Eur J Nucl Med Mol Imaging (2015) 42:818830

DOI 10.1007/s00259-015-2995-8

ORIGINAL ARTICLE

18

F-FLT PET/CT as an imaging tool for early prediction

of pathological response in patients with locally advanced breast
cancer treated with neoadjuvant chemotherapy: a pilot study

Flavio Crippa & Roberto Agresti & Marco Sandri & Gabriella Mariani &
Barbara Padovano & Alessandra Alessi & Giulia Bianchi & Emilio Bombardieri &
Ilaria Maugeri & Mario Rampa & Maria Luisa Carcangiu & Giovanna Trecate &
Claudio Pascali & Anna Bogni & Gabriele Martelli & Filippo de Braud

Received: 4 September 2014 / Accepted: 14 January 2015 / Published online: 12 February 2015
# Springer-Verlag Berlin Heidelberg 2015

M. L. Carcangiu
Pathology Unit, Fondazione IRCCS Istituto Nazionale dei
Tumori Milan (Italy), 20133 Milan, Italy

at the end of NCT. All patients subsequently underwent surgery. Variables from FLT PET examinations were correlated
with postoperative histopathological results.
Results At baseline, median of maximum standardized uptake values (SUVmax) in the groups showing a complete
pathological response (pCR) + residual cancer burden
(RCB) I, RCB II or RCB III did not differ significantly
for the primary tumour (5.0 vs. 2.9 vs. 8.9, p=0.293) or
for axillary nodes (7.9 vs. 1.6 vs. 7.0, p=0.363), whereas
the Spearman correlation between SUVmax and Ki67 proliferation rate index was significant (r=0.69, p< 0.001).
Analysis of the relative percentage change of SUVmaxin
the primary tumour (SUVTmax(t1)) and axillary nodes
(SUVNmax(t1)) after the first NCT cycle showed that
the power of SUVTmax(t1) to predict pCR + RCB I responses (AUC =0.91, p<0.001) was statistically significant, whereas SUVN max (t 1 ) had a moderate ability
(AUC = 0.77, p = 0.119) to separate subjects with
SUVTmax(t1) > 52.9 % into two groups: RCB III patients and a heterogeneous group that included RCB I
and RCB II patients. A predictive score based on
SUVT m a x (t 1 ) and SUVN m a x (t 1 ) parameters is
proposed.
Conclusion The preliminary findings of the present
study suggest the potential utility of FLT PET scans
for early monitoring of response to NCT and to formulate a therapeutic strategy consistent with the estimated
efficacy of NCT. However, these results in a small patient population need to be validated in a larger independent cohort.

G. Trecate
Radiology-RMI Unit, Fondazione IRCCS Istituto Nazionale dei
Tumori Milan (Italy), 20133 Milan, Italy

Keywords Breast cancer . Neoadjuvant chemotherapy . FLT

PET . Pathological response predictive rule

Abstract
Pur pos e We ev alu ate d w h eth er 1 8 F-3 -d eo xy- 3 fluorothymidine positron emission tomography (FLT PET)
can predict the final postoperative histopathological response
in primary breast cancer after the first cycle of neoadjuvant
chemotherapy (NCT).
Methods In this prospective cohort study of 15 patients with
locally advanced operable breast cancer, FLT PET evaluations
were performed before NCT, after the first cycle of NCT, and

Flavio Crippa and Roberto Agresti contributed equally to this work.

F. Crippa : B. Padovano : A. Alessi : E. Bombardieri : C. Pascali :
A. Bogni
Nuclear Medicine Unit, Fondazione IRCCS Istituto Nazionale dei
Tumori Milan (Italy), 20133 Milan, Italy
R. Agresti (*) : I. Maugeri : M. Rampa : G. Martelli
Breast Surgery Unit, Fondazione IRCCS Istituto Nazionale dei
Tumori Milan (Italy), Via Venezian 1, 20133 Milan, Italy
e-mail: roberto.agresti@istitutotumori.mi.it
M. Sandri
Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei
Tumori Milan (Italy), 20133 Milan, Italy
G. Mariani : G. Bianchi : F. de Braud
Medical Oncology Unit, Fondazione IRCCS Istituto Nazionale dei
Tumori Milan (Italy), 20133 Milan, Italy

Eur J Nucl Med Mol Imaging (2015) 42:818830

Introduction
Neoadjuvant chemotherapy (NCT) followed by surgery is a
standard therapeutic strategy for operable, locally advanced
breast cancer. Several clinical trials have demonstrated good
results, with an objective response rate of about 70 % and a
complete pathological response rate of up to 30 % [14].
These findings have led to improved surgical planning, an
increased rate of breast-conserving surgery even in patients
with an unfavourable tumour/breast size ratio before therapy,
and have enabled further evaluation of the biology of the
response of the primary tumour to chemotherapy and of the
efficacy of chemotherapy [5, 6]. It is well known that the type
of pathological response after NCT is a fundamental prognostic factor [512]. However, to date, the efficacy of NCT is
evaluated only upon final histology of the postsurgical specimen. Clinical examination and instrumental diagnostic tools,
such as mammography or ultrasound examination, based on
size and morphological criteria fail to predict early a complete
pathological response (pCR) to NCT and later to efficiently
distinguish responders from nonresponders [13]. Thus, there
is a need for a reliable method to assess the early response to
NCT in order to avoid ineffective and expensive therapy and
to provide alternative management options for consideration.
The use of MRI to search for new surrogate markers of
disease response has yielded interesting results in several studies that have evaluated the functional response to NCT in
breast cancer [14, 15]. Some of these studies investigated the
utility of MRI in monitoring response to treatment by using
combined parameters such as diffusion-weighted MRI (DWMRI), dynamic contrast enhancement (DCE-MRI), volumetrics, and spectroscopy [1619].
PET using different tracers is playing an increasing role in
predicting the response of breast cancer to NCT [20]. At present, 18F-fluorodeoxyglucose (FDG) PET is the most commonly used method for monitoring response of breast cancer to
treatment, according to published results [2129].
In the present study, we used 18F-fluorothymidine (FLT), a
tracer of proliferation [3032] tested in other pilot studies of
breast cancer [3236], to assess the value of FLT PET in the
early prediction of response of locally advanced operable
breast cancer to NCT and to identify its potential predictive
value based on relative changes in standardized uptake values
(SUV) of FLT PET in primary tumours and axillary nodes
after the first cycle of NCT.

Patients and methods

Patient population and treatment
This prospective cohort pilot study comprised patients with
potentially operable, locally advanced T2-3 breast cancer

819

treated with NCT followed by surgery at our institute.

Pretreatment histological diagnosis of invasive breast carcinoma was done by core needle biopsy in all patients and further
characterized for hormone receptor status, HER2 status and
proliferative index. Initial routine staging procedures
consisted of clinical examination and mammographic and ultrasound evaluation of tumour size and axillary nodal status.
Chest plain radiography, whole-body bone scan and ultrasound examination of the liver and abdomen were used to
assess distant metastases.
NCT consisted of an anthracycline/taxane-based regimen
for six cycles, with trastuzumab administered to those with
HER2-positive breast cancer. Specifically, the NCT regimen
included doxorubicin (60 mg/m2) + paclitaxel (200 mg/m2)
every 3 weeks for three cycles followed by cyclophosphamide/methotrexate/fluorouracil (600/40/600 mg//m2) on days
1 and 8 every 4 weeks for three cycles, and trastuzumab in
patients with HER2-positive breast cancer (8 mg/kg loading
dose decreased to 6 mg/kg) every 4 weeks for three cycles
concomitant with cyclophosphamide/methotrexate/
fluorouracil.
Patients underwent breast-conserving surgery or total mastectomy according to the clinical and instrumental response to
NCT, site of tumour inside the breast, cosmetic evaluation,
presence of extensive intraductal component and/or
multifocality. Axillary surgery (complete dissection or sentinel node biopsy) was performed based on clinical nodal status
before and after NCT. Nuclear medicine physicians, surgeons
and pathologists performed their work blinded to other results.
The institutional review board or equivalent approved this
study, and all subjects signed written informed consent.
PET evaluation
18

F-3-Deoxy-3-fluorothymidine (FLT) was synthesized and

prepared by the Radiochemistry and Cyclotron Facility of our
Institute as previously described [37]. To minimize potential
pitfalls due to FLT metabolism, patients fasted for at least 6 h
before receiving approximately 3.5 MBq/kg of FLT administered intravenously as a bolus, followed by 10 ml of normal
saline (0.9 % NaCl). Images were obtained 80 min after injection of FLT, in accordance with the work of Smyczek-Gargya
et al. [38] and with our previous unpublished studies on different tumours. A hybrid PET/CT system (64 TOF Gemini;
Philips Medical Systems) was used in the present study.
During the waiting period, patients were asked to drink 0.5 l
of water to reduce bladder activity and radiation exposure to
the bladder and to use the bathroom 30 min after FLT administration and immediately before the start of the PET study.
The imaging protocol included a CT scout scan to define the
axial imaging range (from upper thigh to skull base), a lowdose CT scan without contrast enhancement, and lastly, a
three-dimensional PET scan (3 min per bed position). CT

820

images, obtained with the patient breathing shallowly,

were used for attenuation correction of the PET data
and anatomical positioning of the FLT findings. Each
patient was studied before therapy (time-point t0), after
one cycle of chemotherapy (time-point t 1) and after
completion of therapy (time-point t2), about 1 month
before surgery. Figure 1 shows the flow chart of the
study with the three FLT PET evaluations.
Using dedicated PET workstations (Philip Extended
Brilliance Workspace), nuclear medicine physicians
blinded to each patients history and clinical and conventional imaging findings visually analysed the PET/
CT images, looking especially for areas of focally increased FLT uptake in the breast tumour and ipsilateral
axillary regions. FLT uptake was evaluated semiquantitatively using the SUVmax defined as the highest SUV
in a region of interest and calculated as: SUV=(tissue
activity in megabecquerels per gram)/(injected activity
in megabecquerels)/(body weight in grams). SUVmax
was measured in the primary breast tumour (SUVTmax)
and, when detectable, in the dominant axillary lymph
node (SUVNmax) lesions, the latter defined as the largest
lymph node and the highest FLT uptake among the detectable nodes in the fused PET/CT images. During the
course of NCT, changes in SUVmax in target lesions
were monitored by comparing FLT uptake at time points
t0, t1 and t2, and the results are expressed as absolute
value and relative percentage change in SUVmax:

Eur J Nucl Med Mol Imaging (2015) 42:818830

measurements of the primary tumour (size and cellularity)

and nodal metastases (number and size):

0:17  

0:17
4 10:75LN d met
RCB index 1:4 f inv d prim

To assess pathological response, we opted for the web-based

MD Anderson Residual Cancer Burden (RCB) calculator
[39]. This method, proposed by Symmans et al. [8], allows
calculation of an index that combines pathology

where finv is the proportion of the primary tumour bed that

contains invasive carcinoma, LN is the number of axillary
lymph nodes containing metastatic carcinoma, dmet is the diameter ofpthe
largest metastasis in an axillary lymph node, and

d prim d 1 d 2 where d1 and d2 are the bidimensional diameters of the primary tumour bed in the resection specimen [8].
Using two cut-off points, the authors proposed four RCB
categories (RCB 0, RCB I, RCB II and RCB III) corresponding to pCR, minimal residual disease (near-complete response), moderate residual disease, and extensive residual disease, respectively (Table 1).
In addition, based on our previous experience, we chose a
cut-off level of 3 for the RCB index, since this value,
contained in the RCB II class (moderate response), may represent the threshold separating patients with RCB II into those
who show a partial response and are closer to RCB I patients
and those with a partly nonresponding large tumour in progression. Hence, this cut-off allowed the study population to
be split into two groups: complete/partial responders and
complete/partial nonresponders.
All pathological assessments and immunohistochemistry
were performed at the Pathology Unit of our institute.
Surgical specimens were fixed in neutral formalin and embedded in paraffin, and sections were stained with haematoxylin
and eosin at 4 C. Tumour grade was assessed according to the
procedure of Elston and Ellis [40]. Immunohistochemistry for
oestrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) was performed on 4-m-thick sections of breast cancer resection
specimens, whereas immunostaining for Ki67 was performed
on both breast cancer resection specimens and metastatic
axillary lymph nodes. Staining for ER (clone SP1,
Ventana), PgR (clone SP2, Ventana), HER2 (p185,
Dako, diluted 1:1,000), Ki67 (MIB1, Dako, diluted

Fig. 1 Flow chart of the study. The neoadjuvant chemotherapy (NCT)

regimen comprised doxorubicin + paclitaxel for three cycles (grey
squares) followed by cyclophosphamide/methotrexate/fluorouracil for

three cycles (white squares). SUVmax(t1) and SUVmax(t2) are

relative percentage changes in SUVmax measured at t1 and t2 for the
primary breast tumour and for the dominant axillary lymph node

.
SUVmax ti 100  SUVmax ti SUVmax t0  SUVmax t 0

where i is 1 or 2.
Pathological examination and evaluation criteria

Eur J Nucl Med Mol Imaging (2015) 42:818830

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Table 1 RCB categories for classification of residual disease in the

breast and axillary nodes after neoadjuvant chemotherapy

Results

RCB class

RCB index

Amount of disease

0
I
II
III

0
0 1.36
1.36 3.28
> 3.28

Pathological complete response

Minimal residual disease
Moderate residual disease
Extensive residual disease

From October 2011 to January 2013, 15 T2-3 N0-1 patients

were accrued to the present study. The median age was
42 years (range 29 63 years). All patients underwent the
three scheduled scans. Figure 2 shows an example of the imaging of the breast tumour and axillary involved nodes on the
baseline FLT PET scan (time t0; Fig. 2a), and the progressive
response to NCT on the interim (time t1) and final (time t2)
scans compared with baseline (Fig. 2b). All patients tolerated
the PET scanning protocol well and all breast lesions were
visualized by FLT PET.
All but one patient, who exhibited progressive disease after the first three cycles of the doxorubicin/
paclitaxel regimen, completed the scheduled NCT.
Consistent with protocol indications, five patients with
HER2 amplification received trastuzumab. All patients
underwent breast surgery and axillary surgery. Eight patients underwent total mastectomy (in two due to
microcalcifications with multicentric foci of intraductal
disease), two underwent nipple-sparing mastectomy
(based on breast size and the site of the tumour in order
to achieve the best cosmetic result), and five underwent
quadrantectomy). Ten patients underwent complete axillary dissection, two sentinel node biopsy followed by
axillary dissection, and three sentinel node biopsy only.
Pathological evaluation of surgical specimens according
to previously described classifications [39] identified six
high responders (RCB 0 or RCB I), five weak responders (RCB II) and four nonresponders (RCB III).
Table 2 summarizes the clinical, radiological and pathological characteristics of the entire patient cohort and
of the three RCB groups at t0. Furthermore, breast cancer subtype in all patients was also classified according
to the consensus of the St Gallen International Expert
Panel members [41]. The baseline characteristics of primary tumours did not differ significantly among the
RCB groups, except for the Ki67 proliferation index.
The mean postoperative pathological tumour size was
15.5 mm (range 0 40 mm) in the entire patient cohort, whereas, consistent with the RCB classification,
tumour size differed greatly between the RCB 0 +
RCB I group (3.5 mm) and the other two RCB groups
(RCB II 23.6 mm, RCB III 23.3 mm), as did the rates
of postoperative histologically negative axillary nodes,
where rates were higher in the RCB 0 + RCB I group
(66.7 %) than in the RBC II group (40.0 %) and the
RCB III group (0.0 %).
Table 3 summarizes the median FLT PET SUVmax at t0, t1
and t2 and the relative percentage changes at t1 and t2 in the
primary tumours and in the axillary nodes in the three RCB
groups. The median time (with range) of the interval between
first chemotherapy and PET at baseline was 15 (1 29) days;

1:100) was detected using an OPTIview DAB detection

system on a Ventana Ultra benchmark Autostainer.
Positive staining for ER and PgR was defined as nuclear staining in 1 % of the tumour cells, whereas HER2
was assessed based on the intensity of tumour cell
membrane staining, scored as 0 (negative), 1+ (weak),
2+ (moderate) and 3+ (strong) staining in at least 30 %
of the tumour cells. The Ki67 proliferation index of
each case was evaluated based on the percentage of
Ki67-positive cells among at least 200 tumour cells.
Our pathological reports included an explicit statement
concerning assessment for the presence and degree of
response to chemotherapy.

Statistical analysis
The hypothesis that k independent samples come from populations with the same median was tested using the KruskalWallis nonparametric rank test. Differences were considered
significant at p0.05.
A linear score based on relative percentage change in
maximum SUV at t1 in the primary tumour and in the dominant axillary node was proposed for predictive purposes:
 SUVTmax t 1  SUVNmax t 1
where the coefficients , and were estimated using a linear
regression model with SUVTmax(t1) and SUVNmax(t1) as
explanatory variables and the RCB index as outcome variable.
The predictive powers of SUVTmax(t1), SUVNmax(t1)
and of the score were assessed by estimating the area under
the ROC curve (AUC). The 95 % confidence intervals (CI) for
AUC and the P value of the test of the null hypothesis that
AUC=0.5 (no predictivity) were estimated using bootstrap
methods with 1,000 replications. Overall accuracy, sensitivity,
specificity, and positive and negative predictive values (PPV
and NPV, respectively) were also estimated for some cut-off
values of the two parameters.
Statistical analyses were performed using Stata13
(StataCorp, College Station, TX) and R (version 3.1.2; R
Foundation for Statistical Computing, Vienna, Austria)
software.

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Eur J Nucl Med Mol Imaging (2015) 42:818830

Fig. 2 Example baseline, interim

and final imaging of breast
tumours and axillary involved
nodes. a Coronal fused PET/CT
slices show abnormal areas of
FLT uptake corresponding to a
primary breast tumour (T) and a
dominant axillary metastatic node
(N). b, c Transaxial CT, PET and
fused PET/CT images in a NCTresponding patient show FLT uptake changes in the primary breast
tumour (b) and in the dominant
axillary metastatic node (c).
Postsurgery histology revealed a
complete pathological response
both in the mammary gland and in
the removed axillary nodes (0/10
nodes). d Axial fused PET/CT
slices in a nonresponding patient.
In the interim PET image, the abnormal area of FLT uptake corresponding to the primary cancer
(arrow) is larger than in the baseline PET image, in spite of a partial reduction in tracer accumulation (SUVmax =26%). In the
final PET image, there is a further
increase in the area of FLT uptake
with appearance of a photopenic
central area, attributable to tumour necrosis

the median time of PET t1 after the first cycle of chemotherapy

was 18 days (12 21 days); the median time of PET t2 after
the last cycle of chemotherapy (before surgery) was 12 days
(5 27 days).
At baseline, median SUVmax values of the primary
tumours for the RCB 0 + RCB I, RCB II and RCB
III groups were 5.0, 2.6, and 8.4, respectively, and of
the axillary nodes were 7.9, 0.8, and 3.2, respectively
(Table 3). Spearmans correlation at t 0 between
SUVTmax and Ki67 proliferation index was positive,
strong and statistically significant (r=0.69, p<0.001).
Figure 3a shows, for each patient, the relative percentage
changes in SUVmax measured at t1 and t2 for the primary
breast tumours [SUVTmax(t1) and SUVTmax(t2)], and the

median values in the three RCB groups. Similarly, Fig. 3b

shows the relative changes in SUVmax measured at t1 and t2
for the dominant axillary lymph nodes [SUVNmax(t1) and
SUVNmax(t2)], together with their median values in each
RCB group. Figure 4 shows, for each patient, a twodimensional scatter plot of SUVTmax(t1) and
SUVNmax(t1) together with the corresponding RCB category and the RCB index value.
The discriminative ability of the FLT PET parameters
SUVTmax(t1) and SUVNmax(t1) was investigated considering three classification problems (Table 4):
1. RCB 0+I responders vs. RCB II+III patients
2. RCB III nonresponders vs. RCB 0+I+II patients

Eur J Nucl Med Mol Imaging (2015) 42:818830

Table 2

823

Baseline patient characteristics by RCB group

Variables

Total

Patients, n (%)
Age (years), median (range)
Mammographic evaluation (mm), median (range)
Echographic evaluation (mm), median (range)
Ki67 index, median (range)
Tumour size (cT), n (%)
T2
>T2
Oestrogen receptor, n (%)
Negative
Positive
HER2, n (%)
Negative
Positive
Grade, n (%)
2
3

15 (100.0)
42.0 (29.0
40.0 (30.0
40.0 (30.0
50.0 (12.0

Nodal involvement (cN), n (%)

Negative
Positive
Breast cancer subtype, n (%)a
Basal-like
Luminal-like
a

63.0)
55.0)
70.0)
90.0)

pCR + RCB I

RCB II

6 (40.0)
40.5 (35.0
52.5 (30.0
47.5 (30.0
65.0 (15.0

4 (26.7)
47.5 (42.0
40.0 (35.0
37.5 (30.0
32.5 (12.0

59.0)
55.0)
70.0)
90.0)

RCB III

63.0)
40.0z)
45.0)
70.0)

5 (33.3)
39.0 (29.0
40.0 (30.0
30.0 (14.0
30.0 (14.0

10 (66.7)
5 (33.3)

3 (50.0)
3 (50.0)

3 (75.0)
1 (25.0)

4 (80.0)
1 (20.0)

9 (60.0)
6 (40.0)

5 (83.3)
1 (16.7)

2 (50.0)
2 (50.0)

2 (40.0)
3 (60.0)

10 (66.7)
5 (33.3)

2 (33.3)
4 (66.7)

4 (100.0)
0 (0.0)

4 (80.0)
1 (20.0)

6 (40.0)
9 (60.0)

1 (16.7)
5 (83.3)

2 (50.0)
2 (50.0)

3 (60.0)
2 (40.0)

6 (40.0)
9 (60.0)

4 (66.7)
2 (33.3)

2 (50.0)
2 (50.0)

0 (0.0)
5 (100.0)

9 (60.0)
6 (40.0)

5 (83.3)
1 (16.7)

2 (50.0)
2 (50.0)

2 (40.0)
3 (60.0)

59.0)
50.0)
90.0)
90.0)

Classified according to the consensus of the St Gallen International Expert Panel members [29]

3. Patients with an RCB index below the threshold level 3

(RCB 0+I+partly-responding RCB II) vs. patients with
RCB index >3

The results showed that the two parameters play a

different role in the identification of the RCB response
(Fig. 5). On the one hand, the predictive power of
SUVT max (t 1) was good and statistically significant
for identifying RCB 0 + RCB I responses (AUC 0.91,
95 % CI 0.72 1.00, p<0.001; Fig. 5a); indeed, when
the cut-off level for identifying RCB 0 + RCB I subjects was set at SUVTmax(t1) 52.9% (i.e. a reduction in SUVTmax greater than or equal to 52.9 %), the
overall accuracy was 93.3 %, the sensitivity was
83.3 %, the specificity was 100.0 %, and the PPV and
NPV were 100.0 and 90.0 %, respectively. However, the
ability of SUVTmax(t1) to identify RCB III patients
and patients with RCB values below/above 3 (AUC
0.66 and 0.63, respectively) was poor (Table 4, Fig. 5b).
On the other hand, in the subset of subjects with
SUVTmax(t1)>52.9% (i.e. a reduction in SUVTmax less

than 52.9 %), SUVNmax(t1) showed excellent accuracy in

identifying subjects with a RCB index above 3 (AUC 1.00,
Table 4). In addition, SUVNmax(t1) had an interesting (although not statistically significant) ability to separate subjects
with SUVTmax(t1)>52.9% into two groups: the group of
RCB III patients and a heterogeneous group that included
RCB I and RCB II patients with delayed response to chemotherapy, clinically node-negative patients, and those with a
low-proliferating luminal A tumour (AUC 0.77, p=0.119).
The cut-off value SUVNmax(t1)18.0% (i.e. a reduction
in SUVNmax greater than or equal to 18.0 %) can be used in
the subgroup with SUVTmax(t1)>52.9% for the identification of RCB III patients.
Lastly, a predictive score based on SUVTmax(t1) and
SUVNmax(t1) parameters was calculated:
= 0.043 SUVT max (t 1 ) 0.013 SUVN max (t 1 ) +
3.060
This score showed good predictive power for all three discrimination problems considered, with AUC 0.94 (p<0.001,
Fig. 5c) for RCB 0 + RCB I patients, AUC 0.82 (p=0.026,
Fig. 5d) for RCB III patients, and AUC 0.85 (p=0.003) for
subjects with RCB values below/above 3 (Table 4).

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Eur J Nucl Med Mol Imaging (2015) 42:818830

Table 3 SUVmax (median and range) in primary tumours (SUVTmax)

and axillary nodes (SUVNmax) after FLT PET scans at t0, t1 and t2 by RCB
group, and relative percentage changes in SUVmax (median and range) at
Predictor
SUVTmax
t0
t1
t2
SUVTmax
t1
t2
SUVNmax
t0
t1
t2
SUVNmax
t1
t2
a

t 1 and t 2 in primary tumours (SUVT max ) and axillary nodes

(SUVNmax) by RCB group

Total

RCB 0 + RCB I

RCB II

RCB III

p valuea

4.0 (1.5 12.1)

2.6 (1.4 10.5)
0.9 (0.0 9.9)

5.0 (3.4 10.9)

2.2 (1.5 4.6)
0.0 (0.0 0.0)

2.6 (1.5 4.4)

2.2 (1.4 4.0)
1.3 (0.0 3.4)

8.4 (2.9 12.1)

6.2 (1.6 10.5)
5.8 (0.9 9.9)

0.105
0.305
0.005

26.2 (80.8 12.9)

69.0 (100.0 18.2)

55.8 (80.8 11.8)

100.0 (100.0 100.0)

7.9 (18.8 5.3)

45.0 (100.0 22.7)

26.2 (44.8 12.9)

26.2 (69.0 18.2)

0.019
0.007

2.8 (0.0 25.6)

2.1 (0.0 9.8)
1.0 (0.0 7.4)

7.9 (1.4 11.2)

2.7 (1.1 5.7)
0.7 (0.0 2.3)

0.8 (0.0 2.8)

0.0 (0.0 3.5)
0.0 (0.0 2.6)

3.2 (1.0 25.6)

1.7 (0.0 9.8)
2.2 (0.0 7.4)

0.128
0.201
0.285

46.9 (100.0 25.0)

69.4 (100.0 37.5)

55.5 (69.5 0.0)

92.3 (100.0 0.0)

1.0 (100.0 25.0)

3.1 (100.0 1.0)

46.9 (100.0 18.8)

69.4 (100.0 37.5)

0.321
0.343

Kruskal-Wallis test

Discussion
PET may be a major noninvasive imaging modality for evaluating cancer cell proliferation, one of the most important
biological features of malignancies [42]. In this regard, the
most promising PET tracer is currently FLT, a fluorinemodified thymidine analogue. FLT is phosphorylated by thymidine kinase-1 and is not incorporated during DNA synthesis, instead becoming trapped within proliferating cells using
the salvage pathway for DNA synthesis [30, 31]. It is widely
accepted that FLT is a marker of cells in the S-phase of the cell
cycle, suggesting its ability to reflect tumour aggressiveness
Fig. 3 Relative percentage
changes in SUVmax at t1 and t2 for
the primary breast tumour
[SUVTmax(t1) and
SUVTmax(t2)] and the dominant
axillary lymph node
[SUVNmax(t1) and
SUVNmax(t2)] in each patient,
together with their median values
for each RCB group. a
SUVTmax (grey lines) and
median SUVTmax (black lines).
b SUVNmax (grey lines) and
median SUVNmax (black lines)

and response to therapy [30, 31, 43]. The uptake of FLT has
been linked to cell proliferation rate and has been used to
study proliferation in lymphomas, and breast and lung tumours [4446]. We have also found a significant association
between FLT uptake and Ki67 proliferation index [47] in our
series.
FLT PET has often been compared with FDG PET in the
visualization, diagnosis and staging of several tumour types,
including those of the lung, head and neck, stomach, oesophagus, brain and breast [4853]. In general these clinical studies
demonstrated that tumour uptake of FLT is generally lower
than that of FDG, but it can provide high sensitivity and major

Eur J Nucl Med Mol Imaging (2015) 42:818830

825

Fig. 4 Scatter plot of the relative

percentage changes in SUVmax at
t1 for the primary breast tumour
(SUVTmax) and for the
dominant axillary lymph node
(SUVNmax) in each patient,
with their corresponding RCB
group and RCB index (in
parentheses)

specificity because, unlike FDG, it does not accumulate in

areas of inflammatory alteration potentially associated with
cancer therapy, leading to false-positive findings [32, 54].
For staging FLT is of limited value because of its high physiological uptake in the liver and bone marrow and consequent
possible false-negative results in the detection of distant metastases at those sites [32, 55].
The potential value of PET in monitoring response to NCT
in breast cancer has been evaluated. However, these studies
were mainly done with FDG PET, with only a few involving
FLT PET in heterogeneous series of patients with different
disease stages, chemotherapeutic regimens and scan

acquisition protocols. In one of these studies, Pio et al. evaluated 14 patients with primary or metastatic breast cancer who
were starting a novel chemotherapy or hormonal therapeutic
regimen, and who underwent sequential FLT PET scans.
Although there were strong clinical and therapeutic differences, FLT PET uptake after the first course of chemotherapy
was significantly correlated with overall response in terms of
late changes in CA27.29 tumour marker levels and tumour
size as measured by CT [33]. In another study in 13 patients
with stage IIIV breast cancer, the response to the 5-fluorouracil/epirubicin/cyclophosphamide (FEC) regimen was evaluated by FLT PET performed at baseline and 1 week after the

Table 4 Predictive ability of SUVTmax(t1), SUVNmax(t1) and of the proposed score for three discriminating problems: identification of pCR +
RCB I patients, RCB III patients, and patients with RCB index below/above 3
RCB 0+I vs. RCB II+III

SUVTmax(t1)
SUVNmax(t1)
SUVNmax(t1) in the subset
SUVTmax(t1)> 52.9 %
scoreb

RCB III vs. RCB 0+I+II

RCB index <3 vs. RCB index >3

AUC

p valuea

AUC

p valuea

AUC

p valuea

0.91 (0.72 1.00)

0.61 (0.30 0.92)

<0.001
0.469

0.66 (0.34 0.98)

0.60 (0.27 0.93)
0.77 (0.45 1.00)

0.326
0.550
0.119

0.63 (0.31 0.95)

0.70 (0.42 0.99)
1.00

0.420
0.160

0.94 (0.82 1.00)

<0.001

0.82 (0.54 1.00)

0.026

0.85 (0.62 1.00)

0.003

Testing the null hypothesis absence of predictive power (AUC 0.5)

=0.043SUVTmax(t1)0.013SUVNmax(t1)+3.060

826

Eur J Nucl Med Mol Imaging (2015) 42:818830

Fig. 5 ROC curves of

SUVTmax(t1) (a, b) and score
(c, d; = 0.043SUVTmax(t1)
0.013SUVNmax(t1)+3.060)
for discriminating pCR+RCB I
subjects (a, c) and not RCB III
(i.e. pCR+RCB I and II)
subjects (b, d); the main cut-off
values are plotted on the curves

first cycle, considering uptake at 90 min and the irreversible

trapping of FLT as the reference parameters. FLT PET was
able to discriminate between clinical response and stable disease [34]. In a further study the value of FLT PET was investigated by measuring the early response to docetaxel chemotherapy. This study was performed in 20 patients with stage
IIIV breast cancer who were unresponsive to first-line chemotherapy or progressing on previous therapy, and FLT PET
was performed after the first or the second cycle. Although the
patient population was nonhomogeneous, the PET response
was assessed after two cycles in six patients (compared with
one cycle in the rest), and clinical and instrumental criteria
were used to define response rather than histopathology,
changes in the FLT PET signal were nonetheless predictive
of response to therapy (sensitivity 0.85, specificity 0.80) [35].
More recently, Woolf et al. evaluated 20 patients with locally
advanced breast cancer who underwent FLT PET before and
after the first cycle of a FEC or FEC + docetaxel (FEC-T)
chemotherapy regimen [36]. However, in this series, although
a significant and strong correlation between Ki67 and SUVmax
was found, neither baseline SUVmax nor SUVmax from FLT

PET was predictive of response after the first cycle of treatment. Homogeneous SUVmax reduction in most patients was
the main concern in that study.
At present, there are no definitive indications regarding the
optimal timing of PET after FLT injection [36, 38, 56]. In our
study, we used static PET imaging acquisitions at 80-min intervals and the semiquantitative SUV method to measure tumour FLT uptake. This is a simple and widely adopted procedure for PET imaging in clinical protocols, providing a sum
total measure of the tumour tracer uptake by metabolic trapping [57]. SUV can be less precise than PET analysis based on
dynamic image acquisitions and kinetic modelling, because it
does not account for the possible contribution of labelled metabolites and perfusion. Nevertheless, in several clinical studies FLT SUV was significantly correlated with cell proliferation [5860] and our study aimed to evaluate a method available in a clinical setting.
This pilot study showed that FLT PET might serve in the
construction of a prediction rule able to discriminate early
between two different populations of patients undergoing
NCT based on the probability of response to the therapy. In

Eur J Nucl Med Mol Imaging (2015) 42:818830

particular, a joint analysis of variations in SUVmax in the primary tumour and the axillary lymph nodes appears to identify
early mammary tumours will achieve pCR or near pCR (RCB
I) on final histology. The ability to predict early response after
one or two cycles of NCT would avoid unnecessary toxicities
in patients with an unsatisfactory response, with a potential
impact on clinical outcomes and quality of life.
Our results showed that at t1, FLT PET identified three
different patient populations: a group with a high rate of response both in tumour and axillary nodes; a group with an
initial response at the axillary node level without a similar
final response in the tumour; and a heterogeneous group with
a low rate of response at both levels due to a low-proliferating
luminal A tumour or a clinically node-negative tumour with
delayed response to NCT. Notwithstanding this interesting
ability to identify different populations, SUVTmax(t1) and
SUVNmax(t1) showed substantially different predictive
roles: SUVTmax(t1) seemed able to discriminate between
responders and all other patients, whereas SUVNmax(t1) discriminated between complete nonresponders and moderate/
partial responders within the nonresponder group. In addition,
the two parameters may be used to develop a single score with
a significant ability to identify responders, partial responders
and nonresponders.
One crucial issue is the optimal time for performing the
PET scan to evaluate response to NCT. A recent metaanalysis has shown that the predictive value of FDG PET early
after therapy (after the first or second cycle of NCT) is significantly better than after three or more cycles [61], possibly
because PET detects metabolic or proliferative changes in
the tumour before other clinical or radiological tools are able
to detect tumour size shrinkage. In our study, after the baseline
FLT PET at t0, we chose to perform the subsequent PET scans
3 weeks after the first cycle of NCT (time t1, immediately
before the second cycle) and at the end of therapy (time t2).
Another crucial point is the definition of a method to evaluate the response after NCT. It is well known that the pathological measurement of residual cancer after NCT is an important prognostic factor that can influence patient outcome [20].
Recently, a meta-analysis of almost 12,000 patients accrued in
12 international trials has shown that pCR is a valid surrogate
endpoint of long-term clinical benefit, particularly in aggressive tumour subtypes [62]. Consequently, the definition of an
effective method for measuring the percentage of residual disease [12] and the correct interpretation of the results [63] is
fundamental. In the study NSABP B18, classification was
based on the simple dichotomy between pCR and pINV (histological evidence of less residual invasive carcinoma) [1],
whereas Sataloff et al. [7] graded the response according to
four categories ranging from a total or near-total therapeutic
effect (grade A) to no therapeutic effect (grade D) as assessed
based on microscopic changes such as necrosis, calcifications,
fibrosis and inflammatory infiltration. Ogston et al. [64]

827

proposed a five-step scale of response based on the progressive reduction in tumour cellularity in the breast only. Finally,
Chollet et al. [65] proposed a new classification based on
residual disease in the breast and nodes (RDBN), which however is only applicable to patients who have undergone axillary dissection and for discriminating between responders and
nonresponders [66]. As discussed above, we opted for the
web-based MD Anderson RCB calculator [39], which was
previously defined by Symmans et al. [8] for the evaluation
of response to NCT using routine pathological features such as
the largest two dimensions (measured in millimetres) of the
tumour bed, percentage of cellularity of the tumour and
intraductal disease, number of nodal metastases, and size of
the largest nodal metastasis. In our opinion, this four-group
RCB classification, that gives an accurate calculated value for
each patient, allowed the best evaluation of response for statistical analysis.
Until now, response of breast cancer to NCT has usually
been evaluated in clinical studies with FDG PET. One of the
first studies evaluated 47 patients treated with different chemotherapy regimens, and FDG PET predicted pCR with a
sensitivity of 91 % and a specificity of 86 % [21].
Interestingly, in multivariate analysis SUV appeared to be
the only predictive factor of pCR. A further study confirmed
the significant value of FDG PET in predicting response to
NCT (sensitivity 77 %, specificity 80 %, AUC 0.82) in 50
patients treated with a more homogeneous chemotherapy regimen, considering the changes in SUVmax between baseline
and after the second cycle of chemotherapy [22].
More recently, several studies have assessed the predictive
value of FDG PET in different molecular subtypes of breast
cancer. Groheux et al. studied the response to NCT in terms of
FDG uptake in patients with stage II/III HER2-positive breast
cancer [23] and triple-negative breast cancer [24]. In the first
group of 30 HER2-positive patients, using a homogeneous
chemotherapy regimen, ROC analysis showed that the best
predictive result in terms of pCR after two cycles of chemotherapy was characterized by an AUC of 0.91, with a sensitivity of 86 % and a specificity of 94 %. This highlights the
ability of FDG PET to identify the group of patients without
pCR [23]. Similarly, in a second group of 50 triple-negative
patients, FDG PET was performed before treatment and after
two cycles of chemotherapy, and the SUVmax in the primary
tumour was the most effective parameter for predicting pCR
(AUC 0.84) [24].
Koolen et al. have also found that FDG PET is of value in
predicting pathology outcomes after NCT in triple-negative
breast cancer [25], but this was not confirmed by Humbert
et al. [26]. In the former study, 98 T2-3 breast cancer patients
underwent FDG PET before treatment and after 6 8 weeks of
NCT, and FDG PET showed different predictive power in the
three molecular subtypes: HER2-positive (AUC 0.35), ERpositive/HER2-negative (AUC 0.90) and triple-negative

828

(AUC 0.96). These results in triple-negative patients have also

been confirmed in a larger series [27]. Humbert et al. examined 136 T2-3 breast cancer patients who were treated with
wide variety of chemotherapy regimens, and investigated the
levels of FDG uptake and metabolic changes after the first
cycle of NCT in patients with the same molecular subtypes.
They found that pCR has significant predictive value only in
HER2-positive breast cancer [26].
In a substudy of the NEOALTTO trial in Her2-positive
breast cancer patients, Gebhart et al. found that early metabolic assessment using FDG PET (2 and 6 weeks after the start of
anti-HER2 treatment) is able to identify patients with an increased likelihood of pCR [28]. Increased likelihood of pCR
was associated with higher SUVmax at both time-points used
in the study. However, it is noteworthy that there was marked
heterogeneity in response in terms of FDG uptake according
to hormonal receptor status, with hormonal receptor-positive
tumours showing a significantly lower frequency of pCR.
The value of FDG as a metabolic tracer may be more affected by several different molecular crosslinks and crosstalk,
as well as metabolic differences in molecular breast cancer
subtypes, and especially in glycolytic metabolism [6769].
The metabolic differences among the distinct immunohistochemical breast cancer subtypes has been related to specific
metabolic patterns [70].
MRI, alone or associated with FDG PET, has been also
investigated as a non-invasive technique for monitoring the
response to NCT and for assessment of residual disease. A
meta-analysis of the diagnostic performance of DW-MRI
and DCE-MRI in terms of the pathological response to NAC
in patients with breast cancer has been performed. The estimated sensitivity and specificity of DW-MRI were 93 % and
82 %, respectively. In contrast, the sensitivity and specificity
of CE-MRI were 68 % and 91 % [71]. Based on these results,
the combined use of DW-MRI and CE-MRI as a
multiparametric evaluation has the potential to improve the
diagnostic performance in monitoring NCT. In this regard,
by applying multiparametric DCE-MRI and DW-MRI some
authors have found a sensitivity, specificity and AUC of 92 %,
78 % and 0.88, respectively, in predicting pathological response after the first cycle of chemotherapy [16].
Furthermore, several studies have investigated the usefulness
of MR spectroscopy for predicting response to therapy. The
discriminative value of this technique is still under investigation due to the difficulty in quantifying choline [18, 19], especially following the central changes in morphology and vascularity induced by chemotherapy.
More recent studies have investigated the possible value of
the combination of MRI and FDG PET [17, 72, 73]. Both
techniques can provide functional information with MRI,
when evaluating angiogenesis, mainly giving information
about volume, perfusion and the permeability index, and
FDG PET reflecting the metabolic changes of breast cancer.

Eur J Nucl Med Mol Imaging (2015) 42:818830

In particular, in a single institutional study of 93 breast cancer

patients treated with NCT, Pengel et al. showed that FDG PET
and MRI have a complementary predictive ability. Using FDG
PET and MRI together in a multivariate analysis combined
with breast cancer subtypes, the AUC was 0.90 [72].
Despite its better accuracy than conventional radiological
techniques, even MRI may encounter difficulties especially in
patients with a non-mass lesion. In these patients, there is no
boundary volume at presentation and the response to therapy cannot follow a concentric shrinkage, although residual disease is most likely represented by scattered foci of
enhancement [74].
Our pilot study with FLT PET was performed in a small
patient population and clearly needs to be repeated in a larger
cohort to definitively assess and validate the estimated cut-off
values and the proposed prediction rule. Further studies are
also needed to evaluate the impact of different molecular
breast cancer subtypes and subgroups with different proliferation rates, if any, on the predictive ability of FLT PET.
However, our study provided interesting results in terms of
sensitivity, specificity and AUC compared with previous studies of FDG PET and/or MRI. The preliminary findings suggest
the potential utility of PET scans for early monitoring of the
response to NCT in order to choose a therapeutic strategy with
a greater probability of efficacy rather than unexpected futility.

Conflicts of interest None.

Funding This work was supported by a grant from Associazione
Italiana Ricerca sul Cancro (Study INT/35/10).

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