Modern Pathology (2014) 27, 765–774
& 2014 USCAP, Inc. All rights reserved 0893-3952/14 $32.00
Renal cell carcinoma with smooth muscle
stroma lacks chromosome 3p and VHL
alterations
Guido Martignoni1,2, Matteo Brunelli1, Diego Segala1,2, Stefano Gobbo1,2, Ioana Borze3,
Lilit Atanesyan4, Suvi Savola4, Luisa Barzon5, Giulia Masi5, Regina Tardanico6,
Shaobo Zhang7, John N Eble7, Marco Chilosi1, Tom Böhling3, Liang Cheng7,
Brett Delahunt8 and Sakari Knuutila3
1Department
of Pathology and Diagnostics, University of Verona, Verona, Italy; 2Anatomia Patologica,
Pederzoli Hospital, Peschiera, Verona, Italy; 3Department of Pathology, Hartmann Institute and HUSLab,
University of Helsinki, Helsinki, Finland; 4MRC-Holland, Amsterdam, Netherlands; 5Department of
Histology, Microbiology and Medical Biotechnologies, University of Padua, Padua, Italy; 6Department of
Anatomic Pathology, Spedali Civili, Brescia, Italy; 7Department of Pathology and Laboratory Medicine,
Indiana University School of Medicine, Indianapolis, IN, USA and 8Department of Pathology and Molecular
Medicine, Wellington School of Medicine and Health Sciences, University of Otago, Wellington, New Zealand
Renal cell carcinoma with prominent smooth muscle stroma is a rare neoplasm composed of an admixture of
epithelial cell with clear cytoplasm arranged in small nest and tubular structures and a stroma composed of
smooth muscle. In the epithelial component, loss of chromosome 3p detected by fluorescence in situ
hybridization (FISH) has been reported and on this basis these neoplasms have been viewed as variants of clear
cell renal cell carcinoma. To test the validity of this classification, we have evaluated the chromosome 3 and VHL
status of three of these tumors using FISH, array comparative genomic hybridization, gene sequencing, and
methylation-specific multiplex ligation-dependent probe amplification analysis. None of the tumors showed
deletion of chromosome 3p, VHL mutation, a significant VHL methylation, or changes in VHL copy number and all
three tumors demonstrated a flat profile in the comparative genomic hybridization analysis. We conclude that
renal cell carcinoma with smooth muscle stroma should be considered as an entity distinct from clear cell renal
cell carcinoma.
Modern Pathology (2014) 27, 765–774; doi:10.1038/modpathol.2013.180; published online 8 November 2013
Keywords: chromosome 3p; clear cell; cytokeratin 7; 34bE12; renal cell carcinoma with smooth muscle stroma; VHL
In 2006, Kuhn et al1 described five cases of what
appeared to be clear cell renal cell carcinoma, Fuhrman grade 2, associated with a peculiar stromal
proliferation having angioleiomyoma-like features
which were particularly prominent at the interphase
between the edge of the tumor and the surrounding
renal parenchyma. They postulated that the
angioleiomyoma-like change is an epiphenomenon
caused by the overproduction of hypoxia-inducible
factor, vascular endothelial growth factor, and other
growth factors by the neoplastic epithelial cells in
Correspondence: Professor G Martignoni, MD, Anatomia Patologica, Università di Verona, Piazzale L. Scuro n. 10, Verona 37134,
Italy.
E-mail: [email protected]
Received 15 January 2013; revised 17 June 2013; accepted 7
August 2013; published online 8 November 2013
www.modernpathology.org
which the inactivation of the Von Hippel-Lindau
gene (VHL) located on the short arm of the
chromosome 3 had occurred.1
Loss of heterozygosity of chromosome 3p has been
frequently shown in both sporadic and hereditary
clear cell renal cell carcinomas2,3 and the VHL gene
has been found to be inactivated by several
mechanisms, including deletions and abnormal
DNA methylation.4–6 Several other recurrent copy
number changes, such as gains of 5q, have been
reported in clear cell renal cell carcinomas.7–9 In
2009, Shannon et al10 described three more of these
tumors and found that they were extensively
positive for cytokeratin 7 by immunohistochemistry. They also performed fluorescence in situ
hybridization (FISH) analysis of chromosome 3
and found loss of the entire chromosome in two
tumors and loss of 3p in the third case. They
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G Martignoni et al
concluded that these neoplasms are low-grade
variants of clear cell renal cell carcinoma, which
have induced a metaplastic stromal reaction.10
In this study, we evaluated the morphological,
immunohistochemical, and molecular features of
three additional examples of these tumors, focusing
on the status of chromosome 3p and the VHL gene
using FISH, methylation-specific multiplex ligationdependent probe amplification analysis, and sequencing analyses of the entire VHL gene. In addition,
chromosomal copy number changes were analyzed
using array comparative genomic hybridization to
examine genome-wide changes and exclude the
presence of recurrent copy number changes, such
as losses of 3p and 5q gains, which are known to
occur frequently in clear cell renal cell carcinoma.
Materials and methods
Patient Recruitment
Three renal cell carcinomas with smooth muscle
stroma were recruited from the kidney tumor
database of the Department of Pathology, University
of Verona (GM) on the basis of their morphological
features. From 2 to 5 paraffin-embedded blocks per
case were available. Sections of 3-mm thick were cut
from tissue blocks and stained with hematoxylin
and eosin and reviewed by three pathologists (GM,
MB, and SG).
Immunohistochemical Analysis
The immunohistochemical profile was investigated
using a panel of antibodies including: CD10 (Novacastra, Burlingame, CA, USA; clone 56C6; 1:10
dilution); cytokeratin 7 (Biogenex, Fremont, CA,
USA; clone OV-TL 12/30; 1:400 dilution); cytokeratin 5 (Novacastra; clone XM26; 1:100 dilution);
cytokeratin 34bE12 (Dako, Carpinteria, CA, USA;
clone 34bE12; 1:40 dilution); cytokeratin AE1/AE3
(Dako; clone AE1/AE3; 1:100); parvalbumin (Sigma
Chemical Company, St Louis, MO, USA; clone P19;
1:400 dilution); SLC2A1 (GLUT1) (Dako; polyclonal,
rabbit; 1:100 dilution); vimentin (Biogenex; clone
V9; 1:50 dilution); alpha-methylacyl-CoA racemase
(P504S) (Dako; clone 13H7; 1:50 dilution); S100A1
(Dako; clone S100A1/1; 1:50 dilution); desmin
(Dako; clone D33; 1:500 dilution); a-smooth muscle
actin (Dako; clone 1A4; 1:250 dilution); caldesmon
(Dako; clone h-CD); androgen receptor (Dako; clone
AR441; 1:20 dilution); estrogen receptor (Dako; 1:20
dilution), progesterone receptor (Dako; clone PgR
636; 1:20 dilution); HMB45 (Dako; clone HMB45;
1:300 dilution) and cathepsin K (Abcam, Cambridge,
UK; clone 3F9; 1:2000 dilution). Immunoreactions
were developed using a non-biotin, highly sensitive
system (Envision peroxidase detection system,
Dako) preventing possible false-positive staining
owing to endogenous biotin present in the tissue.
Modern Pathology (2014) 27, 765–774
Normal renal parenchyma adjacent to the tumors
was used as a control.
Array Comparative Genomic Hybridization and Data
Analysis
Microdissection from the three formalin-fixed paraffin-embedded kidney tumors was performed
manually, with accurate attention in obtaining
material from the epithelial zones of tissue slides.
Genomic DNA was isolated using the QIAamp
DNA mini kit (Qiagen Nordic, Finland) and quantified on the NanoDrop spectrophotometer (NanoDrop
Technologies Inc, DE, USA). As a reference, we used
DNA from pooled peripheral blood leukocytes of
healthy males. Using the Agilent Human 244K array
format containing B240 000 oligonucleotide probes,
covering both coding and non-coding genome regions (Agilent Technologies, Santa Clara, CA, USA),
we screened copy number alterations in all three
tumors. Briefly, 1.5 mg of tumor and reference DNA
were digested, labelled, and hybridized according to
the Agilent protocols. The array images obtained
after scanning (Agilent scanner G2565BA) were
processed with the Feature Extraction software
(version 10.5), and the output data files were
analyzed with the Agilent Genomic Workbench. To
identify copy number alterations, we used aberration
detection method 2 (ADM-2) algorithm and to
exclude the small variances in the data, we set up
a custom aberration filter identifying alterations in
copy number if a minimum of eight probes gained or
lost were identified and with a minimum absolute
average log ratio for the region being 0.5. Regions
with small copy number variations were excluded by
comparing and visualizing the copy number variant
regions tool of the Genomic Workbench software.
Fluorescence in situ Hybridization
FISH analysis was performed using a centromericspecific probe for the chromosome 3 centromere
(SpectrumGreen CEP3; Abbott) and a subtelomeric
probe for 3p25 (SpectrumGreen 3p-LSI; Abbott) to
evaluate 3p deletion. From the whole-tissue sections, 3-mm sections were cut from paraffin-embedded blocks. The paraffin was removed from the
sections with two 10-min washes with xylene. After
hydrating in 100, 85, and 70% ethanol solutions
(10 min), rinsing in distilled water (10 min), and
twice in phosphate-buffered solution (pH 7, 10 min
each), the slides were fixed in methanol–acetic acid
3:1 for 10 min and air-dried. Next, the sections were
treated in a 2 XSSC (standard saline citrate) solution
for 15 min at 37 1C, and then dehydrated in consecutive 70, 85, and 100% ethanol solutions for
1 min each and then dried. Next, the sections were
bathed in 0.1 mM citric acid (pH 6) solution at 85 1C
for 1 h. Then, they were again dehydrated in a series of
ethanol solutions and dried. The tissue was digested
Renal cell carcinoma with smooth muscle stroma
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G Martignoni et al
by applying 0.75 ml of pepsin (Sigma) solution
(4 mg/ml in 0.9% NaCl, pH 1.5) to each slide and
incubating them in a humidified box for 30 min at
37 1C. Next, the slides were rinsed with distilled water
for a few seconds, dehydrated again in graded ethanol
solutions, and dried. Centromeric probes for chromosomes 3 and the locus-specific sub-telomeric probe 3p
were used. Each probe was diluted 1:20 in t-DenHyb-2
buffer (LiStar-FISH, Milan, Italy). Ten microliters of
diluted probe was applied to each slide and coverslips
were placed over the slides. Denaturation was
achieved by incubating the slides at 80 1C for 10 min
in a humidified box, and then hybridization was
carried out at 37 1C for 16 h. The coverslips were later
removed and the slides were immersed at room
temperature in 0.5 XSSC for 2 min, in 50% formamide/1 XSSC for 5 min, and in 2 XSSC for 2 min. The
slides were air-dried and counterstained with 10 ml of
DAPI/Antifade (DAPI in Fluorguard, 0.5 mg/ml; Insitus). The slides were examined using an Olympus
BIX-61 microscope (Olympus, Hamburg, Germany)
with filters for SpectrumGreen, and the UV Filter for
the DAPI nuclear counterstain. The signals were
recorded with a CCD camera (Olympus Digital
Camera). Fluorescent signals were evaluated as reported previously.11-13 Signals from 100 to 200 nuclei
were counted, focusing only on neoplastic nuclei from
the epithelial components. The control distribution
of signals was assessed on non-neoplastic renal
parenchyma adjacent to the tumors. The value of the
ratio (3p/3) of the normal renal parenchyma þ 3s.d.
set to 1.03 þ 3s.d. 0.05 ¼ 1.19. þ 3s.d. was used for
each fluorescent score number.
VHL Sequencing Analysis
Five 10-mm thick sections of tumor tissue were cut
from formalin-fixed paraffin-embedded blocks.
Epithelial and smooth muscle components were
separated by manual microdissection. DNA was
extracted separately from each component. PCR for
the VHL gene analysis was performed using primer
sequences from the literature.14,15 Normal tissues of
the same patients were used as a reference. The
reaction conditions were as follows: 12.5 ml of
HotStart Taq PCR Master Mix (QIAgen, Hilden,
Germany), 10 pmol of each primer, 100 ng of
template DNA, and distilled water up to 25 ml.
Amplification program for all fragments, except the
marker D3S666, consisted of denaturation at 95 1C
for 15 min, then 40 cycles of denaturation at 95 1C
for 1 min, annealing at 55 1C for 1 min, and extension
at 72 1C for 1 min. The program was finished by
72 1C incubation for 7 min. Annealing temperature
for fragment D3S666 was 58 1C. PCR products of the
VHL gene were purified with Montage PCR Centrifugal Filter Devices (Millipore, Billerica, MA, USA)
and sequenced using a Big Dye Terminator
Sequencing kit (PE/Applied Biosystems, Foster
City, CA, USA). Samples were then run on an
automated sequencer ABI Prism 310 (PE/Applied
Biosystems) at a constant voltage of 11.3 kV for
20 min. PCR products of STR markers were mixed
with a size marker and run on an automated
sequencer ABI Prism 310 (PE/Applied Biosystems)
at a constant voltage of 15 kV for 28 min.
Genomic DNA was isolated from three 5 mM-thick
paraffin sections of each renal carcinoma sample
using the Ex-Wax DNA Extraction Kit (Chemicon
International, Temecula, CA, USA) according to the
manufacturer’s instructions.
Bidirectional sequencing of PCR products was
performed by using an ABI PRISM BigDye terminators v3.1 cycle sequencing kit (Applied Biosystems),
and sequences were run on an Applied Biosystems
3130 Genetic Analyzer (Applied Biosystems) and
compared with the reference sequence CCDS 2597.1.
The PCR amplicon carrying the mutation was
subcloned into a pGEMs-T Easy vector (Promega,
Madison, WI, USA), transformed in competent
DH5a cells and plated onto LB agar with ampicillin
and X-gal selection. Then, 12 distinct blank (white)
colonies were picked, plasmid DNA was extracted
and submitted to amplification and sequencing of
VHL exon 3 as above described.
Methylation-Specific Multiplex Ligation-Dependent
Probe Amplification
Microdissection from the three formalin-fixed
paraffin-embedded kidney tumors was manually
performed, with accurate attention in obtaining
material from the epithelial zones of tissue slides.
Genomic DNA extracted from the three samples
was subjected to MS-MLPA using ME001-0808-C1
and ME002-0809-B1 probemixes (MRC-Holland,
Amsterdam, The Netherlands) with 20–100 ng of
DNA per sample. The standard MS-MLPA protocol,
which was used here, has been described.16 Both
probemixes contain one specific MLPA probe for the
exon 1 of VHL gene that has a recognition site for a
CpG methylation-sensitive endonuclease HhaI. The
MS-MLPA product fragments were analyzed by ABI
model 3130 capillary sequencer (Applied Biosystems, Nieuwerkerk aan den Ijssel, The Netherlands)
using Genescan-ROX 500 size standards. As the
same probemixes are intended to detect simultaneously both copy number and methylation changes
of the target genes, both methylation and copy
number status were analyzed using the Coffalyser
software (MRC-Holland, Amsterdam, The Netherlands).17 The data were first normalized by dividing
the peak area of a single probe by a cumulative peak
area of all control probes (not degraded by HhaI).
Then, the normalized peaks from the HhaI digestion
reaction were compared with the normalized peaks
from the undigested control reaction. Final
methylation value for each sample was obtained by
subtracting the background methylation values of
the control samples (male and female DNA samples;
Promega). The following criteria were used for
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Figure 1 Gross and microscopic features of renal cell carcinoma with smooth muscle stroma. The tumors were well circumscribed, tanbrownish semitraslucent in appearance and surrounded by an irregular pseudocapsule; (a) fresh cutting surface of case 2; (b) formalinfixed appearance of case 3. (c, d) The neoplasm displayed an admixture of epithelial cells with clear cytoplasm and a stromal
proliferation composed of mature smooth muscle cells, organized in bundles (case 1); (e, f) the tumor showed the epithelial component
with clear cytoplasm and hyperchromatic nuclei (Fuhrman grade 2), arranged in nests, cords, and small cysts (case 2); (g) the stromal
proliferation was composed of mature smooth muscle cells organized in bundles (case 3); (h) the tumor showed a large focus of stromal
osseous metaplasia and a focal microcystic pattern (case 3).
determining the methylation status: 0.00–0.25
(absent), 0.25–0.50 (mild), 0.50–0.75 (moderate), and
40.75 (extensive methylation). For copy number
analysis, the following cutoff values were used:
o0.7 and 41.3 gain. One DNA sample (labelled
8656) was excluded from the analysis due to the low
amount of DNA and for another sample (labelled
10684) we were able to include only results using
ME002-0809-B1 probemix as no DNA was available
for the further analysis using the ME-001-0808 mix.
Results
Clinical and Pathological Findings
The three patients (all females) were respectively 79,
74, and 74 years old. The tumors presenting a
Modern Pathology (2014) 27, 765–774
diameter ranging from 2 to 3 cm and were all staged
as pT1a N0 M0 (according to AJCC, 2010). Two
neoplasms were treated with simple enucleation of
the tumor and the third was treated with nephrectomy. All patients were well and alive after 52
months (case 1), 30 months (case 2). and 27 months
(case 3).
The three tumors showed similar gross and
microscopic features. They were well circumscribed, tan or brownish, somewhat translucent,
and surrounded by an irregular pseudocapsule
(Figures 1a and b). One of them showed focal cystic
changes. Necrosis was not identified. Histologically,
the neoplasms were composed of a mixture of two
distinct components (Figures 1c and d): the epithelial cells with clear cytoplasm and hyperchromatic
nuclei (Fuhrman grades 1 and 2), arranged in nests,
cords, and small cysts (Figures 1e and f) and a
Renal cell carcinoma with smooth muscle stroma
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G Martignoni et al
Figure 1 (Continued)
stromal proliferation composed of mature smooth
muscle cells (Figure 1g), organized in bundles and
merging with the peripheral discontinuous pseudocapsule. The percentage of tumor volume occupied
by the stromal component was respectively of 20%
(case 1), 40% (case 2), and 30% (case 3). In case 3,
there was a large focus of stromal osseous metaplasia (Figure 1h). Neither necrosis nor mitotic activity
was observed in either of the components.
Immunohistochemical Findings
The epithelial component of all three tumors showed
diffusely positive reactions with antibodies to cytokeratin 7 (100, 80, and 90% of the cells) (Figure 2a)
and cytokeratin AE1-AE3 (100% of the cells in all
three tumors). The reactions with antibody to high
molecular weight cytokeratin 34bE12 were positive in
all three tumors, with the percentage of positive cells
ranging from 10 to 60% (Figure 2b). The reaction
with antibody to carbonic anhydrase IX was positive
in two tumors, sometimes showing lack of labelling
on the luminal aspect of individual cells (a ‘cuplike’
staining pattern). All of the tumors gave positive
reactions with antibody to SLC2A1 (GLUT1)
(Figure 2c). The reaction for CD10 was focally positive
in one tumor, while two tumors gave positive reactions with antibody to S100A1. None of the tumors
gave positive reactions for alpha-methylacyl-CoA
racemase (P504S), cytokeratin 5, parvalbumin, or
vimentin. The reactions with antibodies to progesterone, estrogen and androgen receptors, as well as
HMB45 and cathepsin K were also negative.
The stromal component of the tumors showed
strong and diffusely positive reactions with antibodies to a-smooth muscle actin (Figure 2d) and
caldesmon (100% of the cells). Antibody to desmin
gave a positive reaction only in 30% of the cells in
one of the tumors.
Array Comparative Genomic Hybridization Findings
We did not observe any gene copy number abnormalities. A flat profile was observed in each tumor
(Table 1; Figure 3).
Fluorescence In Situ Hybridization Findings
For chromosomes 3 and the locus-specific subtelomeric chromosome 3p, the percentages of single
Modern Pathology (2014) 27, 765–774
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G Martignoni et al
Figure 2 Immunohistochemical characters of renal cell carcinoma with smooth muscle stroma. High power magnification of (a)
cytokeratin 7, (b) cytokeratin 34bE12, and (c) SLC2A1 (GLUT1) immunoexpression in the epithelial component of case 1; (d) positivity of
a-smooth muscle actin in pseudocapsule and stromal component of case 2.
Table 1 Molecular findings of the three cases of renal cell
carcinoma with smooth muscle stroma, using three different
methods investigating chromosomal imbalances (aCGH), chromosome 3p status (FISH), and VHL status (gene sequencing)
Case
Chromosomal
imbalances
3p
status
VHL
status
1
2
3
Normal status
Normal status
Normal status
Disomy
Disomy
Disomy
WT
WT
WT
signals greater than 42 and 41% were respectively
considered to indicate loss; scoring triple signals
greater than 9 and 11% respectively were considered to indicate gain.
Centromeric chromosome 3 probe. Case 1 showed
single, double, and three or more fluorescent signals
in respectively 35, 62, and 3% of neoplastic
Modern Pathology (2014) 27, 765–774
epithelial nuclei; case 2 showed single, double,
and three or more fluorescent signals in respectively
32, 63, and 5% of neoplastic epithelial nuclei; case 3
showed single, double, and three or more fluorescent signals in respectively 21, 72, and 7% of
neoplastic epithelial nuclei.
Locus-specific sub-telomeric 3p probe. Case 1
showed single, double, and three or more fluorescent signals in respectively 39, 60, and 1% of
neoplastic epithelial nuclei; case 2 showed single,
double, and three or more fluorescent signals in
respectively 40, 57, and 3% of neoplastic epithelial
nuclei; case 3 showed single, double, and three or
more fluorescent signals in respectively 31, 61, and
5% of neoplastic epithelial nuclei.
The value of the ratio on the normal renal
parenchyma þ 3s.d set to 1.03 þ 3s.d. 0.05 ¼ 1.19.
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Table 2 Percentages of nuclei with different numbers of signals and ratio 3/3p from neoplastic cells and results for 3p deletion analysis
Case
1
2
3
CEP3
Locus 3p25
3p
1 Signal
(%)
2 Signals
(%)
Z3 Signals
(%)
1 Signal
(%)
2 Signals
(%)
Z3 Signals
(%)
Signals
CEP3 /Signals 3p25
35
32
21
62
63
72
3
5
7
39
40
31
60
57
61
1
3
5
1.04
1.07
1.11
Results
Not Deleted
Not Deleted
Not Deleted
Table 3 Methylation status and gene copy number of the three
cases of renal cell carcinoma with smooth muscle stroma
Methylation status
Copy number
Gene (map view position)
Case
1
2
3
VHL (03010158426)
VHL (03010158544)
VHL (03010158426)
VHL (03010158544)
n.a.
0.00
0.00
0.33
0.15
0.00
n.a.
1.10
1.36
0.76
1.19
1.39
Ratio was respectively 1.04 in case 1 (not deleted),
1.07 in case 2 (not deleted), and 1.11 in case 3 (not
deleted). The details of the FISH analyses are
presented in Table 2.
VHL Gene Mutation
No mutation of the coding sequence of the VHL gene
was found in either the epithelial or smooth muscle
cell components of any of the tumors (Table 1).
Methylation-Specific Multiplex Ligation-Dependent
Probe Amplification Findings
Methylation-specific ligation-dependent probe amplification analysis of formalin-fixed paraffin-embedded tissue DNA samples showed absent or mild
methylation of the VHL gene (Table 3). In one tumor,
it detected a gain of VHL gene copy number (Table 3).
Discussion
Figure 3 Molecular findings of renal cell carcinoma with smooth
muscle stroma. (a) A flat profile for chromosome 3p by aCGH was
observed in all cases. (b) FISH lacks to find any alteration of
chromosome 3p (case 1); (c) no mutation of coding sequence of
the VHL gene was found by sequencing analysis.
In the three tumors which we studied, we found that
deletion of chromosome 3p, VHL mutation, and a
significant VHL methylation which are molecular
hallmarks of clear cell renal cell carcinoma are
lacking in renal cell carcinoma with smooth muscle
stroma and that all three tumors demonstrated a flat
profile by array comparative genomic hybridization
analysis and failed to show recurrent copy number
changes such as 5q gains, which have been reported
in clear cell renal cell carcinoma. Although the
small size of the study precludes any statistical
analysis, we conclude on the basis of our observations that these tumors should be considered as a
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type of renal cell tumor distinct from the common
clear cell renal cell carcinoma.
Clear cell renal cell carcinoma represents the most
common type of renal cell carcinoma, comprising
60% of all renal tumors. It usually presents as a
yellowish neoplasm composed by cells with clear
cytoplasm, due to their high lipid content, arranged
in a solid or acinar growth pattern, with the clear
cells separated by thin hypervascular, fibrous septa.18 Clear cell renal cell carcinoma typically is
positive for cytokeratin 8-18, vimentin, CD10,
epithelial membrane antigen, RCC marker, and
PAX8 and PAX2 and negative for cytokeratin 7,
alpha-methylacyl-CoA racemase (P504S) and
cathepsin K.19–23 It displays a diffuse membranous
staining for carbonic anhydrase IX,24,25 reflecting
inactivation of the VHL gene and constitutive
activation of the hypoxia-inducible factor pathway.
A deletion on chromosome arm 3p, where the VHL
gene resides, is present in most sporadic and familial
clear cell renal cell carcinoma and recently,
Nickerson et al4 identified VHL gene mutations in
82% of clear cell renal cell carcinomas whereas an
additional 8% showed hypermethylation in the VHL
promoter CpG islands. The VHL gene product,
pVHL, regulates transcription of genes through HIF.
Normal pVHL targets HIF1-a for degradation in
normoxemic states. When the VHL gene is mutated
or the pVHL is absent, conditions of hypoxia are
simulated and HIF1-a accumulates. HIF1-a activates
multiple downstream genes, including vascular
endothelial growth factor, the glucose transporter
SLC2A1 (GLUT1), and carbonic anhydrase IX.24–28
The three cases of renal cell carcinomas with
smooth muscle stroma analyzed in this study did
not reveal the 3p deletion, nor VHL mutations, nor
VHL methylation abnormalities that are frequent in
clear cell renal cell carcinomas. Further, all of them
demonstrated a flat profile by comparative genomic
hybridization analysis and lacked other recurrent
copy number changes such as 5q gains which have
been reported in clear cell renal cell carcinoma.
Therefore, their molecular characters differ from
those usually observed in clear cell renal cell
carcinomas and from those identified in three
similar tumors by Shannon et al.10 They observed
in two tumors the concurrent losses of one signal for
VHL and of 3q, used as a control, in B40% of the
cells of the epithelial component. They interpreted
these results as indicating the loss of one entire
chromosome 3. In their third tumor, they found the
loss of one signal for VHL, but the retention of both
control signals for 3q in B70% of cells and
interpreted that to indicate the loss of 3p on just
one copy of chromosome 3. We think that the
difference between our findings and those
previously reported by Shannon et al could be due
to the different definitions of monosomy in FISH
studies of paraffin sections. Our definition was
based both on statistical distribution of double
signals for chromosome 3p/3 in the non-neoplastic
Modern Pathology (2014) 27, 765–774
renal parenchyma adjacent to each of the three
tumors and on the results obtained in previous
studies.
We observed a variable detection rate of chromosome 3p/3 abnormalities in different series of clear
cell renal carcinoma;3 loss of signals may be either
artifactual or biological. A high cutoff level overcomes the artifactual bias (nuclear truncation). Moreover, the absence of any chromosome 3 alteration in
our three tumors when investigated by array comparative genomic hybridization confirms our FISH
results. By using aCGH analysis, we established a
custom aberration filter identifying alterations in
copy number if a minimum of eight probes gained
or lost were identified, with a minimum absolute
average log ratio for the region being 0.5. These
cutoffs do not decrease the sensitivity by being too
specific, in respect to the 240 000 oligoprobes used in
the kits.
In this study, the tumours showed small sizes and
a low stage assessment (pT1a) as previously
reported.1 Grossly, they appeared to be different
from the yellowish color characteristic of clear cell
renal cell carcinomas, being tan or brownish with a
somewhat translucent appearance. Moreover, they
constantly express diffuse CK7 and frequently
express high molecular weight cytokeratin (clone
34bE12) by immunohistochemistry, being markers
that are usually negative in clear cell renal cell
carcinoma. The absence of immunostaining for
HMB45, cathepsin K, estrogen and progesterone
receptors both in the clear cell and in the smooth
muscle components rule out any relationship
between this neoplasm and angiomyolipoma as
well as other members of the family of lesions
composed by perivascular epithelioid cells
(PEComas), MiTF/TFE family renal translocation
carcinomas and mixed epithelial–stromal tumors,
respectively.23,29–31
Finally, the immunoexpression of cytokeratin 7 in
the epithelial component of renal cell carcinoma
with smooth muscle stroma and the detection of
high molecular weight cytokeratin with antibody
clone 34bE12, which are usually undetectable in
clear cell renal cell carcinoma, are of interest.
These lend support to our conclusion that renal
cell carcinoma with smooth muscle stroma is a type
of renal cell neoplasm distinct from clear cell
renal cell carcinoma. Further, together with our
genetic findings, they also suggest that these tumors
could be related to clear cell tubulopapillary renal
cell carcinoma (and the synonymous or closely
related renal angiomyoadenomatous tumor (RAT)),
in which smooth muscle metaplasia within intratumoral stroma has been described.32–40
Acknowledgments
We thank Fondazione Cassa di Risparmio di Verona
(Unicredit); Diagnostica Molecolare in Oncologia;
Renal cell carcinoma with smooth muscle stroma
773
G Martignoni et al
Ministero Istruzione, Università e Ricerca. This
work has been accepted in part for presentation at
the 98nd Annual Meeting of the United States and
Canadian Academy of Pathology (USCAP), Boston,
MA, 7–13 March 2009.
Disclosure/conflict of interest
15
16
The authors declare no conflict of interest.
17
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