important evidence to clarify the diagnosis. However, given the inter-tumor mutational
heterogeneity in breast cancer, sufficiently large gene panels need to be employed to define
clonality convincingly in all cases.

Corresponding Author: Colin B Begg, PhD, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center,
485 Lexington Avenue, 2nd floor, New York, NY 10017, Phone: 646-888-8239; Fax: 929-321-1517; [email protected].
Novelty and Impact: Contralateral occurrences of breast cancer have been considered by convention to be independent primaries. The
results of our study support a growing literature that in a small proportion of these cases one of the tumors is a metastasis of the other.
Panel sequencing is highly informative in diagnosing metastases, but not always definitive. This study is the largest study to date of
this issue.
 Begg et al. Page 2

Keywords
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Clonality; bilateral breast cancer; next generation sequencing

Introduction
Women with unilateral breast cancer have a significantly elevated risk of developing a
cancer in the contralateral breast.1 Contralateral breast cancer (CBC) is generally considered
to be a second primary rather than a metastasis from the initial primary.2 This view is
supported by studies showing frequent discrepancies in conventional histopathologic
features such as histologic type, grade or estrogen receptor status between the two primaries,
although assignment of histologic grade may vary among pathologists.3 However, a
substantial body of evidence suggests that bilateral metachronous breast cancer is associated
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with poor prognostic features of the initial cancer.4–8 In particular, studies have shown that
patients with bilateral cancer more frequently present with advanced disease than those
presenting with unilateral cancer.9 These findings suggest that some CBCs may represent
first site metastases.

Numerous studies have investigated clonal relationships between primary and contralateral
breast cancers with most reaching the conclusion that the vast majority of CBCs have an
independent origin.10–20 However, many of these studies were based on technologies with
limited resolution for assessing clonality. The preponderance of the early studies involved
assessment of a small number of markers of loss of heterozygosity at genetic regions that
commonly exhibit allelic losses in breast cancer. Later studies employed genome-wide copy
number arrays permitting, in principle, greater resolution and a more definitive test for
clonal relatedness, but were often hampered by the use of deoxyribonucleic acid (DNA)
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obtained from paraffin-embedded tissues. 21,22 Three recent studies used mutational data
derived from whole-exome sequencing for clonality analyses,23–25 and an additional study
examined chromosomal rearrangements in 10 cases using low coverage whole genome
sequencing.26 In the largest of these studies, Klevebring et al. examined 25 metachronous
breast cancers and concluded that 3 (12%) were clonally related.25

In this study we used massively parallel sequencing targeting the 254 genes most frequently
mutated in breast cancer or related to DNA repair to address the question of clonality in
patients with synchronous and metachronous bilateral breast cancer. We also employed a
novel statistical approach that has been developed to address the specific computational
challenges in this setting.27
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Material and Methods

Patients
After approval by the Memorial Sloan Kettering Cancer Center Institutional Review Board
(WA0388-13), the medical records of patients previously identified as having bilateral breast
cancer between 1995 and 2006 were reviewed to ascertain if both surgeries were performed
at Memorial Sloan Kettering Cancer Center. Archival formalin-fixed paraffin-embedded

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(FFPE) tissue blocks from both tumors were obtained from the pathology department.
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Clinical information including patient age, tumor histology and hormone receptor status was
obtained from the medical records.

DNA Extraction
Freshly cut hematoxylin and eosin sections were reviewed to ensure the adequacy of the
specimen. Representative 8µm-thick FFPE sections were stained with nuclear fast red.
Microdissection was performed using a sterile needle under a stereomicroscope (Olympus
SZ61, Center Valley, PA) to ensure >80% of tumor cell content and that the normal tissue
was devoid of any neoplastic cells as previously described.28,29 Genomic DNA extraction
from each tumor and matched normal tissue was performed using the DNeasy Blood and
Tissue Kit (Qiagen, Valencia, CA), and quantified using the Qubit Fluorometer assay
(Invitrogen, Thermo Fisher Scientific, Waltham, MA) following manufacturers’ instructions.
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Targeted-Capture Massively Parallel Sequencing

DNA from all tumor and normal specimens were subjected to targeted capture massively
parallel sequencing using a library of baits targeting exons of the 254 genes most frequently
mutated in breast cancer and/or related to DNA repair.30 Genes harboring deleterious or
potentially deleterious mutations in The Cancer Genome Atlas (TCGA) and the International
Cancer Genome Consortium were included in the panel, in addition to a number of other
genes that are of interest due to the presence of frequent copy number alterations mapping to
their loci (e.g. CDKN2A, CDKN2B, RB1, FGFR1), germline mutations associated to
hereditary cancer syndromes (e.g. BRCA1, BRCA2, PALB2, RAD51D, RAD51C, BRIP) or
the presence of potentially actionable mutations (e.g. BRAF, KIT, EGFR, ERBB2 and
ERBB3). Barcoded sequence libraries were prepared (New England Biolabs,
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KapaBiosystems) as previously described, and sequenced on an Illumina HiSeq 2000 (San

Diego, CA) following validated protocols at the Memorial Sloan Kettering Cancer Center
Integrated Genomics Operations.30

Reads were aligned to the human reference genome GRCh37 using the Burrows-Wheeler
Aligner.31 Local realignment, duplicate removal and quality score recalibration were
performed using Genome Analysis Toolkit.32 Somatic single nucleotide variants (SNVs)
were identified using MuTect33 and small insertions and deletions (indels) were identified
using Strelka and VarScan 2.34,35 SNVs and indels with mutant allelic fraction (MAF) <1%
and/ or supported by <5 reads were disregarded.29,36 Variants found with >5% global minor
allele frequency in dbSNP (Build 137) or that were covered by <10 reads in the tumor or <5
reads in the germline were disregarded. Variants for which the tumor variant allele fraction
was <5 times than that of the normal variant allele fraction were disregarded. The cancer cell
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fractions of mutations were defined using ABSOLUTE as previously described.28,30

In cases in which multiple matching mutations were observed we performed validation by

repeating the targeted capture massively parallel sequencing on a new DNA sample. In
addition we repeated the sequencing for a few additional cases based on availability of
tissue/DNA to examine the validation rate of observed mutations. The overall validation rate
of observed mutations was 90% (35/39). In cases in which a single match was observed we

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 Begg et al. Page 4

resequenced the match using either amplicon sequencing or Sanger sequencing as previously
described.28,30 The primers used for these methods are provided in Supplementary Table 1.
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For the amplicon sequencing mpileup files generated from samtools mpileup (version 1.2
htslib 1.2.1) were used to determine whether a mutation existed in the validation binary
alignment/map file. Mutations that were found at <1% allele frequency in the germline and
>1% in the tumor, where the MAF in the tumor was at least 5 times that in the normal (the
same filter used in the targeted sequencing analysis) and had at least 50 reads in both the
tumor and the normal were considered validated as somatic. Loci with <50 good-quality
reads in either the tumor or normal were considered to have insufficient depth.

Clinical Interpretations
For cases with matching mutations in both tumors we assigned a subjective clinical
interpretation of whether or not the tumors were independent, based on tumor stage, the
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presence of in situ carcinoma, ER, PR, and HER2 status, and clinical course. Features
considered suggestive of metastases included advanced stage of the initial tumor in the case
of metachronous cancers or of one of the cancers in the case of synchronous cancers, the
absence of in situ carcinoma in the proposed metastatic lesion, or the subsequent
development of other sites of metastatic disease.

Statistical Methods
For each individual case we used a statistical test of the hypothesis that two tumors are of
independent origin that was developed specifically for the purpose of comparing mutational
profiles.27 The method relies strongly on the fact that somatic mutations at some loci are
common in breast cancer while the preponderance of mutations are very rare. Matches at
rare loci provide much stronger evidence for clonal relatedness than matches at common
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loci. These “marginal” loci-specific probabilities of observing a mutation were obtained

from combining the empirical relative frequencies observed in the TCGA breast cancer
dataset (downloaded from the Broad GDAC Firehose at https://gdac.broadinstitute.org) and
the present study.37 Somatic mutations observed in our study that were not previously
observed in TCGA were assigned a marginal probability of 1/1039, where 1039 is the
combined number of patients in this study and in the TCGA breast study.

To derive copy number changes the number of reads that mapped to a locus and met base
and mapping quality thresholds were obtained using the samtools mpileup function.
Candidate loci were obtained from dbsnp build 137 along with pseudo- single nucleotide
polymorphisms (SNPs) to obtain counts from regions that are sparse in SNPs. The genome
was split into consecutive bins of size 200 bases and median count of candidate loci in a bin
used for read depth of the bin. Only bins that had a count of at least 25 in the normal were
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considered. Guanine-cytosine (GC) normalization was accomplished by using locally

weighted regression of the logarithm of the tumor to normal count ratio as a function of GC
percentage of a 1000 base window around the locus. Loci with <25 reads in any of the
normal samples were excluded. Log-ratios were then averaged in blocks of 5 consecutive
markers to the final resolution of 2523 markers in order to smooth out the noise. Data were
segmented using circular binary segmentation38 and gains and losses were called if the mean
log-ratios were above or below 1 median absolute deviation of residuals from the sample’s

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median, where residuals were computed as a difference between log-ratios and the
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segmented value at each locus. We elected not to compare break points from full
segmentation of these data because the targeted panel consisted of genes that are not spread
uniformly in the genome. Consequently we elected to use a conservative approach whereby
we limited attention to gains and losses that spanned an entire chromosome arm defined as a
gain or loss covering at least 90% of the markers on the chromosome arm. We gauge and
compare the similarities of these whole-arm copy number profiles using the log likelihood
ratio measure proposed by Ostrovnaya et al. in which attention is restricted solely to the
matching patterns of gains or losses that covered an entire chromosome arm.22

Results
The study population included 248 patients with bilateral invasive breast carcinoma in whom
both surgeries were performed at Memorial Sloan Kettering Cancer Center, of which 97
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were metachronous and 151 were synchronous. Tissue from both cancers was reported as
archived in 98 of these cases. DNA of sufficient quality and quantity was obtained from both
contralateral breast tumors and matched normal tissue and subjected successfully to targeted
capture massively parallel sequencing for 49 of these cases. The clinical characteristics of
these patients are described in Supplementary Table 2.

Targeted capture massively parallel sequencing of the tumors and matched normal tissue was
performed to a median coverage of 544× (range 77×-1552×) in the tumors and 360× (range
117×-2069×) in the normal samples, and somatic mutations observed in both tumors from a
patient were independently validated (see Methods). The numbers of somatic mutations per
tumor observed in the 254 gene panel analyzed ranged from 0 to 33 with a median of 4 (the
complete list of mutations is provided in Supplementary Table 3). Consistent with previous
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studies, the genes most frequently affected by somatic mutations in the breast cancers
analyzed here included PIK3CA, TP53 and CDH1 (Figure 1). We note that 5 of the 7 genes
found to be mutated and shared between the CBCs from a given patient (designated with a
“●” in Figure 1) affected the top 8 most frequently mutated genes, specifically PIK3CA,
ARID1A, CDH1, TBX3 and MAP3K1. Identical somatic mutations were identified in the
tumors from both breasts in 6 of the 49 cases analyzed (Table 1). Importantly, these shared
mutations were present at similar cancer cell fractions of tumor cells in the right and left
breast cancers (Supplementary Table 3). For 3 of these cases our statistical test for clonality
was significant. In case #36, 3 of 4 observed somatic mutations were matches and in case
#48, 2 of 3 observed somatic mutations were matches. However, for case #8 only 1 of the 7
somatic mutations identified was shared between the two breast cancers. Notably, this
ARID1A mutation was found to have a cancer cell fraction of 1 (i.e. bioinformatically
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inferred to be present in all tumor cells) in both the left and right invasive breast cancers, and
to be the sole mutation in either left or right tumor of this case with a cancer cell fraction of
1 (Supplementary Table 3). The statistical test employed classified the two metachronous
breast tumors of this case as clonal given that the shared mutation occurred at a very rare
residue (e.g. ARID1A E250fs). In 3 additional cases (cases #63, #67 and #75) a single
common PIK3CA H1047R hotspot mutation was found to be shared between the
synchronously diagnosed left and right breast cancers. The statistical test employed was not

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 Begg et al. Page 6

significant for any of these cases. [We note that a validation experiment was not performed
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for the match in case #63.]

We further evaluated the evidence for clonal relatedness by examining the copy number
alterations. These are displayed for the 6 cases with matches in Figure 2. For the 3 cases
designated as “clonally related” by mutational analysis (cases #8, #36 and #48) the copy
number profiles of the tumor pairs displayed multiple common gains and losses. These 3
cases had the 3 highest values of our log likelihood measure characterizing the similarity of
the copy number profiles (Table 1). However several other cases demonstrated multiple
matching gains and/or losses, indicating that there may be cases which are clonal but where
no matches were observed on the panel (Supplementary Plot 1).

Key clinical details and interpretations of the 6 cases with mutational matches are provided
in Table 2 (additional clinical information on all cases is provided in Supplementary Table
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2). For cases #8, #36 and #48 the clinical features are consistent with the possibility of the
second breast cancer being a metastasis, supporting the evidence from both matching
mutations and similar copy number patterns, although distant metastases were not observed
after longer-term follow-up for 2 of these 3 cases. By contrast, the 3 cases with single
matching PIK3CA H1047R mutations all have clinical features consistent with independent
primary tumors.

Discussion
Our findings are consistent with the hypothesis that a small subset of CBCs are clonally
related and constitute metastatic dissemination from one breast to another. This phenomenon
can occur regardless of whether the two breast tumors occur synchronously or
metachronously. We found strong molecular evidence in this study to support the clonal
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relationship between the two invasive breast cancers in 3 out of 49 (6%) CBCs. In these 3
cases, we identified identical mutations at rare residues, all of which were validated, and the
copy number alterations and clinical information supported the interpretation that these
tumor pairs are clonally related. In 3 additional patients a shared single PIK3CA hotspot
mutation was observed. However this evidence for clonality alone is weak due to the high
frequency of mutations at this residue in breast cancers generally and the fact that the
clinical data suggest that the tumors are independent in all 3 patients.

We believe that the clinical features of the 3 cases classified as clonal are consistent with this
diagnosis. The 3-year interval between the initial cancer and the contra-lateral diagnosis for
case #36 is congruent with recent studies that support the premise that cancer cells can
disseminate to distant sites early in tumor development but may remain dormant for long
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periods before clinically detectable metastases emerge.39,40 This prolonged period of

dormancy is particularly common in ER positive tumors, where more than 50% of distant
recurrences occur more than 5 years post diagnosis, particularly in the setting of adjuvant
endocrine therapy.41 Case #36 was still in remission one year later. In case #48 the patient
had synchronous inflammatory cancers and developed a chest wall recurrence 12 years post-
diagnosis. Primary therapy for both of these cases consisted of modified radical mastectomy,
chemotherapy with an anthracycline, cyclophosphamide and a taxane (ACT), post

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mastectomy radiotherapy to the chest wall and node fields, and tamoxifen. The same
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treatment strategy was used for the subsequent contra-lateral tumor in case #36 in addition to
an aromatase inhibitor. The application of what was in effect cytoreductive surgery for a
solitary metastasis for both these cases may have proved to be effective treatment in
providing the long disease-free interval for case #48. Indeed, prolonged survival after
aggressive local and systemic therapy is well documented in stage IV breast cancers. In an
analysis of 21,372 patients with stage IV cancer and an intact primary, survival at 10 years
was observed in 10% of patients who received surgery on the primary tumor.42

In contrast to these two cases, in case #8, after treatment with modified radical mastectomy,
ACT and post mastectomy radiotherapy to the chest wall and node fields, the contralateral
lesion developed 1 year post-diagnosis and despite treatment with neoadjuvant ACT
followed by modified radical mastectomy, chest wall recurrence developed 2 months
postoperatively followed by distant metastases 11 months later. The finding of DCIS in the
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second cancer in this case is somewhat puzzling since the presence of DCIS is generally
regarded as an indicator of a primary tumor. However, a recent large observational study
identified a small but significant number of patients who died of metastatic breast cancer
without local recurrence following diagnosis and treatment of DCIS, suggesting that DCIS
itself represents very early stage breast cancer that nonetheless has metastatic potential.43 It
is notable that the preponderance of observed mutations in this case were not matches.

The knowledge that a proportion of CBCs are in fact metastases is clinically important to
allow individualization of the use of surgery on the contralateral breast and axilla based on a
diagnosis of metastatic disease versus a new primary cancer, and to direct the choice and
duration of systemic therapy. The management of the breast and axilla in patients presenting
with stage IV cancer and an intact primary tumor remains a subject of debate. Although
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multiple retrospective studies suggest a survival benefit following surgery of the primary
tumor in these patients,44 a prospective randomized trial45 and a prospective registry study45
have failed to confirm this. Until this issue is resolved with the reporting of the on-going
Eastern Cooperative Oncology Group randomized trial (NCT01242800), it is unclear if the
finding that a subset of women with CBC actually have metastases to the contralateral breast
rather than a new primary tumor has the potential to alter clinical management of CBC.

To implement the approach employed here in a clinical setting when a new contralateral
breast tumor is diagnosed would require the availability of tumor tissue from both tumors as
well as matched normal tissue. This may be challenging for metachronous CBCs if the first
tumor was diagnosed several years previously or at a different hospital. In the era of
precision medicine, however, and with the implementation of routine panel mutation testing
such as the Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT)47 at
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our institution and other similar assays at other institutions, genomic analysis of tumors is
likely to become increasingly common. Our success rate in extracting DNA from archived
tissue samples and successfully sequencing the samples was 75% in this study. Whole-
exome and/ or targeted capture massively parallel sequencing analyses have been proven to
constitute a successful strategy to resolve the clonality of synchronous neoplasms in other
contexts,48–50 and to define the origin of metastatic deposits in the presence of independent
primaries in distinct anatomical sites.51 Given the increasing robustness of sequencing

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assays based on the analysis of DNA extracted from archival FFPE tissue samples,
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genotyping approaches may constitute a useful ancillary method to distinguish independent

primary cancers from metastatic disease.

Our study has important limitations. When the DNA samples from both tumors are
successfully sequenced there remain challenges in making the differential diagnosis of
independence versus metastasis. Our targeted sequencing panel contained 254 genes,
including all the known driver genes in breast cancer. Yet the number of mutations observed
in any given tumor was frequently small. The median number of somatic mutations per
tumor was 4, and in 1 of the 98 tumors no mutation was observed at all. As a result there
will frequently be considerable uncertainty in calling cases as clonal versus independent. For
example, even if no shared genetic alterations are observed between two tumors using a
given targeted capture massively parallel sequencing assay, there may be clonal mutations
present in both breast cancers in genomic locations that were not included in the sequencing
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panel or in non-coding regions of the genome. Indeed for a few cases in our study in which
no mutational matches were observed the copy number profiles showed considerable
similarities (Supplementary Plot 1), suggesting that CBCs may be determined to be clonally
related based on their patterns of gene copy number alterations rather than on their
mutational profiles if no matches are observed after testing a limited panel of genes.
Conversely, the presence of a mutation shared between two tumors does not necessarily
indicate a clonal origin for the tumors, since mutations at the same locus could occur by
chance in independent tumors, especially if the mutation is common, such as the PIK3CA
hotspot mutation that occurred in both breasts in three cases in this study, #63, #67 and #75.
Whole-exome sequencing has the potential to greatly reduce the diagnostic uncertainty, but
it would not necessarily eliminate it. Indeed in an earlier study by our group that used
whole-exome sequencing to assess the evidence of clonal relationships between pre-
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malignant lesions (lobular carcinoma in situ) and invasive breast cancers several cases were
observed with a single shared somatic mutation, rendering the evidence for clonality
equivocal.52 Finally, our statistical testing is based on the premise that the probability of a
matching mutation in two independently occurring tumors is the square of the relative
frequiency of the given mutation in breast cancers generally. However, matches are more
likely if host characteristics of the patient influence these probabilities, a phenomenon that
will occur if somatic mutational patterns are influenced by the germline. While there are, to
our knowledge, no published studies addressing this issue, there has been an anecdotal
report of a pair of monozygotic twins with breast cancer whose tumors had unusually similar
copy number patterns.53

Despite these limitations, our study confirms that a small but substantial proportion of CBCs
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may actually represent metastases from the original primary cancers. Formal clonality
testing of tissue samples from both tumors has the potential to alter the clinical management
for these cases and represents a paradigm shift in our thinking about CBC.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

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Acknowledgments
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We are grateful to the following individuals who provided assistance with specimen processing or data
management:- Nicola Fusco, Elena Guerini-Rocco, Caterina Marchio, Russell Towers.

Grant Sponsor: The study was funded in part by the Alan and Sandra Gerry Metastasis Research Initiative at
Memorial Sloan Kettering Cancer Center, by award CA08748 from the National Cancer Institute, and in part by a
Cancer Center Support Grant of the National Institutes of Health/National Cancer Institute (grant No
P30CA008748). J.S. Reis-Filho is funded in part by the Breast Cancer Research Foundation. S. Piscuoglio is
funded by Swiss National Science Foundation (Ambizione grant PZ00P3_168165)

Abbreviations
CBC contralateral breast cancer

FFPE formalin-fixed paraffin-embedded

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TCGA The Cancer Genome Atlas

SNV single nucleotide variants

MAF mutant allelic fraction

GC guanine-cytosine

DNA deoxyribonucleic acid

SNP Single nucleotide polymorphism

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Figure 1. Somatic Mutations Observed

Heatmap showing the 30 genes most frequently affected by mutations in 98 tumors
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subjected to targeted massively parallel sequencing from 49 patients with contralateral breast
cancer. Samples are shown in rows, genes in columns. The top 6 cases are those with
observed matching mutations, identified by ●. The histogram on the right displays the total
numbers of mutations observed, per case, and the color code distinguishes different types of
mutation, as indicated: indel, small insertion and deletion; SNV, single nucleotide variant.

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 Begg et al. Page 14
Author Manuscript
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Figure 2. Copy Number Alterations

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Each row represents whole chromosome arm copy number gains (in blue) and losses (in red)
for an individual case. Pairs of tumors for all 6 cases with matching mutations are presented.
Copy number plots for all cases included in this study are presented in Supplementary Plot
1.
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Table 1

Results Summary

Somatic Mutations Copy Number

Begg et al.

Case # Matches**
Left Right Shared P-Value* Similarity/Rank***

1 9 7 0 1.0 −1.78 / 43

2 3 3 0 1.0 0.76 / 20

3 2 7 0 1.0 0.57 / 22

4 8 10 0 1.0 −1.75 / 42

6 6 5 0 1.0 0.55/ 23

8 6 2 1 0.004 ARID1A E250fs 3.74 / 2

9 2 3 0 1.0 −1.16/ 35

12 14 3 0 1.0 −4.09 / 49
13 3 3 0 1.0 −0.22 / 28

15 8 5 0 1.0 −1.92 / 44

16 10 8 0 1.0 −2.65 / 47

17 6 8 0 1.0 −0.14/ 26

18 8 2 0 1.0 2.37 / 5

21 4 3 0 1.0 0.29 / 24

23 10 4 0 1.0 −1.47 / 40

24 4 3 0 1.0 1.76/ 8

25 4 6 0 1.0 −0.32 / 30

Int J Cancer. Author manuscript; available in PMC 2019 January 15.

26 6 5 0 1.0 1.57 / 11

27 4 5 0 1.0 1.38 / 13

29 3 1 0 1.0 −0.28 / 29

30 6 5 0 1.0 −1.18 / 36

31 6 5 0 1.0 −1.21 / 37

32 5 4 0 1.0 −0.98 / 34

33 6 4 0 1.0 1.80 / 7

35 5 4 0 1.0 −0.01 / 25
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Somatic Mutations Copy Number

Case # Matches**
Left Right Shared P-Value* Similarity/Rank***

36 3 4 3 <0.001 CDH1 S111fs; TBX3 T267fs; EPPK1 R2337H 4.46 / 1

Begg et al.

38 8 2 0 1.0 −1.60 / 41

40 10 1 0 1.0 2.68 / 4

41 0 9 -- 1.0 1.12 / 17

43 4 4 0 1.0 −0.69 / 33

44 9 21 0 1.0 −2.32 / 46

45 3 4 0 1.0 1.24 / 15

48 2 3 2 <0.001 MLH3 M346R; MAP3K1 R248* 3.01 / 3

52 5 7 0 1.0 2.23 / 6

56 2 5 0 1.0 1.51 / 12

58 3 4 0 1.0 −1.25 / 38
59 2 3 0 1.0 0.83 / 19

62 4 4 0 1.0 −0.17 / 27

63 3 9 1 0.08 PIK3CA H1047R 0.98 / 18

64 5 4 0 1.0 0.63 / 21

66 33 3 0 1.0 1.34 / 14

67 4 1 1 0.08 PIK3CA H1047R 1.75 / 9

70 5 2 0 1.0 −2.95 / 48

71 3 1 0 1.0 1.21 / 16

72 1 3 0 1.0 −2.19 / 45

Int J Cancer. Author manuscript; available in PMC 2019 January 15.

74 2 1 0 1.0 −0.66 / 32

75 4 3 1 0.08 PIK3CA H1047R −1.36 / 39

76 7 5 0 1.0 1.70 / 10

77 3 1 0 1.0 −0.60 / 31

*
P-value for the test of clonal relatedness of the tumor pair.
**
Matches were validated either by re-sampling the tumor and resequencing if sufficient tumor was available or by targeted sequencing (MiSeq), or by Sanger sequencing.
***
Log likelihood ratio measure of similarity of copy number profiles and ranking
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Table 2

Clinical/Pathological Details of Potentially Clonal Cases

Case# Initial Histology Interval to 2nd 2nd Tumor Outcome Clinical

Begg et al.

Primary Histology Interpretation

8 T= 6.8cm, 22 positive nodes, extensive LVI, DCIS 1yr Inflammatory, 12 positive nodes, DCIS present, ER Distant metastases 11 Consistent with metastases
present, ER+/PR−/HER2− −/PR−/HER2− months after second
cancer

36 T>5cm, 19 positive nodes, extensive LVI, no DCIS, ER 3yrs T-diffuse, >5cm, 12 positive nodes, extensive LVI, no NED years after initial Consistent with metastases
+/PR−/HER2− DCIS, ER+/PR−/HER2− diagnosis

48 Inflammatory, 5 positive nodes, no DCIS, ER+/PR+/ Synchronous T= 5cm, 5 positive nodes, extensive LVI, no DCIS, Chest wall recurrence 12 Consistent with metastases
HER2− ER+/PR+/HER2− yrs later

63 T= 1.0cm, 0/5 nodes, DCIS present, no LVI, ER+/PR+/ Synchronous T= 1.8cm, 0/2 nodes, DCIS present, no LVI, ER+/PR NED 4 years 9months Independent primaries
HER2− +/HER2−

67 T= 1.6cm, 0/1 nodes, DCIS present, no LVI, ER+/PR+/ Synchronous T= 1.4cm, 1/3 positive nodes, DCIS present, no LVI, NED 3 years Independent primaries
HER2− ER+/PR+/HER2−

75 T= 1.1cm, 0/1 nodes, DCIS present, no LVI, ER+/PR+/ Synchronous T= 1.4cm, 0/1 nodes, DCIS present, no LVI, ER+/PR NED 1year Independent primaries
HER2− +/HER2−

Abbreviation: DCIS, ductal carcinoma in situ; LVI, lymphovascular invasion; NED, no evidence of disease.

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