Breast Cancer Res Treat
DOI 10.1007/s10549-010-1264-6
PRECLINICAL STUDY
Acquisition of metastatic tissue from patients with bone
metastases from breast cancer
J. F. Hilton • E. Amir • S. Hopkins • M. Nabavi • G. DiPrimio
A. Sheikh • S. J. Done • D. Gianfelice • F. Kanji • S. Dent •
D. Barth • N. Bouganim • A. Al-Najjar • M. Clemons
Received: 11 October 2010 / Accepted: 12 November 2010
Ó Springer Science+Business Media, LLC. 2010
Abstract Biopsies of metastatic tissue are increasingly
being performed. Bone is the most frequent site of metastasis
in breast cancer patients, but bone remains technically
challenging to biopsy. Difficulties with both tissue acquisi-
tion and techniques for analysis of hormone receptor status
are well described. Bone biopsies can be carried out by either
by standard posterior iliac crest bone marrow trephine/
aspiration or CT-guided biopsy of a radiologically evident
J. F. Hilton
F. Kanji
S. Dent
N. Bouganim
A. Al-Najjar

M. Clemons (&)
Division of Medical Oncology, The Ottawa Hospital Cancer
Centre, University of Ottawa, Box 912, 501 Smyth Road,
Ottawa, ON K1H 8L6, Canada
e-mail: mclemons@toh.on.ca
E. Amir
D. Barth
Division of Hematology and Medical Oncology,
The Princess Margaret Hospital, University of Toronto,
Mississauga, ON, Canada
S. Hopkins
Department of Pharmacy, The Ottawa Hospital,
University of Ottawa, Ottawa, ON, Canada
M. Nabavi
Department of Pathology, The Ottawa Hospital,
University of Ottawa, Ottawa, ON, Canada
G. DiPrimio
A. Sheikh
Department of Radiology, The Ottawa Hospital,
University of Ottawa, Ottawa, ON, Canada
S. J. Done
Laboratory Medicine Program,
University of Toronto, Ottawa, ON, Canada
D. Gianfelice
Department of Radiology, The Princess Margaret Hospital,
University of Toronto, Mississauga, ON, Canada
•
bone metastasis. The differential yield of these techniques is
unknown. Results from three prospective studies of similar
methodology were pooled. Patients underwent both an out-
patient posterior iliac crest bone marrow trephine/aspiration
and a CT-guided biopsy of a radiologically evident bone
metastasis. Samples were assessed for the presence of
malignant cells and where possible also for estrogen (ER)
and progesterone receptor (PgR) expression. 40 patients
were enrolled. Bone marrow aspiration/trephine biopsy was
completed in 39/40 (97.5%) and CT-guided biopsy was
completed in 34/40 (85%) of patients. Sufficient tumor cells
for hormone receptor analysis were available in 19/39
(48.8%) and 16/34 (47%) of and bone marrow aspiration/
trephine and CT-guided biopsies, respectively. Significant
discordance in ER and PgR between the primary and the
bone metastasis was also seen. Nine patients had tissue
available from both bone marrow and CT-guided bone
biopsies. ER and PgR concordance between these sites was
100 and 78%, respectively. Performing studies on human
bone metastases is technically challenging, with relatively
low yields regardless of technique. Given resource issues and
similar success rates when comparing both techniques, bone
marrow examination may be utilized first and if inadequate
tissue is obtained, CT-guided biopsies can then be used.
Keywords Metastatic breast cancer
Hormone receptor
discordance
Bone metastases
Abbreviations
ER Estrogen receptor
PgR Progesterone receptor
HER2 Human epidermal growth factor receptor 2
IHC Immunohistochemistry
FISH Fluorescent in-situ hybridization
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 Breast Cancer Res Treat

Introduction unknown whether the unguided or CT-guided method is the

There are a number of reasons for biopsying metastatic
lesions. These biopsies can be used for histological con-
firmation of metastatic disease in patients where the clin-
ical and/or radiological features are in doubt or where it is
possible that there may be a new primary tumor [1]. It has
also been shown that tumor characteristics, specifically
estrogen receptor (ER), progesterone receptor (PgR) and
epidermal growth factor receptor 2 (HER2) status can
change between the primary and metastatic sites (Table 1).
In a pooled analysis of two large prospective studies, dis-
cordance rates in ER, PgR and HER2 were found to be 16,
40, and 10%, respectively. Biopsy results altered man-
agement in 15.9% of patients (95% CI 11.7–20.9%,
P = \0.0001) [2]. In addition, an increasing number of
clinical trials are mandating tissue collection as part of on
going translational sub-studies to understand the in vivo
mechanism of a range of ‘‘targeted’’ agents.
Even though bone is the most frequent site of metastasis
in breast cancer patients [15], it remains one of the most
technically difficult areas to biopsy. The choice of tech-
nique for acquiring bone samples usually includes either
unguided posterior iliac crest bone marrow trephine/aspi-
ration or CT-guided biopsy of a radiologically evident bone
metastasis. The difficulties of obtaining tissue from bone
are well described [16, 17]. At the current time, it is
best for acquiring tissue from bone.
The purpose of this study was to assess whether bone
marrow trephine/aspiration biopsy can be utilized in place
of CT-guided biopsy of bone metastases in patients with
metastatic breast cancer and report the overall feasibility of
obtaining utilizable malignant cells by either biopsy tech-
nique. In addition, in patients where metastatic bone tissue
was obtained by either technique, the hormone receptor
status of the metastasis was compared to that of the primary
tumor. In those cases where both biopsy techniques yielded
sufficient cells, the receptor status of both these sites was
compared to see whether there was any significant dis-
cordance between different bone sites in the same patient.
Materials and methods
Three prospective studies were conducted in patients with
metastatic breast cancer to bone who were being treated at
The Princess Margaret Hospital, Toronto and The Ottawa
Hospital Cancer Centre, Ottawa, Canada. Eligibility
included: histologically confirmed breast cancer and
radiological evidence of at least one bone metastasis that
was amenable to CT-guided biopsy. Both patients with
stable and progressive disease were eligible. There was no
restriction with regard to the number of lines of hormonal

Table 1 Review of studies assessing hormone receptor/HER2 discordance

Author Design Number Number ER PR HER2 Method for Method
Enrolled Assessed discordance discordance discordance ER/PgR for HER2
(%) (%) (%) determination determination

Amir et al. [2] Prospective 271 271 13 28 5 IHC FISH

Locatelli et al. [3] Retrospective 255 255 16 29.8 13.1 IHC IHC or FISH
Karlsson et al. [4] Retrospective – 486 for ER, 456 35 43 NR Biochemical –
for PR and IHC
Simmons et al. [5] Prospective 40 29 12 28 8 IHC IHC or FISH
Broom et al. [6] Retrospective 100 100 17.7 37.3 0 IHC IHC or FISH
Lower et al. [7] Retrospective 200 173 30 39 NR IHC –
Lipton et al. [8] Prospective 240 240 NR NR 26 – HER2
immunosorbent
assay
Regitnig et al. [9] Retrospective 31 31 NR NR 48.4 – IHC and FISH
Li et al. 1994 [10] Retrospective – 83 for ER, 32 for 29 44 NR IHC –
PR
Mobbs et al.[11] Retrospective – 129 12–19 24–33 NR Biochemical –
Raemaekers et al. Prospective – 75 for ER, 50 for 18.7 28 NR Biochemical –
[12] PR
Holdaway et al. Prospective 28 28 53.6 36.8 NR Biochemical –
1983 [13]
Hull et al. [14] Retrospective 232 232 16.5 NR NR Biochemical –

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Breast Cancer Res Treat
therapy, chemotherapy, or bisphosphonate therapy.
Patients with a hematologic condition or who were at a
significant risk of bleeding were excluded from the study.
The study protocol and consent process was reviewed and
approved by the Research Ethics Board at both institutions.
Following informed consent, patients underwent two
biopsy procedures. The first involved a standard bone
marrow aspirate and trephine biopsy from either the left or
right posterior iliac crest. Between 1 and 2 cc of marrow
aspirate were collected; the goal for trephine biopsies was
to obtain a sample measuring at least 10 mm. CT-guided
biopsies in this study were performed by specialist inter-
ventional radiologists (GD, AS, and DG). In cases where
patients had multiple metastases to bone, the site of biopsy
was left to the radiologist’s discretion. To maximize yield,
periosseous soft tissue and lytic metastases were prefer-
entially sampled rather than blastic metastases. Sites of
disease which had received prior radiation were avoided if
possible. Specimens were obtained with a 13-gauge needle
yielding two samples with a maximal length of 20 mm. All
biopsies were stored in 10% formalin prior to pathologic
evaluation. Core biopsy specimens from bone metastases
were not decalcified, in order to ensure that interpretation
of ER and PgR was not compromised. Samples were
analyzed by a pathologist (SD or MN) to confirm meta-
static disease and to verify adequate cellularity for the
purposes of ER and PgR analysis. While ER and PgR
testing is technically feasible on any number of tumor cells,
to achieve confidence, especially for negative results,
adequate cellularity was defined as the presence of a few
hundred tumor cells. Immunostaining for ER and PgR
proteins were conducted using the Ventana Ultraview
Detection System at both study locations. Receptor anal-
ysis for ER and PgR was carried out using the Ventana
6F11/Vector clone 6F11 and Ventana clone 16/DAKO
clone PGR636 for each receptor, respectively. The pres-
ence of positive and negative external laboratory controls
was checked. In line with guidelines available at the time
of the studies [18], results were dichotomised with a
positive result defined as 10% or more of tumor cell nuclei
staining positively with any intensity. Biopsies were not re-
analyzed using a lower cut-off as due to low cellularity in
many samples leading to poor accuracy (i.e. confidence
intervals for receptor expression \10% were very wide).
Results
In total, 40 patients consented to participate in the three
studies: 25 in Toronto and 15 in Ottawa. Patient demo-
graphics and tumor characteristics are presented in Table 2.
With regards to the primary tumor, 39/40 patients (97.5%)
and 38/40 patients (95%) had their ER and PR status
Table 2 Baseline characteristics of participants
Characteristic Mean (range)
Age 55.3 years (34–76)
Treatment history
Number of lines of hormone treatment 1.79 (0–6)
Number of lines of chemotherapy 0.88 (0–3)
Previous radiotherapy to CT biopsy site 35.3%
Bisphosphonate exposure 79.4%
Average duration of bisphosphonate use 2 years (0.5–4.0 years)
Hormone receptor status of primary
ER (?) PR (?) 65%
ER (?) PR (–) 22.5%
ER (-) PR (?) 0%
ER (-) PR (-) 2.5%
ER unknown 2.5%
PR unknown 7.5%
available respectively. One patient included in this analysis
was diagnosed initially with DCIS and was found subse-
quently to have distant ER-/PR- metastatic bone disease.
Acquisition of tumor cells
Of all 40 patients consented to the study, 39 underwent
bone marrow aspiration/trephine biopsy; one participant
withdrew consent prior to the procedure being performed.
19 of the samples (48.8%) contained sufficient tumor to
allow for hormone receptor analysis. In total, 34/40
patients underwent CT-guided biopsy of their metastatic
bone disease. In the six cases where the biopsy was not
performed, five patients withdrew consent, and one was
unable to assume a position to allow for a biopsy to be
safely performed. 16 of the samples (47%) contained suf-
ficient tumor to allow for hormone receptor analysis.
Despite the low success rate of each individual technique,
when utilized in combination, the chance of obtaining
sufficient tumor tissue from metastatic disease bone
improved to 26/39 patients (67%). Table 3 shows the
proportion of biopsies with tumor cells present and the
analyzable yield for ER and PgR. Details of adequate
cellularity of biopsies for immunohistochemistry and for
molecular analysis have been reported previously [16].
Concordance of ER/PgR between the primary
and bone metastases
For the 26 patients where sufficient tumor tissue was
acquired to permit hormone receptor analysis, discordance
between the primary and metastatic samples was seen in 14
patients (53.8%). In terms of ER, the receptor changed
123

Table 3 Acquisition of tumor cells per technique
Technique Samples with tumor Samples with adequate
cells present number of tumor cells
(40 samples) for receptor analysis
(40 samples)
Bone marrow 24 (60%) 16 (40%)
trephine/aspirate
CT-guided biopsy 21 (52.5%) 19 (47.5%)
from positive to negative 10 cases (38.5%) and negative to
positive in one case (2.5%). For PgR, the receptor changed
from positive to negative in 11 cases (42.3%) and from
negative to positive in one case (2.5%). It should be noted
that changes from ER-negative and PgR-negative to
receptor positive occurred in the same patient. These dis-
cordance rates are consistent with those reported previ-
ously (Table 1).
Concordance of ER/PgR between bone metastases
in the same patient
Nine patients (22.5%) had adequate numbers of malignant
cells identified in both bone metastasis and bone marrow
samples to allow for receptor analysis. Among these, ER
and PgR were concordant in 100 and 78% of cases,
respectively.
Discussion
In recent years, there has been an increasing drive to obtain
metastatic tissue from patients with breast cancer. This
tissue can be used in a number of ways including histo-
logical confirmation of metastatic disease, obtaining tissue
for receptor analysis and increasingly as part of clinical
trial protocols. In addition, given the expanding role of
novel bone targeted agents and the potential adjuvant role
of adjuvant bisphosphonate therapy, there is increasing
interest in bone research on human specimens. Central to
such research is the acquisition of tissue from this site.
The first goal of this study was, therefore, to assess the
feasibility with regards to the acquisition of tissue. We
compared two methods: standard bone marrow trephine/
aspiration from the posterior iliac crest performed at the
bedside versus a CT-guided biopsy. Logically, it would
appear that CT-guided biopsy should have a major sam-
pling advantage as it allows for specific sampling of
known, radiologically evident lesions; in contrast, bone
marrow aspiration/trephine biopsy is an unguided proce-
dure and is only likely to be successful if by chance met-
astatic disease is present in either the marrow or bone at the
site of biopsy. The major advantage to bone marrow
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Breast Cancer Res Treat
aspiration/trephine biopsy is one of speed and it is signif-
icantly less resource intensive. Bone marrow aspiration/
trephine biopsy may have the additional advantage that it
can be repeated with changing clinical situations.
Despite the differences in resource cost, there does not
appear to be a large difference in terms of success rate
between the two techniques; in fact, tumor cells were
numerically more likely to be observed with bone marrow
biopsies (48.8%) than with CT-guided biopsy samples
(47%).
As a means of quality control measure to ensure that our
population was representative of that from other studies,
the primary and metastatic sites were compared for hor-
mone receptor discordance. In our population, we found a
significant rate of discordance between the primary and
metastatic sites in bone in breast cancer in terms of ER and
PgR. We have detected a general discordance rate of 53.8%
with specific discordance rates of 42.3% for ER and 46.2%
for PR. These results are similar to what was found in other
metastatic biopsy studies and suggests that our findings can
be generalized for future work.
Finally in order to ensure that there were no significant
discrepancies in the data obtained by each technique, sig-
nificant hormone receptor discordance between bone
metastases in the same patient may indicate that the sam-
ples may be affected by the method of acquisition.
Unfortunately, due to the low yield of each technique,
respectively, it was difficult to obtain a large number of
patients who had tissue available for analysis by both
techniques. Nevertheless, there does not appear to be
substantial differences. In the nine patients where this
analysis was possible, ER and PgR were concordant in 100
and 78% of the metastases, respectively. This suggests that
data obtained by bone marrow aspiration/trephine biopsy is
reproducible when compared to guided techniques.
This study clearly has a number of limitations. The suc-
cess rate of the CT-guided biopsy appears to be relatively
low. However, similar yield was observed in the two par-
ticipating centres. In addition, they were also performed in
dedicated interventional radiology departments. Further-
more, bone marrow trephine/aspiration was only performed
unilaterally in the iliac crest. By performing this procedure
bilaterally, or by combining it with cell sorting or other
enhancement techniques, it may be possible to increase the
yield of tumor cells. The discordance of receptor results may
be due, at least in part, to the very small number of cells being
tested in the bone biopsies which limits the reliability of
results. Finally, for those biopsies where there was an
absence of tumor cells, it is unclear whether these were true
positives (absence of metastatic disease) or false positives
(target miss). Nonetheless, the aim of the study was to assess
analyzable yield for receptor analysis and therefore, this
limitation would have little effect on the overall conclusions.
Breast Cancer Res Treat
In conclusion, this multi-centre pooled analysis suggests
that bone marrow aspiration/trephine biopsy and CT-gui-
ded biopsy are equivalent in terms of obtaining metastatic
tissue from bone. In general, acquisition of metastatic bone
disease is difficult and technically challenging. Given that
CT-guided biopsy demands higher resource allocation, we
would recommend that bone marrow aspiration/trephine
biopsy first be utilized when studying metastatic bone
disease. In cases where tissue must absolutely be acquired,
we would recommend that both techniques be utilized
sequentially.
Acknowledgments Funding for this study was provided by The
Ottawa Cancer Research Foundation and the ‘‘Tina and her Angels of
Hope Fund’’.
Conflict of interest None.
References
1. Amir E, Clemons M (2009) Should a biopsy be recommended to
confirm metastatic disease in women with breast cancer? Lancet
Oncol 10:933–935
2. Amir E, Clemons M, Freedman OC, Miller NR, Coleman RE,
Purdie C, Jordan L, Quinlan P, Thompson AM (2010)Tissue
confirmation of disease recurrence in patients with breast cancer:
pooled analysis of two large prospective studies. J Clin Oncol
228:s1007
3. Locatelli MA, Curigliano G, Fumagalli L, Bagnardi V, Aurilio G,
Della Vigna P, Monfardini L, Giudici S, Viale G, Goldhirsch A
(2010) Should liver metastases of breast cancer be biopsied to
improve treatment choice? J Clin Oncol 28:s1008
4. Karlsson E, Lindström LS, Wilking U, Skoog L, Johansson U,
Bergh J (2010) Discordance in hormone receptor status in breast
cancer during tumor progression. J Clin Oncol 28:s1009
5. Simmons C, Miller N, Geddie W, Gianfelice D, Oldfield M,
Dranitsaris G, Clemons MJ (2009) Does confirmatory tumor
biopsy alter the management of breast cancer patients with distant
metastases? Ann Oncol 20:1499–1504
6. Broom RJ, Tang PA, Simmons C, Bordeleau L, Mulligan AM,
O’Malley FP, Miller N, Andrulis IL, Brenner DM, Clemons MJ
(2009) Changes in estrogen receptor, progesterone receptor and
Her-2/neu status with time: discordance rates between primary
and metastatic breast cancer. Anticancer Res 29:1557–1562
7. Lower EE, Glass EL, Bradley DA, Blau R, Heffelfinger S (2005)
Impact of metastatic ER and PR status on survival. Breast Cancer
Res Treat 90:65–70
8. Lipton A, Leitzel K, Ali SM, Demers L, Harvey HA, Chaudri-
Ross HA, Evans D, Lang R, Hackl W, Hamer P, Carney W
(2005) Serum HER-2/neu conversion to positive at the time of
disease progression in patients with breast carcinoma on hormone
therapy. Cancer 104:257–263
9. Regitnig P, Schippinger W, Lindbauer M, Samonigg H, Lax SF
(2004) Change of HER-2/neu status in a subset of distant
metastases from breast carcinomas. J Pathol 203:918–926
10. Li BD, Byskosh A, Molteni A, Duda RB (1994) Estrogen and
progesterone receptor concordance between primary and recur-
rent breast cancer. J Surg Oncol 57:71–77
11. Mobbs BG, Fish EB, Pritchard KI, Oldfield G, Hanna WH (1987)
Estrogen and progesterone receptor content of primary and sec-
ondary breast cancer: influence of time and treatment. Eur J
Cancer Clin Oncol 23:819–826
12. Raemaekers JM, Beex LV, Koenders AJ, Pieters GF, Smals AG,
Benraad TJ, Kloppenborg PW (1984) Concordance and discor-
dance of estrogen and progesterone receptor content in sequential
biopsies of patients with advanced breast cancer: relation to
survival. Eur J Cancer Clin Oncol 20:1011–1018
13. Holdaway IM, Bowditch JV (1983) Variation in receptor status
between primary and metastatic breast cancer. Cancer
52:479–485
14. Hull DF, Clark GM, Osborne CK, Chamness GC, Knight WA III,
McGuire WL (1983) Multiple estrogen receptor assays in human
breast cancer. Cancer Res 43:413–416
15. Coleman RE (1997) Skeletal complications of malignancy.
Cancer 80:1588–1594
16. Cawthorn TR, Amir E, Broom R, Freedman O, Gianfelice D,
Barth D, Wang D, Holen I, Done SJ, Clemons M (2009) Mech-
anisms and pathways of bone metastasis: challenges and pitfalls
of performing molecular research on patient samples. Clin Exp
Metastasis 26:935–943
17. Amir E, Miller N, Geddie W et al (2010) Can bone biopsies be
used to confirm metastatic disease recurrence and receptor status
in breast cancer patients? In: 10th International Conference.
Cancer-induced bone disease, 22–25 September, Sheffield, UK,
Abstract P16
18. Diaz LK, Sneige N (2005) Estrogen receptor analysis for breast
cancer: current issues and keys to increasing testing accuracy.
Adv Anat Pathol 12:10–19
123