Research Article

Research Article
Received: 18 April 2008,
Revised: 12 February 2009,
Accepted: 4 April 2009,
Published online in Wiley InterScience: 2009
(www.interscience.wiley.com) DOI:10.1002/nbm.1408
Classification of prostatic diseases by means
of multivariate analysis on in vivo proton MRSI
and DCE-MRI data
Mariacristina Valerioa *, Valeria Panebiancob, Alessandro Sciarrac,
Marcello Osimanib, Stefano Salsicciac, Lorena Casciania,
Alessandro Giulianid, Mariano Bizzarrie, Franco Di Silverioc,
Roberto Passariellob and Filippo Contiay
Multivariate analysis has been applied on proton magnetic resonance spectroscopic imaging (1H-MRSI) and dynamic
contrast enhanced MRI (DCE-MRI) data of patients with different prostatic diseases such as chronic inflammation,
fibrosis and adenocarcinoma. Multivariate analysis offers a global view of the entire range of information coming from
both the imaging and spectroscopic side of NMR technology, leading to an integrated picture of the system relying
upon the entire metabolic and dynamic profile of the studied samples. In this study, we show how this approach,
applied to 1H-MRSI/DCE-MRI results, allows us to differentiate among the various prostatic diseases in a non-invasive
way with a 100% accuracy. These findings suggest that multivariate analysis of 1H-MRSI/DCE-MRI can significantly
improve the diagnostic accuracy for these pathological entities. From a more theoretical point of view, the
complementation of a single biomarker approach with an integrated picture of the entire metabolic and dynamic
profile allows for a more realistic appreciation of pathological entities. Copyright ß 2009 John Wiley & Sons, Ltd.
Keywords: cancer; differential diagnosis; dynamic contrast enhanced MRI; medical imaging; metabolomics; multivariate
analysis; prostatic diseases; proton magnetic resonance spectroscopic imaging
INTRODUCTION
Differential radiological diagnosis of both benign and malignant
prostatic diseases, such as benign prostatic hyperplasia (BPH),
prostatitis and prostate cancer (PrC) is often difficult despite their
epidemiological and clinical relevance (1,2). The non-invasive
differential diagnosis of prostate cancer, BPH, prostatisits and
normal tissue is of utmost importance for cancer staging and for
follow-up after therapy (3).
The serum prostate-specific antigen (PSA) has been identified
as a sensitive biological marker for prostate cancer diagnosis in
recent times. However, the PSA levels, although significantly
linked to prostate cancer, are devoid of any discriminatory power
for deciding among different prostatic diseases (4,5). In fact,
elevated serum PSA levels can be caused by benign conditions
that are mainly prevalent in older men (6). To date, histological
analysis of biopsy specimens has been the only reliable
procedure to distinguish normal, benign and malignant prostatic
tissues. However, due to the heterogeneous and frequent
multifocal nature of prostate cancer, biopsy methods may not
include an adequate specimen sampling of the prostate. In fact,
clinical studies have indicated that the usual systematic sextant
biopsy technique shows a positive predictive value of only 30%
for detection of prostate cancer (7). The use of novel biopsy
schemes significantly increases the diagnostic yield of prostate
biopsy in finding the malignant disease (8–10), but still does not
reach a fully satisfactory accuracy of cancer mapping within the
prostate (11).
* Correspondence to: M. Valerio, Department of Chemistry, University of Rome
‘La Sapienza’, P.le. A. Moro 5, 00185 Rome, Italy.
E-mail: [email protected]
a M. Valerio, L. Casciani, F. Conti
Department of Chemistry, University of Rome ‘La Sapienza’, Rome, Italy
b V. Panebianco, M. Osimani, R. Passariello
Department of Radiological Sciences, University of Rome ‘La Sapienza’, Rome,
Italy
c A. Sciarra, S. Salsiccia, F. Di Silverio
Department of Urology, University of Rome ‘La Sapienza’, Rome, Italy
d A. Giuliani
Department of Environment and Health, Istituto Superiore di Sanità, Rome,
Italy
e M. Bizzarri
Department of Experimental Medicine, University of Rome ‘La Sapienza’,
Rome, Italy
y
Deceased
Abbreviations used: AC, adenocarcinoma; BPH, benign prostatic hyperplasia; Cho, choline; CI, chronic inflammation; Cit, citrate; CO, control; Cr, creatine;
DCE-MRI, dynamic contrast-enhanced magnetic resonance imaging; FB,
fibrosis; 1H-MRSI, proton magnetic resonance imaging; LVs, latent variables;
OT, onset time; PCA, principal component analysis; PCs, principal components;
PE, peak enhancement; PLS-DA, partial least square discriminant analysis; PrC,
prostate cancer; PSA, prostate-specific antigen; ROI, region-of-interest; SI-T,
signal intensity-time; TRUS, transrectal ultrasonography; TTP, time to peak;
VOI, volume of interest.
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Copyright ß 2009 John Wiley & Sons, Ltd.
M. VALERIO ET AL.
Improvements in prostate imaging provide more accurate
mapping of cancer allowing for biopsy optimized plans. Needle
biopsy guided by transrectal ultrasonography (TRUS) is the most
commonly used method for histological diagnosis of prostate
cancer. On the other hand, this technique is limited by the lack of
accuracy in the estimation of cancer tissue extension (12).
High-resolution endo-rectal/pelvic phased array MRI has
demonstrated an enhanced sensitivity compared to clinical
data, systematic biopsy, TRUS and MRI when considered alone,
but it provides low specificity in detection and localization of
prostate cancer due to other benign pathologies which cause low
signal intensity on T2-weighted images similar to that of prostate
cancer (13–15). Moreover, this conventional imaging method
sometimes cannot distinguish accurately between healthy and
malignant tissues subsequent to resection or treatments for
prostate cancer because of induced changes in tissue structure
(16). The ability to identify prostate tissue types can be
significantly improved by the combined use of MRI and magnetic
resonance spectroscopic imaging (MRSI) (17,18). Specifically,
proton MRSI (1H-MRSI) allows the recognition of some relevant
metabolites like citrate (Cit), choline (Cho) and creatine (Cr),
endowed with the discrimination power for different prostate
diseases. In particular, the ratio choline-plus-creatine to citrate
([Cho þ Cr]/Cit) has been widely investigated in the differential
diagnosis of prostate diseases. The rationale at the basis of the
use of this index stems from the observed increase of
Cho-containing compounds and on the reduction or absence
of Cit in PrC compared to BPH and the surrounding healthy
peripheral zone tissue. However, in some cases, it has been
reported that glandular BPH and normal, healthy peripheral zone
tissues display similar Cit levels; on the other hand, stromal BPH
regions can show reduced Cit levels similar to those observed in
peripheral zone cancers (19–26). In a recent paper, Li et al. (27)
analysed the spectral differences between PrC and BPH,
evaluating the (Cho þ Cr)/Cit and Cho/Cr ratios measured in
each voxel with proven-biopsy cancer or BPH. The specificity,
sensitivity and accuracy for the discriminant function were 98.6,
85.7, 92.9%, respectively. Finally, the addition of metabolic
information provided by MRSI to morphologic information
provided an enhanced specificity up to 95% for localizing cancer
and has become important for detecting the extent of cancer
within the prostate and its aggressiveness (17,18,28). Despite the
very high accuracy of the MRI/MRSI combined approach in the
detection of prostate cancers, this mixed technique is not very
accurate in identifying cancers (only 30% are found) in both the
central gland (29) and small (<0.5 cm3) cancers within the
peripheral zone (21,30).
Recent studies have shown that accuracy can be improved by
performing MRI/MRSI at higher magnetic field strengths and
through the addition of dynamic contrast enhanced MRI
(DCE-MRI) (31–33).
DCE-MRI is known to be a powerful tool for visualizing the
vascularity of prostate gland tumour and for providing additional
information useful for both the detection and the staging of
prostate cancer (31,34). Dynamic MRI was demonstrated to be
able to differentiate cancer from normal prostatic tissue (35,36),
an earlier and stronger enhancement in PrC versus normal tissue
was found in these studies. DCE-MRI was also demonstrated to
differentiate the cancer tissue from benign lesions (37,38).
Recently, Ren et al. (39) reported that, based on T2-weighted
imaging, DCE-MRI curves can discriminate PrC and BPH with a
sensitivity, specificity and accuracy of 79.31, 66.67 and 74%,
respectively. In particular, PrC showed stronger enhancement
with an earlier peak time, higher enhancement and enhancement rate than those of BPH.
In this study, we developed a computational approach to the
analysis of 1H-MRSI and DCE-MRI combined results, that was
demonstrated to outperform both the classical ([Cho þ Cr]/Cit)
ratio and purely image-based parameters in the differential
diagnosis of prostatic diseases.
Our data set was composed of 11 prostatic tissue samples
coming from healthy control subjects, and of 40 pathologic tissue
samples coming from patients affected by one of the following
pathologies: (a) chronic inflammation, (b) fibrosis or (c) adenocarcinoma. The discrimination between healthy and disease samples
and the differential diagnosis of the various prostatic diseases
was the aim of the present study. Given the paucity of the data
set, our work has mainly methodological value, giving a
proof-of-concept to the feasibility of an integrative systemic
view, made possible by multivariate approach, to the MRI/MRSI
non-invasive differential diagnosis of prostatic diseases.
We adopted a data analysis strategy that mixed unsupervised
and supervised approaches. Unsupervised is the general heading
of techniques such as principal component analysis (PCA) and
cluster analysis, whose final result is not guided by the
maximization of ‘externally’ imposed classification goals, like
the discrimination between different classes of disease or
placebo and drug treated patients. The goal of unsupervised
algorithms is to maximize some purely syntactical internal
features of the data set at hand, like the obtaining of the most
faithful projection of an initially high dimensional data set with
the least number of axes (PCA), or the allocation of the statistical
units to classes that are the most internally compact and
separated (cluster analysis) (40). Unsupervised methods allow for
an unbiased (not driven by the goal of diagnosis) description of
the natural correlation structure present in the data. In contrast,
supervised methods that we adopted in our strategy, like partial
least squares (PLS) or discriminant analysis (DA), have the goal of
maximizing an ‘externally imposed task’, such as the separation of
two a priori classes (like healthy/disease) inside a given data field.
In fact, supervised methods are driven by a specific goal, external
to the intrinsic nature of the collected data, assuring the ‘best
possible discrimination’ of the classes, at the expense of the
appreciation of the natural correlations present in the data, which
we exploited with the unsupervised approach.
We decided to complement the unsupervised and supervised
approaches, with a classical strategy of data analysis (41) so as to
get the maximal global efficiency of the model on both
descriptive and diagnostic sides. The initial unsupervised
extraction of principal components from the original data set
allows for noise filtering of the data, maintaining only the
correlated (and thus more reliable) portion of information and
permitting a biological interpretation of the obtained results.
Subsequently, the use of the extracted principal components as
initial variables for the supervised portion of the procedure,
avoids possible inconsistencies coming from the regressors’
mutual collinearity components which are orthogonally constructed. However, besides statistical subtleties, the important
thing to stress is that the adopted strategy allows for all the
information embedded in our data to be potentially exploited for
the task of classification.
All in all, the obtained results allowed for both an efficient
discrimination of the different diseases and for a biologically
sound general picture of the system at hand.
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METABOLOMIC ANALYSIS OF PROSTATIC DISEASES
METHODS
Patient population
In this study, we retrospectively reviewed a total of 51 prostate
MR examinations, including morphologic imaging, 1H-MRSI and
DCE-MRI protocols, carried out from June 2007 to February 2008.
The MR investigations were performed before TRUS-guided
needle biopsy. The mean time interval between MR examination
and TRUS-guided biopsy was 7 3 days. For each patient,
medical histories including digital rectal examination, serum PSA
level and a confirmed biopsy report were obtained. Our study
population can be subdivided into four groups based on the kind
of prostate disease clinically and histopathologically diagnosed.
The first group consisted of 13 patients with biopsy-proven PrC
who subsequently underwent radical retropubic prostatectomy
performed within 3 weeks (mean ¼ 9 4 days). Histolopathological examination of the radical prostatectomy specimen
revealed a mean Gleason score of 7 2 (range ¼ 6–10) and
the absence of BPH nodules. In the second group, we included 14
patients with biopsy-proven BPH who subsequently underwent
trans-urethral resection. Pathologic assessment of the resected
tissue confirmed TRUS results. The mean period between MR
examinations and trans-urethral resection was 12 7 days. The
third group was composed of 13 patients with a biopsy-proven
fibrosis performed for a TRUS suspicious prostate nodule. A
control group of 11 patients with no pathological findings of PrC,
BPH and fibrosis was also evaluated. In this group, a prostate
TRUS-biopsy was performed following a suspicious clinical
examination and/or rising serum PSA level (mean ¼
4.9 7.0 ng/mL; median ¼ 1.7 ng/mL; range ¼ 0.5–18 ng/mL).
Furthermore, digital rectal examination did not reveal any
prostate abnormalities.
The following exclusion criteria were used in the selection of
patient population: clinical diagnosis of acute prostatitis,
coexistent clinically proven cancer, hormonal therapies (including
five a reductase inhibitors), radiotherapy, chemotherapy,
previous prostate surgery.
The 51 included patients had a mean age of 65 (range 48–75
years); age was checked for its possible confounding effect on the
MR-based parameters, without finding any significant correlation
with the descriptors under study (data not shown).
This study was approved by the local ethics committee and
signed informed consent was obtained from all patients.
MRI, 1H-MRSI and DCE-MRI data acquisition and processing
Acquisition of imaging data
All examinations were performed on a commercially available
1.5 T scanner (Magnetom Avanto, Siemens Medical Solutions,
Erlangen, Germany), equipped with surface phased-array (Body
Matrix, Siemens Medical Solutions) and endo-rectal coil (e-Coil,
Medrad, Pittsburgh, PA, USA, combined with Endo-Interface,
Siemens Medical Solutions). The balloon-mounted disposable
endo-rectal coil was first lubricated with a local anaesthetic gel
and then placed while the patient was in the left lateral decubitus
position. Then the patient was turned supine and the balloon was
inflated with up to 70 mL of room air based on the patient’s tolerance.
Before scanning, 20 mg butyl scopolamine (Buscopan, Boehringer, Ingelheim, Germany) was injected to suppress peristalsis.
First, localizer images in the sagittal, axial and coronal planes
were obtained to ensure endo-rectal coil position and to select
locations for the transverse images. Following this, T2-weighted
images in the three orthogonal planes were acquired providing
coverage of the entire prostate using turbo spin-echo (TSE)
sequences (TR ¼ 5190 ms; TE ¼ 95 ms; flip angle ¼ 1508;
average ¼ 3; FOV read ¼ 256 mm; FOV phase ¼ 100; slice thickness ¼ 3 mm; interslice gap ¼ 0; matrix size ¼ 512 512; phase
resolution ¼ 100%; bandwidth ¼ 130 Hz; scan time ¼ 3.40 min).
1
H-MRSI data were acquired by two skilled radiologists after a
first review of morphological images to localize suspicious areas
in the prostate, which were subsequently used to position the
spectroscopic acquisition volume. In patients with no MR
morphological evidence of changes, the volumes of interest
(VOIs) were centred on each prostate gland emi-portion (left and
right). The VOI to be studied with spectroscopy was selected in
such a way, as to maximize the coverage of the prostate while
minimizing the inclusion of surrounding structures (muscles, fat,
rectal air and urine). 1H-MRSI was performed using a sectionselected box drawn closely around the prostate fossa and a
point-resolved spectroscopic sequence was obtained by using a
3D chemical shift imaging (CSI) sequence (FOV ¼ 50 50 50 mm3; VOI ¼ 30 30 30 mm3; TR ¼ 700 ms; TE ¼ 120 ms;
ms; flip angle ¼ 908; interpolation ¼ 16; vector size ¼ 512;
TA ¼ 11.50 min; delta frequency ¼ 1.80 ppm; average ¼ 6;
filter ¼ Hamming) (42).
DCE-MRI images were acquired using 3D FLASH T1-weighted
spoiled gradient-echo sequence (TR ¼ 2.44 ms; TE ¼ 0.9 ms; flip
angle ¼ 308; average ¼ 1; thickness ¼ 4 mm; interslice gap ¼ 0;
slice number ¼ 12; matrix size ¼ 256 256; phase resolution ¼
100%; bandwidth ¼ 120 Hz; TA ¼ 4.40 min) performing 90
measurements in rapid succession, immediately after the
completion of an intravenous bolus injection of 0.1 mmol of
gadopentetate dimeglumine (Multihance, Bracco Spa, Milano,
Italy). Contrast liquid was administered with a power injector
(Spectris; Medrad) at 2.5 mL/s and was followed by a 15-mL saline
flush. The 3D volume was acquired with the same positioning
angle and centre as the transverse T2-weighted sequence
covering the entire prostate fossa and the periurethralperianastomotic region. Relative gadolinium chelate concentration curves were calculated in order to derive the three
dynamic DCE-MRI parameters: onset time, time to peak and peak
enhancement (PE).
Processing and analysis of imaging data
MR images were analysed in consensus by two radiologists with 5
and 9 years of experience in uro-genital MRI. They were unaware
of serum PSA levels and TRUS-biopsy results. T2-weighted images
were excluded from retrospective reviewing and the radiologists’
attempts were focused only on spectroscopy and DCE-MRI
findings.
An operator-independent standard post-processing protocol
was applied to the MR spectroscopic imaging data. These data
were acquired as 16 8 8 phase-encoded spectral arrays (1024
voxels) with a nominal spatial resolution >0.3 cm3 before Fourier
transformation in the spatial dimensions. After Fourier transformation, zero- and first-order phase correction and automated
baseline correction (polynomial of 6th order), a frequency
domain curve fitting was used subsequently for quantification
with the assumption of Gaussian line shapes, by using the
standard Syngo Spectroscopic Evaluation software package
(Siemens), provided with the MR imaging system (43). Goodness
of fit of the obtained parameter by means of the classical
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M. VALERIO ET AL.
Gaussian distribution hypothesis was assessed by subtracting the
processed spectrum from the fitted one, and checking that only
signals indistinguishable from the baseline noise remained at the
mentioned ppm in the residual curves. We used areas under the
curve to compute Cho (3.2 ppm), Cr (3.0 ppm) and Cit (2.6 ppm)
values. The average post-processing duration was 25–30 min for
each data set. Cho, Cr and Cit peak areas were evaluated for all
1
H-MRSI voxels not contaminated by inadequately suppressed
water or lipids, and did not contain mixed tissues from the
urethra, seminal vesicles, ejaculatory ducts, and bladder and
rectal wall. In addition, only voxels in which the choline/creatine/
citrate peaks were detectable with a signal-to-noise ratio of 3:1
were assessed (44).
The dynamic MR post-processing procedure lasted 10 min for
each patient. Functional dynamic imaging parameters were
estimated from the gadolinium curve using the procedure of
Fütterer et al.(19). The edge and the contour characteristics of the
lesions were defined using the same sections on which the
region-of-interest (ROI) analyses were performed.
A group of three ROIs were drawn independently by the
radiologists, and differences in the measurements were assessed
by consensus. The selected ROIs referred to three distinct areas:
(1) pelvic muscle (acting as low baseline ROI), (2) most-enhancing
areas within the main PrC foci, BPH nodules, fibrosis nodules or in
these regions with suspect spectroscopic voxels and (3) iliac
vessel (acting as high baseline ROI). In particular, suspect regions
were identified based on higher enhancing values on DCE-MRI
images (qualitative method). Correspondingly, normal tissue was
identified as the one having homogenous enhancing regions.
When multiple suspicious areas were identified, the signal
intensity–time (SI–T) records of the most enhancing lesion were
considered as significant values for subsequent SI–T analyses. The
following parameters were set to describe the SI time curve:
onset time, time to peak and PE. We determined the
enhancement onset time for the data sets by averaging (during
90 measurements) the intensity across the slices and using the
last point before the averaged signal increased 2.5 standard
deviations (SDs) above the running baseline average.
TRUS-biopsy evaluation
TRUS guided biopsies were performed using a biplanar 7.5 MHz
frequency probe according to a 12-core biopsy scheme (standard
sextant scheme, plus laterally directed samples of the prostate
apex (two cores), middle (two cores) and base (two cores)) (10).
No samples in the transitional zone of the prostate were
obtained. The operator was blinded from 1H-MRSI and DCE-MRI
results. All biopsy specimens were obtained under TRUS
guidance using an 18-gauge needle loaded in an automatic
spring action biopsy device, and were fixed overnight in a
solution of 10% neutral buffered formalin. The operator evaluated
the distance from the prostate apex and basis and the distance
from the urethra from which the biopsy specimen was drawn, in
order to provide a method of comparison with T2-weighted MR
images as reference. MRI films were interpreted independently by
a third radiologist with 6 years of experience in uro-genital
radiology, who had no knowledge of 1H-MRSI and DCE-MRI
results and of the final diagnosis. Each axial T2-weighted image
was branched in 12 radial triangles with apex orientation on the
urethra in a clockwise order. Distance from the prostate apex and
basis and the distance from the centre were noted by the
Radiologist. For each sample, all abnormalities were examined in
consensus by the biopsy operator and radiologist.
Statistical analysis
Raw data matrix structure
Multivariate analysis was applied to the data set constituted by
the 1H-MRSI spectral and dynamic DCE-MRI parameters
measured on healthy prostatic tissues of 11 control (CO) as well
as on the pathologic tissues of 40 patients who had one of the
following pathologies: (1) chronic inflammation (CI, n ¼ 14); (2) a
fibrosis (FB, n ¼ 13) or (3) adenocarcinoma (AC, n ¼ 13). This
produced a raw data set constituted by a matrix having as rows
(statistical units) 51 patients and as column (variables) the
6 values relative to choline, creatine, citrate (all these three
variables are expressed in terms of the area of the relative peak),
onset time, time to peak (these dynamic descriptors are
expressed in seconds) and PE parameters (c (mmol/kg contrast
agent)) obtained from 1H-MRSI/DCE-MRI measurements. The
original 51 units in the population were separated into two sets: a
training and a validation (test) set. The training set was made of
45 subjects, while 6 subjects (2 CI, 2FB, 2AC) composed the test
set. The model was built upon the 45 subjects in the training set
and then was checked on its ability to correctly classify the set of
six patients. This procedure known as cross-validation allows for
the testing of the generalization ability of the proposed model,
outside the range of the specific data set it comes from. This
eliminates overfitting and chance correlation problems, which is
particularly important in this case, where the paucity of data
greatly increases the risk of apparent correlations (41).
Principal component analysis
PCA is a projection method used for exploiting the information
embedded in multidimensional data sets (40). The data are
reduced to a few latent variables (LVs) (or principal components)
collecting the information implicit in the original variables’
correlation structure. The extracted components (PCs) are each
orthogonal and ordered in terms of the percentage of explained
variation, with the first components collecting the ‘signal’
(correlated) portion of information, while minor components
can be considered as ‘noise’ components. From an algebraic
point of view, each component is a weighted summation
computed across the original variables in the form of:
PC ¼ aX1 þ bX2 þ cX3. where X1, X2 and X3 are the measured
features and a, b, c numerical constants. Each statistical unit is
assigned a score relative to each extracted component, while the
correlation coefficient between each original variable and
extracted components (loading) allows us to give a meaning
to the PCs.
Partial least square discriminant analysis
While PCA is an unsupervised technique in which each variable
enters with the same role of description of the data set and
the solution is driven by the maximal parsimony principle alone
(maximal amount of explained variation with the minimum
number of components); on the other hand, both PLS and DA are
supervised techniques in which the analysed variables pertain to
two classes: the ‘diagnosis’ (dependent, Y) and ‘symptoms’
(independent, X) variables. The goal of both PLS and DA is to find
the linear combination of X variables that explains the Y
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METABOLOMIC ANALYSIS OF PROSTATIC DISEASES
variable(s) better. In the case of DA, this goal is achieved by the
construction of a set of weights multiplying each X variable so as
to build metrics in which the errors of assignment of each
statistical unit to the correct Y class is minimized. PLS, on the
other hand, works by the generation of mutually orthogonal
linear combinations of X variables maximally correlated with Y
counterparts. PLS-DA was used to build and test a supervised
model that could predict the pathology of a patient based on its
spectral and dynamic data. The ‘leave-one-out’ cross validation
method was used to validate the model and to select the
appropriate number of LV. The identification and the removal of
outliers were performed by using the Q and T2 statistics (45).
Range restriction effect check
The presence of outliers is known to deeply influence the
correlation analyses by means of the so called range restriction
effect (46). This effect has to do with the fact that outliers
(extreme values observations) acquire a disproportionate weight
in the computation of any least-squares- based model. In fact,
optimizing the fit of an extreme point (outlier) has a much greater
influence in the sum of errors (to be minimized by the system)
than the best fitting of an average unit. This statistical effect could
give rise to biased descriptions of the studied data sets. For this
reason, we decided to perform two independent analyses: the
first one on the complete data set (45 units) and the second one
on the same data sets after removal of four possible outliers
(41 units). We considered as outliers the units being located at
more than 3 SD units from the centroid of the data cloud.
RESULTS
Multivariate analysis of 1H-MRSI and DCE-MRI data
Individual and mean values of spectral 1H-MRSI and dynamic
DCE-MRI parameters measured on healthy prostatic tissues of
control as well as on the pathologic tissues of patients with
chronic inflammation, fibrosis or adenocarcinoma are shown in
Fig. 1. The data indicate the presence of a substantial overlap
among groups, even if we must take into account the fact that
the relatively high variances of the considered variables is partly
due to the coil sensitivity profiles. Nevertheless, correction factors
are not routinely used in such kind of analyses. This figure allows
to immediately perceive the huge advancement obtained by
explicitly taking into account the fact that the same statistical unit
(patient) is simultaneously defined over different variables. In fact,
the possibility of distinction relies on the correlation between
variables instead of considering each descriptor in isolation from
all the others (47).
Before entering the actual statistical strategy with the explicit
consideration of the correlation structure of the measured
features, we adopted the classical approach applied in this
category of problems by submitting the descriptors most widely
used for differential diagnosis to DA. The discriminant function
was computed over the (Cho þ Cr)/Cit ratio and OT, TTP and PE.
The specificity, sensitivity and accuracy are reported in Table 1 for
differences between class comparisons. Although all the
considered descriptors displayed a statistically significant
discriminative power, there are some specific inter-group
comparisons that were markedly sub-standard. In contrast, we
will show how the multivariate approach not only reaches
maximal accuracy on the entire set of comparisons, but how it is
also able to correctly predict the test set statistical units, thus
demonstrating a predictive ability outside the realm of the
samples used to build the model.
Data analysis begun with the computation of PCA to identify
any clustering of data related to the types of pathology in an
unsupervised manner, while PLS-DA was subsequently applied to
build a classification model to predict the clinical outcome of a
patient based on its spectral and dynamic data and usable even
for independently analysed patients (test set) (45).
PCA applied to the original 45 units/6 variables data set
([Cho þ Cr]/Cit ratio is a derived variable and its information is
implicit in the original descriptors), gave rise to a four component
solution explaining about 87% of the total variability in the
system. In Table 2 the variance explained by each component is
reported. To compare the controls and patients (as a whole), a
t-test was applied to the component scores, highlighting
significant differences between the two groups on PC1 and
PC3 (see Table 2). This can be appreciated in Fig. 2, where the
component score plot is shown. A linear discriminant analysis
applied to this space allowed for a clear separation of the two
groups (Fisher’s exact test, p < 0.0001 on the classification matrix).
The t-test on PCs is then repeated to compare all the specific
pairs of control and pathology groups (see Table 2). As a result, CI
and AC patients showed significant differences compared to
controls on both PC1 and PC3 (Fisher’s exact test p < 0.0001),
whereas the FB group differed from controls as for PC2 and PC3
(Fisher’s exact test p < 0.0001). Figures 2 and 3 display the
above-mentioned differences in various component planes.
We subsequently analysed the data by performing a t-test on
PCs to compare a single pathology (see Table 2). PC1 was
responsible for the discrimination among pathologies and
pairwise discriminations are also observed on PC2 and PC4
(Fisher’s exact test p < 0.0001) for CI versus FB as well as CI versus
AC groups (see Fig. 4). The above results indicate that even by
using this unsupervised method of analysis, with only a posteriori
computation of the statistical significance over the extracted
components, there is a clear separation between controls and
pathologic groups, controls and single-pathologic groups and
among patients with different types of pathology.
After having checked for the general robustness of the
correlation structure, we considered the possibility of using
multivariate methods to classify patients not used for model
building. This means shifting from a purely descriptive to a
practical diagnostic use of the technique that by definition must
be effective in predicting the diagnosis of samples not explicitly
taken into account for statistical model building. For this goal, we
built a PLS-DA model based on 45 patients data set. This method,
at odds with PCA, is a supervised procedure explicitly driven by
the optimization of discrimination power of the model. The
feasibility of the proposed method as a routine diagnostic
procedure depends on the successful classification of the test set.
The PLS-DA model generated three LVs which explain 70% of
the X-variance (spectral and dynamic data) and 50% of the
Y-variance (which represent the membership class). Table 3
summarizes the features of this model. A clear separation among
the classes was found in the first and second components (Fig. 5)
thus confirming the unsupervised approach (PCA). This model
was used to assess the predictive capabilities for six other
patients with unknown pathology. The model predicted that two
of the unknown samples were from the CI group, two from the FB
and two from the AC group. The actual identities of the samples
coincided with the predicted ones (i.e. the sensitivity and
5
NMR Biomed. (2009)
Copyright ß 2009 John Wiley & Sons, Ltd.
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M. VALERIO ET AL.
Figure 1. Plot of individual (open circle) and mean (filled circle) values of spectral 1H-MRSI (area) and dynamic DCE-MRI (onset time and time to peak in
seconds; peak enhancement in c (mmol/kg contrast agent) parameters measured on healthy prostatic tissue of normal control as well as on the
pathologic tissue of patients with chronic inflammation, fibrosis or adenocarcinoma.
specificity for the PLS model based on pathology are both 100%),
thus providing a validation of the PLS model as a diagnostic tool
(see Table 4).
Having proven the efficacy of the model as a predictor, we
decided to go more in depth into the nature of the metabolic and
dynamic biomarkers which permit discrimination. To this aim, we
derived a PLS model using a reduced data set by eliminating
outliers that could bias the solution by their excessive weight in
computing the correlation. The outliers were removed by means
of the procedure previously outlined in the Method section. The
removal of outliers provided a final 41 statistical units and a raw
data matrix of six variables. The loading plot relative to the
discriminating variables is shown superimposed over a score plot
in Fig. 6. This representation allows us to simultaneously
appreciate the discrimination power (position of the patients
6
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in the plane) and the functional meaning (the loadings
correspond to the correlation coefficients of the original variables
with the axes) of the proposed solution.
From Fig. 6, it is evident that choline concentration and PE are
crucial parameters to discriminate between the benign and
malignant diseases. Choline has a high negative correlation
coefficient with LV1, while PE shows a strong negative correlation
with LV1 and LV2. Since the malignant group displays lower
scores in both LV1 and LV2 compared to benign groups, this
corresponds to the fact that patients with adenocarcinoma have
higher values of choline and PE compared to patients with
benign pathologies. Concerning the differential diagnosis of
benign pathologies, the descriptors endowed with the highest
discrimination power for FB patients are the scoring of a high
level of citrate (loadings ¼ 0.460 and 0.300 on LV1 and LV2,
Copyright ß 2009 John Wiley & Sons, Ltd.
NMR Biomed. (2009)
METABOLOMIC ANALYSIS OF PROSTATIC DISEASES
Table 1. Discriminant function analysis comparing controls versus single pathology and single pathology versus single pathology
with regard to spectroscopic 1H-MRSI or dynamic DCE-MRI or both spectroscopic and dynamic parameters
Control vs.
chronic
inflammation
F-value
Specificity
Sensitivity
Accuracy
34.45
100
100
100
F-value
Specificity
Sensitivity
Accuracy
2.31
64
79
72
F-value
Specificity
Sensitivity
Accuracy
12.19
100
93
96
Control vs.
fibrosis
Control vs.
adenocarcinoma
Chronic
inflammation
vs. fibrosis
Chronic
inflammation vs.
adenocarcinoma
Fibrosis vs.
adenocarcinoma
(Cho þ Cre)/Cit ratio
7.03
46.82
5.54
46
100
31
100
93
100
71
96
67
Onset time, time to peak and peak enhancement parameters
8.003
53.81
9.22
31.43
100
100
85
92
85
100
93
93
92
100
89
93
(Cho þ Cre)/Cit ratio, onset time, time to peak and peak enhancement parameters
6.05
39.12
15.39
22.90
100
100
100
92
85
100
93
93
92
100
96
93
1.34
27
77
54
8.49
46
100
73
49.53
100
100
100
38.24
100
100
100
For each discriminant function the specificity, sensitivity and accuracy are reported.
respectively) and the presence of a comparatively high value of
time-to-peak (loadings ¼ 0.308 and 0.147 on LV1 and LV2,
respectively). It is worth noting that some patients with fibrosis
are characterized by high values of onset time (loadings ¼ 0.630
and 0.133 on LV1 and LV2, respectively). Furthermore, chronic
inflammation provokes an increase of creatine level (high positive
correlation with LV2, loading ¼ 0.605) with respect to other
benign pathologies.
DISCUSSION
Due to the increased use of both serum PSA screening and
TRUS-guided biopsies, prostate cancer is being identified at an
earlier and more treatable stage (48). Therefore there is an
increased interest in routine check-ups, but clinical parameters
alone are not sufficient to predict the course of a benign disease.
In fact, the risk of over-detection has been estimated to vary
between 15 and 84% (49,50). Current classification systems are
able to predict only a binary outcome, i.e. benign or malignant,
with sensitivities of 95 and 73% as well as specificities of 91 and
81% for 1H-MRSI and DCE-MRI, respectively (17,51). The most
widely used metabolic classification method is based on previously
reported differences between cancer and normal prostate tissue;
voxels are considered suspicious for cancer if the [Cho þ Cr]/Cit
ratio is at least 2 SDs above the average ratio for the normal
peripheral zone, and voxels are considered very suspicious for
cancer if [Cho þ Cr]/Cit ratio is more than 3 SDs above the average
ratio (21,22). However, other conditions such as prostatitis or post
biopsy haemorrhage might increase the [Cho þ Cr]/Cit ratio, and
also normal prostatic tissue may show higher [Cho þ Cr]/Cit ratios
within the transitional and periurethral tissue (52). Furthermore,
Table 2. t-test comparing pathologic patients on the whole versus controls, controls versus single pathology and single pathology
versus single pathology
Statistical significance
Patient group
Pathologic patients on the whole vs. controls
Controls vs. chronic inflammation
Controls vs. fibrosis
Controls vs. adenocarcinoma
Chronic inflammation vs. fibrosis
Chronic inflammation vs. adenocarcinoma
Fibrosis vs. adenocarcinoma
PC1 (35.43)
PC2 (21.73)
PC3 (15.85)
PC4 (14.33)
0.004
0.043
0.144
0.0001
0.003
0.0001
0.0001
0.514
0.207
0.050
0.295
0.009
0.039
0.267
0.0001
0.004
0.014
0.001
0.626
0.421
0.859
0.773
0.054
0.378
0.702
0.0001
0.0001
0.397
Threshold p < 0.05.
In parentheses the percent of variance explained by each principal component is reported.
7
NMR Biomed. (2009)
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M. VALERIO ET AL.
Figure 2. PCA score plot of the third PC (PC3) versus the first PC (PC1) of
the 45 patient data set. Differentiation between control and patient (as a
whole) groups and between control and single-pathologic groups (control vs. chronic inflammation and control vs. fibrosis) is shown.
although average values and SDs for [Cho þ Cr]/Cit in healthy
prostate tissue have been available, to date, it remains unclear if
these values are independent of the respective MR scanner and/
or MRS sequence applied. In the Shukla-Dave et al. (53) study, Cho
was elevated in 9 out of 12 patients with histopathologically
confirmed chronic prostatitis, and 86% of the voxels indicated
intermediate or high-grade diseases.
The diagnostic value of DCE-MRI in histologically proven
benign and malignant prostate tissues has been evaluated by
several studies (54,55), which postulated that prostate cancer
showed earlier and stronger enhancement than normal tissue. In
particular, Ren et al. (39) demonstrated the potential of DCE-MRI
to distinguish between BPH nodules and PrC foci; the timeto-peak of PrC lesion occurred earlier than the BPH peak time and
the enhancement degree and rate of PrC were higher than those
of BPH. On the other hand, limitations of the technique including
inadequate lesion characterization, particularly in the differentiation of prostatitis from cancer in the peripheral gland and in
Figure 4. PCA score plot of the first two PCs (PC1 and PC2) versus the
fourth PC (PC4) of the 45 patient data set. Differentiation among pathologies is shown.
the discrimination between BPH and central gland tumours has
been established (34).
The combined use of 1H-MRSI and DCE-MRI techniques could
be able to address the limitations found in the two techniques
when used independently, improving the prediction accuracy
(19). van Dorsten et al. (56) showed that the addition of 1H-MRSI
and DCE-MRI to the conventional MRI protocol has great
potential for improved localization and characterization of
prostate cancer in a clinical setting.
Our results represent a further improvement along this line.
Multivariate analysis is much more efficient in discrimination than
in the use of original variables as they do not allow for an equally
precise discrimination: the ([Cho þ Cr]/Cit) ratio, the most
discriminant 1H-MRSI index, fails to separate between control
and FB groups, whereas dynamic parameters are not able to
separate the CI/CO groups. Furthermore, the classical, combined
Table 3. PLS-DA model summary for discriminating 1H-MRSI/
DCE-MRI data from patients with both benign and malignant
prostatic diseases
Model
LV
R2X
R2Y
Q2
45 units
LV1
LV2
LV3
LV1
LV2
LV3
0.353
0.187
0.158
0.364
0.155
0.151
0.273
0.172
0.099
0.283
0.185
0.102
0.249
0.164
0.078
0.274
0.159
0.069
41 units
Figure 3. PCA score plot of the third PC (PC3) vs. the second PC (PC2) of
the 45 patient data set. Differentiation between control and fibrosis
groups is shown.
R2X, cumulative fraction of the variation of the X variable
explained per component; R2Y, cumulative fraction of the
variation of the Y variable explained per component; Q2, the
cumulative predicted fraction (cross-validation) of the variation joint X and Y.
8
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Copyright ß 2009 John Wiley & Sons, Ltd.
NMR Biomed. (2009)
METABOLOMIC ANALYSIS OF PROSTATIC DISEASES
Figure 5. PLS-DA score plot of the first two LVs (LV1 vs. LV2) of the 45
patient data set. Differentiation among control and pathology groups is
shown.
1
H-MRSI and DCE-MRI analysis showed a lower specificity,
sensitivity and accuracy compared to those obtained for the
PLS model.
The first multivariate data analysis applied was PCA. The
application of this method embodies a sort of ‘natural normalization’ of the studied data set, given that principal components
correspond to the eigenvectors of the correlation matrix that in
turn corresponds by definition to the covariance matrix of the
standardized variables. This is particularly convenient when
dealing with heterogeneous variables defined by completely
different measurement units (57), ruling out all questionable
a priori defined standardization processes.
PCA showed a natural trend to clustering of the prostatic
diseases in the MR space. This is a proof-of-concept of the
possibility of obtaining metabolic and morphological fingerprints
useful for the differential diagnosis of prostatic diseases, even
without specifically imposing the discrimination task to the
model. Principal components are orthogonally constructed, thus
the different relevance of the components for the discrimination
of diverse prostatic diseases is the image in light of the biological
differences between pathologies (58).
Table 4. Membership score (correlation coefficient) for each
of the six samples derived from the PLS model built using the
whole data set (45 units/6 variables)
Chronic
Sample Control inflammation
Fibrosis
Adenocarcinoma
CI
CI
FB
AC
AC
FB
0.126
0.041
0.852
0.028
0.058
0.813
0.212
0.118
0.168
0.873
0.693
0.057
0.233
0.340
0.323
0.048
0.011
0.175
0.428
0.501
0.008
0.107
0.355
0.069
The sample column reports the effective clinical status of the
patient, the other columns the allocation coefficient made by
the system on the sole basis of MR information.
Figure 6. PLS-DA score and loading plots (superimposed) of the 41
patient data set containing no outliers. The score plot provides a map
of how the groups relate to each other showing differentiation among
classes, while the loadings plot reveals which original variables (Cho, Cr,
Cit, OT, TP and PE) are important in separating the four groups.
This clustering tendency of the prostatic diseases in the MR
space was confirmed in terms of diagnostic accuracy, by a PLS
methodology that highlighted choline, creatine and citrate as the
main discriminant metabolites among different prostate diseases. The dynamic parameters endowed with the highest clinical
significance were onset time, time-to-peak and PE. Taken
altogether, metabolic and dynamic descriptors allowed us to
obtain a correct reclassification of an independent test set in
addition to a complete classification of the training set. The
prediction of the six patients in the test set confirms the exact
discrimination already found in the general (45 patients) data set,
adding the dimension of the generalization ability to the pure
internal consistency of the model.
To investigate the metabolic and dynamic biomarkers, we
obtained a PLS model using a reduced data set by removing the
outlier (41 patients). The reproduction of the same correlation
structure, by means of a data set depurated by the most extreme
statistical units, is proof of the fact that we can safely rule out a
‘range restriction effect’ (46) as a possible source of confounding
for our results. The fact that both the PLS analyses gave rise to the
same result is an important proof of the robustness of the
classification.
As we mentioned in the Results section, the two latent
variables (LV1 and LV2) endowed with the highest discrimination
power were mainly related to the opposition between choline
and onset time for LV1 (these two variables are at the opposite
poles of the LV1 axis in Fig. 6) and PE and creatine for LV2
(extreme opposite poles of the LV2 axis in Fig. 6). As for the LV1
axis, the high choline pole matches to adenocarcinoma patients
(black squares in Fig. 6), while the opposite LV pole corresponding
to high values of onset time is the preferred location of fibrosis
patients (black circles in Fig. 6). As for the LV2 axis, near the
creatine (Cr) pole in the LV space we observe the chronic
inflammation patients (black triangles in Fig. 6), whereas the
adenocarcinoma patients shifted toward the direction of the high
PE pole. The increased efficiency of both PCA and PLS-DA
analyses is a natural consequence of the fact that both
9
NMR Biomed. (2009)
Copyright ß 2009 John Wiley & Sons, Ltd.
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M. VALERIO ET AL.
techniques filter out the noisy portion of information into minor
components while concentrating on the most informative
portion of information retained by the major axes (40,45).
Our data are consistent with the well-known increased degree
of vascularization in tumour pathologies that scale with PE, while
the discriminant ability of choline can be related to the changes
in cell membrane synthesis and degradation of tumour tissues
that go hand in hand with an increased choline concentration
(19–26,39).
Beside the mechanistic interpretation of the results, we can
safely affirm that the non-invasive acquisition of 1H-MRSI/
DCE-MRI data is a potentially valid approach in both the
differential diagnosis and treatment evaluation of prostatic
diseases.
Our study can be considered as a pilot study: the paucity of the
considered data set together with the limitation of ‘pure’ diseases
(e.g. patients with mixed syndromes where BPH nodules go hand
in hand with cancer are excluded by the analysis) are strong
caveats to the generalization of our findings. A particularly hard
constraint is the limitation to pure diseases given the high
prevalence of mixed syndromes in nature. Nevertheless, in order
to validate the method, at first, we preferred to rely upon a more
neat case. Further developments of metabolomic research in the
future will need to address this very important point (59).
Acknowledgements
While this paper was in the final stage of preparation, our
co-author Prof. Filippo Conti passed away on 30 May 2009. He
was our principal source of inspiration in the search for a
metabolism-based holistic perspective in medical diagnosis.
His death was both a cause of great sorrow and a potent drive
for all of us for pursuing his dream of an integrated scientific
culture offering a concrete hope of real scientific advancement.
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