Alpha-methyl CoA racemase expression in
renal cell carcinomas
Vincent Molinie ´ MDa,*, Andre ´ Balaton MDa, Samuel Rotman MDb,
Douha Mansouri RNa, Isabelle De Pinieux MDa, Toufik Homsi MDa, Louis Guillou MDb
aDepartment of Pathology, Ho ˆpital Saint Joseph, 75014 Paris Cedex, France
bDepartment of Pathology, University Institute of Pathology, Lausanne, Switzerland
Received 4 December 2005; revised 19 January 2006; accepted 19 January 2006
Summary Alpha-methyl CoA racemase (AMACR), a new molecular marker for prostate cancer, has
been recently reported to be one of the most highly expressed genes in papillary renal cell carcinomas
(RCCs). We tested the diagnostic usefulness of AMACR antibody in a series of 110 renal tumors:
53 papillary RCCs (33 type 1, 20 type 2); 25 conventional RCCs; 6 chromophobe RCCs; 9 oncocytomas;
5 mucinous tubular and spindle tumors; 2 urothelial carcinomas; 7 angiomyolipomas; and 2 Bellini
carcinomas. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue
sections, with a primary prediluted rabbit monoclonal anti-AMACR antibody. Both type 1 and type 2
papillary RCCs exhibited cytoplasmic immunoreactivity for AMACR, with diffuse strong granular
staining in 96.4% (53/55) of tumors, without correlation with type or nuclear grade. The 5 mucinous,
tubular, and spindle cell carcinomas strongly expressed AMACR, and only 5 of 25 clear cell RCCs and
1 of 9 oncocytomas were focally reactive. The remaining 6 chromophobe RCCs, 5 urothelial
carcinomas, and Bellini duct carcinomas showed no immunoreactivity for AMACR. Because high
expression of AMACR is found in papillary RCCs (type 1 and 2) and in mucinous, tubular, and spindle
cell carcinomas of the kidney, immunostaining for AMACR should be used in conjunction with other
markers when histological typing of a renal tumor is difficult.
D 2006 Elsevier Inc. All rights reserved.
Renal cell carcinoma (RCC) is a clinicopathologically
heterogeneous disease, subdivided into clear cell, papillary,
chromophobe, spindle cell, cystic, and collecting duct
carcinoma subtypes based on morphological features
according to the World Health Organization international
histological classification of kidney tumors [1-6]. Papillary
RCC represents the second most frequent RCC and
accounts for 10% to 15%  of cases. Papillary RCCs are
defined by their papillary or tubulopapillary architecture and
can be subdivided into two morphological subtypes: type 1
with small cells arranged in a single layer on delicate papilla
cores and type 2 with a large eosinophilic cytoplasm and
pseudostratified nuclei arranged on broad papillae, which
may have different genetic backgrounds [8-15]. Papillary
RCCs can display not only papillary but also nonpapillary
growth patterns such as trabecular, tubular, and solid
patterns. In case of an equivocal diagnosis, immunohisto-
0046-8177/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
* Corresponding author.
E-mail address: email@example.com (V. Molinie ´).
Renal cell carcinoma;
Mucinous and spindle
Human Pathology (2006) 37, 698–703
chemistry may be a useful adjuvant in the diagnosis of
papillary RCC . The most relevant markers of papillary
RCC are vimentin and CK7 . More recently, alpha-
methyl CoA racemase (AMACR), a peroxymal mitochon-
drial enzyme involved in the b-oxidation of branched-chain
fatty acids and fatty acid derivatives, identified as a
molecular marker for prostate cancer [18-21] on the basis
of complementary (cDNA) microarray technology, has also
been reported in epithelial tumors [22-24] and in kidney
cancers [16,25]. Complementary DNA microarray study has
demonstrated that AMACR overexpression in papillary
RCC is one of the top 10 most highly expressed genes
[26,27]. The purpose of our study was to assess the use-
fulness of AMACR (Menarini Diagnostics, Paris, France) in
110 renal tumors.
2. Materials and methods
2.1. Case selection
One hundred ten cases of renal tumors were selected
for this study from the pathological databases of the
Ho ˆpital Saint Joseph, Paris, France, and the Centre
Hospitalier Universitaire, Lausanne, Switzerland.
Immunohistochemical staining was performed on 5-lm
sections cut from formalin-fixed, paraffin-embedded tissue
blocks. Heat-mediated antigen retrieval was performed in
0.1 mol/L pH 6.0 citrate in a water bath for 30 minutes.
Immunostaining was performed on a Dako autostainer using
a peroxidase-labeled polymer-based detection system (En-
vision plus, Dako France SAS, Trappes, France) and
diaminobenzidine as a chromogen. Rabbit monoclonal
prediluted anti-AMACR antibody (p504s, Menarini Diag-
nostics) was used and incubated for 25 minutes at room
temperature. Slides were then counterstained with hema-
toxylin. Prostatic adenocarcinoma was used as a positive
control. Appropriate negative controls were used.
2.3. Evaluation of immunohistochemistry
Immunoreactivity was evaluated in a semiquantitative
manner that assessed both staining intensity and percentage
of positive cells. Positive AMACR staining was defined as
A-D, Different renal subtype tumors and p504s expression.
AMACR and renal cell carcinomas699
coarse dense cytoplasmic granularity. We divided the
staining intensity into three categories: negative, weak,
and strong. Immunoreactivity was considered negative in
cases with weak staining of less than 5% of cells.
Positivity was divided into focal (5%-50% positive cells)
or diffuse (N50% positive cells). To reduce interobserver
variation in the evaluation of staining patterns, the above
semiquantitative method was used to independently score
immunohistochemical staining of the tumors by two
pathologists (V. M. and A. B.), and a consensus score
3.1. Histological diagnosis
All hematoxylin and eosin saffron slides were reviewed
in this study. The 110 renal tumors were diagnosed as
53 papillary RCCs (33 type 1, 20 type 2); 25 conventional
(clear cell) RCCs; 6 chromophobe RCCs; 9 oncocytomas;
5 mucinous tubular and spindle RCCs; 2 urothelial
carcinomas; 7 angiomyolipomas; and 2 Bellini carcinomas,
according to the current 2004 World Health Organization
consensus pathological definition criteria.
3.2. AMACR in nonneoplastic kidney
In all cases, normal kidney tissue adjacent to tumors was
present. Alpha-methyl CoA racemase immunoreactivity was
noted in proximal convoluted renal tubules, but was absent
or minimal in distal tubules, glomeruli, medullary tubules,
and stromal cells.
3.3. AMACR in papillary RCCs
Both type 1 and type 2 papillary RCCs showed
cytoplasmic immunoreactivity for AMACR, with diffuse
granular AMACR staining in 96.4% (53/55) of tumor cells
(Fig. 1). Only two cases showed strong focal positivity. The
extent and intensity of AMACR expression did not correlate
with type or nuclear grade of the tumors.
3.4. AMACR in mucinous tubular and spindle
All 5 (100%) cases of mucinous tubular and spindle cell
carcinoma displayed diffuse cytoplasmic positivity with
AMACR (Fig. 2), similar to the reactivity observed in
papillary RCC. Immunoreactivity was observed in both
tubular and spindle cells.
3.5. AMACR in other renal tumors
In contrast to papillary RCCs, most other types of renal
tumors did not express AMACR by immunohistochemis-
try. Only 4 of 25 clear cell RCCs were focally reactive and
1 of 25 clear cell RCCs and 1 of 9 oncocytomas were
diffusely reactive for AMACR. The remaining 6 chromo-
phobe RCCs, 5 urothelial carcinomas, and the Bellini duct
carcinomas showed no immunoreactivity for AMACR
(Tables 1 and 2).
A total of 32000 new cases of RCC are diagnosed each
year in the United States, accounting for 3% of all adult
malignancies . In France, there were 8300 new RCCs
diagnosed and 3600 deaths in the year 2000, representing
the eighth most important cancer . Renal cell carcinoma
is a clinicopathologically heterogeneous tumor; histologi-
cally, it has been subdivided into clear cell, papillary (types
Mucinous and spindle cell tumor and p504s expression.
p504s immunoreactivity in renal tumors (%)
Clear cell carcinoma
(n = 25)
Chromophobe (n = 6)
Oncocytoma (n = 9)
Bellini/urothelial (n = 5)
Mucinous and spindle
cell carcinoma (n = 5)
Papillary (n = 55)
3.696.4 100 (55/55)
p504s immunoreactivity in renal tumors (%)
et al 
V. Molinie ´ et al.700
1 and 2), chromophobe, collecting duct, and unclassified
subtypes [1,4,8-10,12-14,24]. As such, the diagnosis of
RCC can occasionally be challenging if, for example, the
tumor contains mixed types of tissues or when the tumor
does not resemble any of the known types (ie, unclassified)
. Recently, cytogenetic analysis and gene expression
microarray analysis profiled the major RCC histological
subtypes: papillary, clear cell, and chromophobe subtypes
were described [2,11,13,30]. Papillary RCCs are character-
ized by a gain of two or more chromosomes, 3q, 7, 8, 12,
16, 17, or 20 and no 3p loss [13,26,30]. However,
cytogenetic and gene expression microarray analyses are
reserved for research and are not usually performed .
Using RNA subtraction hybridization and cDNA micro-
array analysis of prostatic carcinoma, Xu et al  found
that AMACR and its RNA messenger (p504s) were over-
expressed in prostatic carcinoma. Alpha-methyl CoA race-
mase has been located in peroxisomes and mitochondria in
human cells . Expression of the enzyme is strongly
expressed in normal liver and kidney cells, and over-
expression has been detected in many human carcinomas
such as those of the colon and breast and in melanomas
[20,23]. In normal renal parenchyma, AMACR expression
is present in the proximal convoluted tubular epithelium
 and strong immunostaining is present in proximal
tubules adjacent to the renal neoplasm . Therefore, it is
likely that some of the tumors derived from these structures
may express AMACR .
In renal tumors, 5.2-fold expression of AMACR com-
pared with normal kidney tissues has been observed . In
their study, Tretiakova et al  found that 6 of their 8 cases
of papillary RCCs tested by cDNA microarrays showed
significant elevation of AMACR and mRNA levels, and
only one papillary RCC did not reveal any signs because of
a technical difficulty. Among all other subtypes of kidney
tumors tested, only two clear cell RCCs with prominent
papillary patterns showed significantly elevated AMACR
mRNA levels . The AMACR enzyme plays a role in
b-oxidation of branched-chain fatty acids and fatty acid
derivatives [21,22], and hyperexpression has been found in
prostate, colorectal, and ovarian cancers that have been
linked to a high-fat diet [22,23,33,34]. In epidemiological
studies, prostate and colorectal cancers have been linked to a
high-fat diet. There are several studies indicating that dietary
factors may play a role in the development of RCC as well.
Yet the relationship between fatty acid consumption,
metabolism, and kidney cancer development is not clearly
elucidated. Further studies are necessary to understand the
possible role of AMACR as a fatty-acid protein in renal
Our results showed that diffuse, strong granular AMACR
staining was observed in 96.4% of papillary RCCs. These
results lend support the study of Yang et al  who
confirmed the localization of protein expression in the 41
papillary RCCs tested. As reported in previous studies,
chromophobe RCCs were totally negative . The 16% of
weak positivity of AMACR found in clear cell RCCs is
lower than the 25% positivity reported by Tretiakova et al
. In their series, they found weak (7/52), moderate
(strong focal [3/52]), or strong diffuse (3/52) intensity in
clear cell RCCs . In our 3 cases of urothelial
carcinomas, AMACR immunostaining was always negative.
This differs from the moderate elevations in AMACR found
in 1 of 3 urothelial carcinomas by cDNA microarray and in
2 of 17 by immunohistochemistry by those authors [28,32].
As in the 2 Bellini carcinomas, our 3 collecting duct
carcinomas were negative .
Lin et al , in 2004, published a series of 115 renal
neoplasms tested for AMACR expression on formalin-
embedded tissue. Their results demonstrated that a granular,
cytoplasmic staining pattern for AMACR was observed in
100% of papillary RCCs (15/15), but also in 68.6% of
conventional (clear cell) RCCs (48/70), in 29% of chromo-
phobe RCCs (2/7), and in 25% of oncocytomas (2/8) .
Among the 70 cases of clear cell RCC, positivity of
AMACR was correlated with nuclear grade . In their
series of metastatic papillary carcinomas, 82% (23/28) of
metastatic RCCs were positive for AMACR 20. However,
only 24 (13%) of 189 nonrenal malignancies were positive.
The 24 positive cases included 12 (92%) of 13 colorectal
adenocarcinomas, 6 (20%) of 30 ductal carcinomas of the
breast, and 4 (17%) of 23 adenocarcinomas of the lung .
These findings suggest that AMACR is a useful marker in
diagnosing primary and metastatic RCCs, although it has
little value in differentiating chromophobe RCCs from
More recently, Chen et al , on 63 cases of formalin-
fixed, paraffin-embedded renal tumors, found that AMACR
expression was highest in papillary RCC. Using other
markers, they established a molecular diagnostic algorithm
that accurately classified the cases into 15 in the clear cell,
16 in the papillary, and 32 in the chromophobe/oncocytoma
group, but could only separate some, but not all, oncocy-
tomas from chromophobe RCCs . All these findings
suggest that AMACR is significantly overexpressed in
papillary RCCs, with high sensitivity (100%) and high
specificity (85%), and that it could be used like many other
tumor markers in classifying renal tumors.
Our results are similar to the 87.2% of AMACR
immunostaining observed in 86 papillary RCCs from a
tissue microarray analysis that included 310 renal cell
tumors from 4 Parisian centers (personal communication
from Bazille et al, 20th European Congress of Pathology
[ECP]). This study was performed with another mono-
clonal AMACR antibody (p504s, Zeta Corporation, Sierra
Madre, Calif) and a different immunohistochemistry pro-
tocol (Ventana Nexex R).
The positivity found in our cases of mucinous, tubular,
and spindle cell tumors supports the point of view that
mucinous tubular and spindle cell carcinomas and papillary
RCCs are related. They may stem from ontogenetically si-
milar precursor cells that manifest divergent morphological
AMACR and renal cell carcinomas 701
differentiation: tubular and spindle cell tumors could be a
variant of papillary renal cell tumors [16,31,35,36]. This
confirms results recently published by Paner et al , who
observed fairly consistent expression of AMACR in 93% of
mucinous tubular and spindle cell carcinomas.
An accurate diagnosis and classification of renal tumors
is important for predicting the prognosis and designing
effective treatment [11,16,26]. Because high expression of
AMACR is found in papillary RCCs (even type 1 or 2) and
in mucinous tubular and spindle cell carcinomas of the
kidney, AMACR should be used in conjunction with other
markers in case of non-evident renal neoplasm [6,16,24,36].
This study confirms the specificity of AMACR for papillary
RCCs among the RCC subtypes. But pathologists must
remember that weak focal AMACR staining could be
present in case of clear cell and chromophobe carcinomas
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