Breast Cancer Res Treat

DOI 10.1007/s10549-016-3737-8

EPIDEMIOLOGY

Study of breast cancer incidence in patients of lymphangioleiomyomatosis

Olivier Nun˜ ez1 Antonio Roma´ n2 Simon R. Johnson3 Yoshikazu Inoue4
Masaki Hirose4 A´ lvaro Casanova5 Gorka Ruiz de Garibay6 Carmen Herranz6
Gema Bueno-Moreno7,8 Jacopo Boni6 Francesca Mateo6 Anna Petit9
Fina Climent9 Teresa Soler9 August Vidal9 Jose´ Vicente Sa´ nchez-Mut10 Manel Esteller10,11,12 Jose´ Ignacio Lo´ pez13 Nadia Garc´ıa6 Anna Guma` 14 Rau´ l Ortega14 Mar´ıa Jesu´ s Pla` 15 Miriam Campos16 Emilio Anso´ tegui17
Mar´ıa Molina-Molina18,19 Claudia Valenzuela20 Piedad Ussetti21
Rosal´ıa Laporta21 Julio Ancochea20 Antoni Xaubet19,22 Marina Polla´ n1
Miguel Angel Pujana6

Received: 4 January 2016 / Accepted: 27 February 2016

© The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract Molecular evidence has linked the pathophys- iology of lymphangioleiomyomatosis (LAM) to that of metastatic breast cancer. Following on this observation, we assessed the association between LAM and subsequent breast cancer. An epidemiological study was carried out using three LAM country cohorts, from Japan, Spain, and the United Kingdom. The number of incident breast cancer

Electronic supplementary material The online version of this article (doi:10.1007/s10549-016-3737-8) contains supplementary material, which is available to authorized users.

& Marina Polla´n mpollan@isciii.es

& Miguel Angel Pujana mapujana@iconcologia.net

1 Cancer and Environmental Epidemiology Unit, National Center for Epidemiology, Carlos III Institute of Health, and Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Sinesio Delgado 6,

28029 Madrid, Spain

2 Lung Transplant Unit, Department of Pulmonology, Lymphangioleiomyomatosis Clinic, Vall d’Hebron University Hospital, 08035 Barcelona, Catalonia, Spain

3 National Centre for Lymphangioleiomyomatosis, Nottingham University Hospitals NHS Trust, Nottingham, Nottinghamshire, UK Division of Respiratory Medicine and Respiratory Research Unit, University of Nottingham, Nottingham NG7 2UH, UK

4 National Hospital Organization Kinki-Chuo Chest Medical

Center, Sakai 591-8555, Osaka, Japan

5 Department of Pneumology, Henares Hospital,

28882 Madrid, Spain

cases observed in these cohorts was compared with the number expected on the basis of the country-specific incidence rates for the period 2000–2014. Immunohisto- chemical studies and exome sequence analysis were per- formed in two and one tumors, respectively. All cohorts revealed breast cancer standardized incidence ratios (SIRs) C 2.25. The combined analysis of all cases or restricted to pre-menopausal age groups revealed signifi- cantly higher incidence of breast cancer: SIR = 2.81, 95 % confidence interval (CI) = 1.32–5.57, P = 0.009; and SIR = 4.88, 95 % CI = 2.29–9.99, P = 0.0007,

6 ProCURE, Breast Cancer and Systems Biology, Catalan Institute of Oncology (ICO), Bellvitge Institute for Biomedical Research (IDIBELL), Gran via 199, L’Hospitalet del Llobregat, 08908 Barcelona, Catalonia, Spain

7 Department of Biochemistry, Autonomous University of Madrid (UAM), Biomedical Research Institute ‘‘Alberto Sols’’ (Spanish National Research Council (CSIC)-UAM), Hospital La Paz Institute for Health Research (IdiPAZ),

28029 Madrid, Spain

8 MD Anderson International Foundation, 28033 Madrid, Spain

9 Department of Pathology, University Hospital of Bellvitge, IDIBELL, L’Hospitalet del Llobregat, 08908 Barcelona, Catalonia, Spain

10 Cancer Epigenetics and Biology Program, IDIBELL, L’Hospitalet del Llobregat, 08908 Barcelona, Catalonia, Spain

11 Department of Physiological Sciences II, School of Medicine, University of Barcelona, 08908 Barcelona, Catalonia, Spain

12 Catalan Institution for Research and Advanced Studies

(ICREA), 08010 Barcelona, Catalonia, Spain

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respectively. Immunohistochemical analyses showed pos- itivity for known markers of lung metastatic potential. This study suggests the existence of increased breast cancer risk among LAM patients. Prospective studies may be war- ranted to corroborate this result, which may be particularly relevant for pre-menopausal women with LAM.
Keywords Breast cancer · Incidence ·
Lymphangioleiomyomatosis · mTOR · TSC1 · TSC2

Introduction

LAM is a rare neoplastic disease that appears predomi- nantly in women of childbearing age and is characterized by cystic lung destruction [1, 2]. LAM lesions are hetero- geneous at the cellular phenotypic level but are typically characterized by the proliferation of estrogen receptor a (ERa)- and progesterone receptor (PR)-positive smooth muscle-like cells with lung metastatic potential whose tissue origin remains unclear [3]. LAM cells commonly carry loss-of-function mutations in the tumor suppressor genes TSC1 or TSC2, and consequently, exhibit abnormal activation of the mechanistic target of rapamycin complex
1 (mTORC1) [4]. Thus, LAM can occur as an isolated

13 Cruces University Hospital, BioCruces Research Institute, University of the Basque Country, 48903 Barakaldo, Spain

14 Department of Radiology, University Hospital of Bellvitge, IDIBELL, L’Hospitalet del Llobregat, 08908 Barcelona, Catalonia, Spain

15 Breast Cancer Functional Unit, Department of Gynecology, University Hospital of Bellvitge, ICO, IDIBELL, L’Hospitalet del Llobregat, 08908 Barcelona, Catalonia, Spain

16 Breast Cancer Functional Unit, Department of Medical

Oncology, ICO, IDIBELL, L’Hospitalet del Llobregat,

08908 Barcelona, Catalonia, Spain

17 Lung Transplant and Cystic Fibrosis Unit, Hospital

Universitario y Politecnico La Fe, 46026 Valencia, Spain

18 Department of Pneumology, University Hospital of Bellvitge, IDIBELL, L’Hospitalet del Llobregat, 08908 Barcelona, Catalonia, Spain

19 Consortium for Biomedical Research in Respiratory Diseases

(CIBERES), 28029 Madrid, Spain

20 Department of Pneumology, Instituto de Investigacio´ n Sanitaria La Princesa, Hospital La Princesa, 28006 Madrid, Spain

21 Department of Pneumology, University Hospital Cl´ınica

Puerta del Hierro, 28222 Madrid, Spain

22 Department of Pneumology, Hospital Clinic of Barcelona, August Pi Sun˜ er Biomedical Research Institute (IDIBAPS),

08036 Barcelona, Catalonia, Spain

disease (termed sporadic LAM) or in association with another rare disorder, tuberous sclerosis complex.
mTORC1 regulates a cancer metastasis transcriptional
program [5]. In breast cancer, low expression of TSC1 or TSC2 is associated with poor clinical outcome [6], and depletion of TSC2 expression promotes lung metastasis [7]. These observations led us to the test whether the mediators of breast cancer metastasis to lung could also play a role in LAM. Thus, we identified molecular positivity in LAM lesions for known metastasis mediators [8]. Low TSC1/2 expression in primary breast tumors was found to be associated with enhanced mTORC1 signaling and lung (but not bone) metastasis. Collectively, the clinical, pathologi- cal, and molecular similarities between LAM and breast cancer prompted us to hypothesize a higher incidence of breast cancer in LAM patients. To assess this hypothesis, we compiled the largest epidemiological LAM study to date.

Methods

LAM cohorts
The cohorts comprised LAM patients from three countries (Japan, Spain, and the United Kingdom). Patients were diagnosed by computed tomography scan. In most cases ([80 % of patients in any cohort), diagnosis was comple- mented by the presence of at least one of the following findings: lymphatic complication, lung biopsy, renal angiomyolipoma, and/or TSC [2, 9, 10]. Collectively, the number of diagnoses per population was consistent with the reported prevalence of the disease in developed countries (1–9 in 106 individuals) [10]: 108, 175, and 175 in Japan, Spain, and the United Kingdom, respectively. The ethics committee of the Hospital de Henares approved the inter- national epidemiological study (approval number PI-753). The data from the Japan and United Kingdom cohorts (irreversibly encoded) were provided for combined analy- sis at the Spanish study center. Informed consent was not required for the epidemiological study (PI-753), as it was based on irreversibly encoded retrospective records; how- ever, approved informed consent was obtained from those patients that provided tumor samples for genetic and immunohistochemical analyses. These studies were
approved by the ethics committees of the Bellvitge Institute for Biomedical Research (IDIBELL; PR082/13), the Instituto de Investigacio´ n Sanitaria La Princesa (SEPAR-
2012), and the Hospital de Henares (PI-753). In addition, all LAM patients in the Japanese cohort provided informed consent for the comprehensive analysis of their clinical data (National Hospital Organization Kinki-Chuo Chest Medical Center, approval number 365).

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Follow-up and breast cancer ascertainment
In all clinical settings, LAM patients underwent regular follow-up evaluations with a periodicity of 3–12 months, depending on the country and each patient’s condition. In addition, follow-up was updated via telephone, and patient conditions, including death, were recorded in each data- base. Given that LAM has only been monitored for a short time [9], and since breast cancer screening programs were not fully implemented in some countries until relatively recently, only patients diagnosed from 2000 were consid- ered in this study, which corresponded to [80 % of the cases in each cohort (Supplementary Table 1). In all cases, follow-up started with LAM diagnosis and finished with the first occurrence of one of the following events: death, breast cancer diagnosis, date of last contact, or end of the study (December 31st, 2014). Breast cancer diagnosis required pathological confirmation. Since national breast cancer rates do not include in situ tumors, only invasive cases were considered.
Statistical analysis
The incidence of breast cancer in the LAM cohorts was compared with the incidence observed in the general population, using standardized incidence ratios (SIRs) [11]. This ratio corresponds to the observed versus expected number of cases, where the expected number is obtained considering age (5-year groups) and period- (2000–2004,
2005–2009, and 2010–2014) specific incidence breast cancer rates in each country. The same analysis was repeated considering pre-menopausal age groups (women younger than 50). Person-years in each stratum were cal- culated using the survival package (version 2.38, R soft- ware), and SIR confidence intervals were computed under the Poisson assumption and using the exact method [12].
Antibodies
The antibodies used in this study were anti-ERa (#IR151, Dako), anti-FSCN1 (#SC-56531, Santa Cruz Biotechnol- ogy), anti-HMB-45 (#SC-59305, Santa Cruz Biotechnol- ogy), anti ID1 (#SC-488, Santa Cruz Biotechnology), anti- PR (#IR168, Dako), anti-phospho-Ser235-236 S6 riboso- mal protein (anti-pS6; clone 91B2, Cell Signaling Tech- nology), anti-SMA (#A2547, Sigma-Aldrich), and anti- SOX9 (#AB5535, Millipore).
Immunohistochemistry
Immunohistochemical assays were performed using stan- dard protocols with the EnVision (Dako) method. Each tissue and biomarker was evaluated in at least two
independent assays and no substantial differences were observed. Equivalent sections were processed to include incubation with immunoglobulin controls (Sigma-Aldrich), which did not reveal staining in any case. The immuno- histochemistry results were evaluated independently by at least two expert pathologists.
Exome sequencing
Breast cancer ([50 % tumor cells) DNA was extracted from a surgical sample following standard protocols and the exome sequence analyzed by GATC Biotech. The coverage was of [109 for at least 90 % of the genome and some TSC1/2 exonic regions were targeted by Sanger sequencing to obtain full annotation. Variant mapping, alignment, calling, annotation, and filtration were per- formed using the genome reference hg19 (GRCh37) and the GATK modules [13].

Results

Analysis of standardized incidence ratios
The three LAM cohorts revealed breast cancer SIRs C 2.25 (Table 1, which also includes the observed and expected numbers per cohort and for the combined analysis). In fact, the United Kingdom cohort revealed a significant SIR of 3.16; 95 % confidence interval (CI) = 1.08–8.15, P = 0.039. Thus, the global estimation of an excess of breast cancer cases was found to be sig- nificant: SIR = 2.81, 95 % CI = 1.32–5.57, P = 0.009 (Table 1).
Since LAM is generally diagnosed in women of fertile age and an increased risk of breast cancer may be associ- ated with earlier age of onset [14], an analysis restricted to pre-menopausal age groups (women younger than 50) was performed. A significantly higher incidence of breast can- cer in the cohorts of Spain and the United Kingdom was observed, with SIRs = 4.98 and 7.09; P values = 0.023 and 0.003, respectively. Moreover, the global analysis was also found to be significant: SIR = 4.88, 95 % CI = 2.29–9.99, P = 0.0007 (Table 1).
Immunohistochemical features of breast tumors from LAM patients
Approximately, two-thirds of the breast tumors that develop in pre-menopausal women in the general popula- tion are hormone receptor-positive [15]. In our study, all tumors with available pathological information (n = 8) were recorded as ERa- and PR-positive. While none pre- sented family history of the disease, relatively uncommon

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Table 1 Number of breast

All cases

cancer cases observed and

expected, and SIRs in the three

LAM cohorts

Country Person-years Observed cases (n)

Expected cases (n)

SIR 95 % CI P value

Japan 605.02 1 0.44 2.25 0.12–12.96 0.36

Spain 899.01 3 1.13 2.64 0.72–7.77 0.11

United Kingdom 809.22 4 1.26 3.16 1.08–8.15 0.039

Combined 2313.26 8 2.84 2.81 1.32–5.57 0.009

Pre-menopausal (\50 years old)


Country Person-years Breast cancer cases/cohort (n)

Expected cases (n)

SIR 95 % CI P value

Japan 453.32 0 0.27 – – – Spain 620.00 3 0.60 4.98 1.36–14.63 0.023

United Kingdom 568.36 4 0.56 7.09 2.42–18.28 0.003

Combined 1641.68 7 1.43 4.88 2.29–9.99 0.0007

clinical and histopathological features were noted. Three Spanish cases were diagnosed with multifocal breast can- cer, one of them was diagnosed at age 37 with rapid pro- gression following 8 months of rapamycin treatment for LAM [10]. Whole exome sequencing did not identify TSC1/2 mutations in a fresh tumor sample for this case; importantly, exonic TSC1/2 mutations are common but not seen in all sporadic LAM patients [16]. The tumor exome analysis did uncover a known oncogenic mutation in PIK3CA (c.3140A [ G, H1047R), which was confirmed by Sanger sequencing (Supplementary Fig. 1); however, mutations in this gene are found relatively frequent in ERa- positive breast cancer [17].
Immunohistochemical analyses of the above depicted tumor and of an additional case showed positivity for both ERa and PR in cells with apparently different phenotypes (Fig. 1). Both cases were also positive for a canonical marker of mTORC1 activity and for the lung metastatic mediators revealed in our original study (FSCN1, ID1, and SOX9; Fig. 2). Heterogeneity was also apparent and pos- itive cells were linked to either an epithelial or a spindle phenotype (Fig. 2). Together, the observations of multifo- cal ERa-positive tumors in pre-menopausal LAM patients without family history of the disease would further suggest a link to a specific breast cancer subtype.

Discussion

In previous analyses, we tested the hypothesis that the metastatic properties of LAM cells could be further depicted using knowledge of breast cancer tropism to lung, and thus identified the expression of metastatic mediators and cancer cell stemness molecular determinants in LAM lesions [8]. Following on from this evidence, and given that
the tissue of origin of LAM cells remains a subject of debate [3, 18, 19], we aimed to assess breast cancer inci- dence in LAM patients. The results of our study of three cohorts in different countries suggest that LAM patients, particularly those in the pre-menopausal age range, are at higher risk of developing invasive breast cancer than women from the general population. This might therefore indicate a shared cell origin and/or shared genetic risk factors between the two diseases. However, we cannot rule out that the retrospective nature of the survey and/or the regular clinical monitoring of LAM patients may have led to an over-estimation of breast cancer incidence.
Apart from brain and kidney malignancies [20], there is no previous evidence of other cancer susceptibilities in patients with tuberous sclerosis complex; indeed, among the LAM patients with breast cancer in our study, only one (1/15; 6, 7 %) was diagnosed with this disease (i.e., carrier of a germline TSC2 mutation), so the others were sporadic LAM cases. Intriguingly, however, a recent study of 1,000 breast cancer patients incidentally identified a pathogenic germline TSC2 mutation [21]; therefore, further germline studies of TSC1/2 may be warranted to assess the potential link with breast cancer risk. In our study, exome analysis of a breast tumor sample did not reveal mutations in these genes, but exonic mutations are not detected in all sporadic LAM patients [16], which further suggests heterogeneity in the biology of LAM.
Hypothetically, multiple tissue or organ origins can co- exist if it is considered that enhanced mTORC1 activity mediates metastatic behavior in different cancer types [5]. Nevertheless, the link between LAM and women at childbearing age [2, 10] suggests tissue and/or cell type specificity. Dependence on hormone signaling is a hall- mark of several cell types in breast tissue [22]. In this scenario, the repeated cycles of vast cell proliferation that

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Fig. 1 Histopathological and immunohistochemical characterization of breast tumors in two LAM patients. a Hematoxylin-eosin (HE) and p63 (patient #1 only) staining results from the corresponding tumors in LAM patients. Arrows mark magnified fields shown in the insets. Three panels are shown for patient #2, which correspond to

(i) invasive, (ii) in situ, and (iii) desmoplastic histologies. The p63 marker was used as evidence of a metaplastic carcinoma. b Immunos- taining results for ERa and PR in the corresponding breast tumors. Red arrows mark magnified fields shown in the insets and black arrows mark positive cells with a spindle-like phenotype

occur in normal breast tissue at reproductive age provide the time window for acquiring somatic genetic mutations by chance. Thus, if a mutation is acquired in a specific
population of ERa/PR-positive cell progenitors [22] lead- ing to an increase in mTORC1 activity, the corresponding cells would possess metastatic behavior with lung tropism.

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Fig. 2 Positivity for mTORC1 signaling and metastatic markers in breast tumors of LAM patients. a Results of phospho-Ser235/236- ribosomal protein S6 (pS6) staining in two available breast tumors. Heterogeneity (i.e., positive and negative tumor cells in case #1) and positive cells with a spindle phenotype (case #2, right panels,

depicted in insets) can be observed (arrows mark magnified regions). b Results of the analysis of the metastatic markers (FSCN1, ID1, and SOX9) in both cases. Heterogeneity (particularly in case #1) and spindle-like phenotypes (particularly in case #2) can be observed (arrows mark magnified regions)

Abnormal activation of mTORC1 would not produce invasive tumors unless additional mutations are acquired, which we speculate might have occurred in the identified cases of LAM and breast cancer. Overall, prospective studies may be warranted to corroborate our findings,
which may be particularly relevant for pre-menopausal women with LAM.

Acknowledgments We wish to thank all patients for their contri- bution to the work of the corresponding centers, clinics, or

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foundations and owe particular thanks to the Spanish Association of LAM (AELAM) for continuous support during the study. We also wish to thank Judy Sheridan for sharing with us the self-reported United States LAM Foundation data, Prof. Francis McCormack for his valuable comments, and Prof. Jose´ Baselga for helping to identify a breast cancer case.

Authors’ contributions OM, MP, and MAP conceived of the study and participated in its design. GRG, CH, GM-B, JP, FM, AP, FC, TS, AV, JIL, and NG conducted analysis of pathology specimens. GRG, JVS-M, and ME conducted genetic analyses. AR, SRJ, YI, MH, AC, AG, RO, MJP, MC, EA, MM-M, CV, JA, AX, PU, and RL collected the clinical data. All authors read and approved the final manuscript.

Grant support This work was supported by grants from the

‘‘Asociacio´ n Espan˜ ola Contra el Ca´ncer’’ (AECC, Hereditary Can- cer), the Generalitat de Catalunya (SGR 2014-364), the ‘‘Instituto de Salud Carlos III’’ (ISCIII), Spanish Ministry of Economy and Com- petitiveness, ‘‘Fondo Europeo de Desarrollo Regional (FEDER), una

manera de hacer Europa’’ (PIE13/00022-ONCOPROFILE, FIS PI12/

01528 and PI15/00854, and RTICC RD12/0036/0008), and the Telemaraton 2014 ‘‘Todos Somos Raros, Todos Somos U´ nicos’’ (P35).

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

Open Access This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which per- mits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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