RESEARCH ARTICLE
Cytogenetic analysis of 130 renal oncocytomas identify
three distinct and mutually exclusive diagnostic classes
of chromosome aberrations
Christopher B. Anderson
1
| Michael Lipsky
1
| Subhadra V. Nandula
2
|
Christopher E. Freeman
3
| Thomas Matthews
3
| Caitlin E. Walsh
3
| Gen Li
4
|
Matthias Szabolcs
3
| Mahesh M. Mansukhani
3
| James M. McKiernan
1
|
Vundavalli V. Murty
3
1
Department of Urology, Columbia University
Irving Medical Center, New York, New York
2
Cancer Genetics, Inc., Rutherford, New Jersey
3
Department of Pathology and Cell Biology,
Columbia University Irving Medical Center,
New York, New York
4
Department of Biostatistics, Columbia
University Irving Medical Center, New York,
New York
Correspondence
Vundavalli V. Murty, Department of Pathology
and Cell Biology, Columbia University Irving
Medical Center, CHC 409, 3959 Broadway,
New York, NY 10032
Email: vvm2@cumc.columbia.edu
Abstract
The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We ana-
lyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities
were identified in 63 (49%) cases, which could be categorized into three classes of
mutually exclusive cytogenetic categories. Class 1 (N = 20) RO had diploid karyotypes
with characteristic 11q13 rearrangement in balanced translocations with 10 or more
different chromosome partners in all cases. We identified recurrent translocation part-
ners at 5q35, 6p21, 9p24, 11p13-14, and 11q23, and confirmed that CCND1 gene
rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO
(N = 25) exhibited hypodiploid karyotypes with loss of chromosome 1 and/or losses of
Y in males and X in females in all cases. The class 3 tumors comprising of 18 cases
showed diverse types of abnormalities with the involvement of two or more chromo-
somes exclusive of abnormalities seen in classes 1 and 2 tumors. Furthermore, karyo-
typically uninformative cases were subjected to FISH analysis to identify classes 1 and
2 abnormalities. In this group, we found similar frequencies of CCND1 rearrangement,
loss of chromosome 1 or Y as with karyotypically abnormal cases. We validated our
results against 91 tumors from the Mitelman database. Correlation of clinical data with
all the three classes of ROs showed no clear evidence of overall patient survival. Our
findings support the hypothesis that RO exhibit three principal cytogenetic categories,
which may have different roles in initiation and/or progression. These cytogenetic
markers provide a key tool in the diagnostic evaluation of RO.
KEYWORDS
11q13 rearrangement, cyclin D1, cytogenetics, loss of chromosome 1, renal oncocytoma
1 | INTRODUCTION
Renal oncocytoma (RO) accounts for 3% to 5% of all adult renal
tumors.
1
RO with male predominance arises from intercalated cells of
the collecting system and presents as both sporadic and familial forms.
Oncocytomas are the most frequent renal tumors in patients with the
Birt-Hogg-Dube syndrome caused by mutations in the folliculin gene.
2
However, the oncogenic etiology of sporadic RO is not well understood.
Received: 2 March 2019 Revised: 15 May 2019 Accepted: 15 May 2019
DOI: 10.1002/gc c.22766
Genes Chromosomes Cancer. 2019;1–7. wileyonlinelibrary.com/journal/gcc © 2019 Wiley Periodicals, Inc. 1
Clinically, RO are benign tumors typically occur as solitary lesions. His-
tological features of RO are usually distinctive but can overlap chromo-
phobe renal cell tumors.
3
Because chromophobe renal cell carcinoma
(RCC) is a malignant tumor, differentiating RO from chromophobe is
clinically important.
Only about 100 cases of RO with conventional karyotype have
been reported in the literature (https://cgap.nci.nih.gov/Chromosomes/
CytList).
4
Most of these studies were either single case reports or small
case series, which identified loss of chromosomes 1, Y, and 14, and
11q13 rearrangements as recurrent chromosome aberrations. However,
the frequency of recurrent cytogenetic changes and their relation with
each other has not been systematically examined.
We sought to identify recurrent chromosomal changes in RO by
analyzing 63 karyotypically abnormal cases. We identified three mutu-
ally exclusive karyotype classes of ROs. Frequency of chromosome
changes in the order of their occurrence was 11q13 rearrangement,
loss of chromosome 1, loss of Y in males and loss of X in females, tri-
somy 7, and loss of chromosomes 14, 21, and 22. The present findings
can be useful in differential diagnosis of RO and in understanding the
role of genetic mechanisms in their pathogenesis.
2 | MATERIALS AND METHODS
2.1 | Patient cohort
A total of 130 consecutive RO specimens were subjected to karyo-
type analysis for diagnostic purposes at Columbia University Medical
Center, New York, between 1999 and 2016. All patients had a radical
or partial nephrectomy for a renal mass. We performed chart review
to obtain clinical and histologic information on all the patients.
2.2 | Karyotype and fluorescence in situ
hybridization analyses
We routinely perform karyotype and fluorescence in situ hybridization
(FISH) analyses of all surgically removed renal m asses at our institution.
Touch imprints were made and fixed immediately in methanol-acetic acid
in majority of cases for FISH testing. Chromosome analysis was per-
formed using standard methods. Briefly, fresh tumor specimens collected
after surgery were washed with antimycotic solution (0.5 μg/mL
Amphotericin B) and antibiotics (200 IU/mL penicillin and 200 μg/mL
streptomycin), minced with scalpel blade into fine pieces, and digested
2-12 hours with collagenase (200 U/mL) in complete Dulbecco's Modi-
fied Eagle's Medium (DMEM medium). Dissociated cells were washed
twice and then cultured in complete DMEM medium supplemented with
epidermal growth factor, insulin-transferrin-sodium selenate (Invitrogen).
The cultures were monitored daily and appropriately confluent cells were
subjected to metaphase preparations using standard methods after addi-
tion of colcemid for 12 hours. Metaphase preparations were subjected
to Giemsa (GTG) banding and analyzed by standard methods. Karyotypes
were described by the Standard Cytogenetic Nomenclature. FISH was
performed either on touch imprints or cells processed for karyotype anal-
ysis using CCND1/CEP 11, CCND1 break apart, chromosome
enumeration probes CEP X, CEP Y, CEP 1, and CEP 7 (Vysis, Downers
Grove, IL) by standard methods. The cyclin D1 locus at 11q13 is labeled
in SpectrumOrange fluorochrome in CCND1/CEP 11 cocktail spanning a
378-kb physical distance equally covering on both 5
0
and 3
0
ends of the
gene. When 11q13 rearrangement occurs, there is a spit in orange signal
while SpectrumGreen labeled chromosome 11 serve as control to enu-
merate chromosome number. Dual color break apart CCND1 probe con-
sists of two probes flanked by a centromeric 700-kb probe labeled with
SpectrumGreen and a telomeric 500-kb probe labeled with
SpectrumOrange. This probe shows as orange-green signals together
due to their close proximity in normal chromosome 11 and orange signal
separated from green signal when 11q13 (CCND1) rearranged.
2.3 | Data analysis
Mitelman Database of Chromosome Aberrations and Gene Fusions in
Cancer relates chromosome aberrations to tumor characteristics based
on individual cases or associations. All ROs tumors reported until April
2016 in this database were retrieved by case quick searcher (http://
cgap.nci.nih.gov/Chromosomes/Mitelman).
4
The database includes
91 karyotypically abnormal RO cases, excluding our previously publi-
shed cases.
5
Based on chromosomal abnormalities, we identified sub-
groups of patients. Pearson Chi-squared test and analysis of variance
was performed to evaluate associations between clinical and demo-
graphic features, and different genetic characteristics.
3 | RESULTS
Of the 130 tumor specimens subjected to karyotype analysis,
63 (49%) cases showed clonal chromosome abnormalities which
include 11 previously reported cases.
5
Normal karyotypes were found
in 30 (23%) cases and 37 (29%) were failed to yield metaphases. The
clinical, histologic, and cytogenetic features of karyotypically abnormal
cases are shown in Table S1. Nearly all tumors (61 of 63) exhibited
near-diploid range or pseudo-diploid karyotypes in their stem-line,
while one had triploid karyotype and the other had 53 chromosomes.
Ten tumors showed evidence for whole genome doubling (WGD). In
general, RO exhibited simple karyotypes (less than four aberrations)
with numerical loss of one or two chromosomes or 11q13
rearrangement. The most frequent chromosome aberration was the
loss of a sex chromosome in 24 (38%) cases (-Y in 20/44, 46% males;
-X in 4/19, 21% females). Other recurrent chromosomal abnormalities
include loss or partial deletion of chromosome 1 (40%), 11q13
rearrangement (32%), trisomy 7 (18%), monosomy 22 (11%), mono-
somy 14 (10%), and monosomy 21 (10%) (Table 1).
3.1 | Chromosome aberrations identify three
mutually exclusive karyotypic classes of ROs
The types of clonal chromosome abnormalities identified were allowed
us to classify three karyotypically distinct classes of ROs (Table 2,
Figure 1). Class 1 tumors showed 11q13 translocation involving
2 ANDERSON ET AL.
10 known and two unknown chromosome partners in all 20 tumors.
Both recurrent (6p21 four cases, 5q35 and 9p24 in three cases each,
and 11q23 in two cases) and nonrecurrent (1p13, 4q27, 6q13, 7q11.2,
11p14, 15q21, and two unidentifiable chromosome regions) transloca-
tion partners were identified (Table 2, Figure 2). Seven (four with single
abnormalities and three with two or more abnormalities) of the class
1 RO with 11q13 rearrangement also exhibited other chromosome
abnormalities. These additional abnormalities were in the main line in
four cases and three with loss of Y in subclones. However, no recurrent
copy number changes or structural chromosome rearrangements were
seen among the additional abnormalities. Evidence for WGD was seen
in 2 of 20 (10%) tumors.
All 25 tumors in class 2 showed chromosome 1 abnormalities.
Twenty-three tumors showed loss of chromosome 1, and two tumors
showed deletion 1p21 and 1p22-p34, respectively. Among the other
changes associated with monosomy 1 were loss of Y chromosome in
12 of 17 (71%) males and loss of X chromosome in 3 of 8 (38%) female
patients. Other recurrent abnormalities in this group were trisomy
7 and monosomy 14 in six cases each, and loss of chromosomes 21 and
22 in five cases each (Table 2, Figure 1). Five cases showed additional
isolated single chromosome abnormalities and three cases exhibited
additional complex abnormalities in the form of chromosome gains or
structural rearrangements associated with loss of chromosome 1. How-
ever, no evidence of recurrent structural rearrangements could be iden-
tified among these additional changes. Evidence for WGD was found in
8 of 25 (32%) tumors.
Class 3 ROs consisted of 18 cases that had neither 11q13 translo-
cations nor chromosome 1 abnormalities. This group exhibited diver-
gent types of chromosome abnormalities either as simple chromosome
changes in 13 cases or complex chromosome abnormalities in 5 cases.
The only recurrent numerical abnormalities were gain of chromosome
7 and loss of sex chromosomes in five cases each (Table 2, Figure 1).
Among the structural abnormalities, 2q37 rearrangements were seen in
three cases and telomeric associations in three cases. No evidence of
WGD was seen in this group.
3.2 | FISH analysis of recurrent chromosome
changes in classes 1 and 2 ROs
We validated the karyotypic changes of loss of chromosomes 1, Y, X,
and 11q13 (CCND1) rearrangements by FISH. We tested 42 of 63 kar-
yotypically abnormal cases by FISH based on the availability of mate-
rials. CCND1 rearrangement was found in all 15 class 1 RO cases with
11q13 translocation. None of the 10 class 2 or 3 RO lacking 11q13
translocation were positive for CCND1 rearrangement. This high con-
cordance between karyotype and FISH suggests that karyotype is reli-
able in identifying 11q13 translocations. Loss of Y chromosome was
confirmed by FISH in all six cases and loss of X chromosome in a sin-
gle case (Table 1). All five cases with loss of chromosome 1 were con-
firmed by FISH. Four cases that were karyotypic negative for loss of
Y was also negative by FISH. However, FISH analysis identified
TABLE 1 Recurrent chromosome alterations identified in oncocytoma in the present study and compared to Mitelman database
Abnormality Present study (N = 63) Mitelman database (N = 91) All (N = 154)
Loss of Y in males 20/44 (46%) 26/52 (50%) 46/96 (48%)
Loss of X in females 4/19 (21%) 8/37 (22%) 12/56 (21%)
Loss of 1 or del 1p/1q 25 (40%) 27 (30%) 52 (34%)
Trisomy 7 11 (18%) 13 (15%) 24 (16%)
Monosomy 14 6 (9.5%) 13 (15%) 19 (12%)
11q13 rearrangement 20 (32%) 14 (16%) 34 (22%)
Monosomy 21q 6 (9.5%) 8 (9%) 14 (9%)
Monosomy 22q 7 (11%) 10 (11%) 17 (11%)
TABLE 2 Comparison of types of chromosome abnormalities in
various cytogenetic classes of renal oncocytoma in the present study
and Mitelman database
Abnormality Present study Mitelman database
Class 1 N = 20 N = 16
11q13 rearrangement 20/20 (100%) 16/16 (100%)
Simple karyotype 7/20 (35%) 4/16 (31%)
Complex karyotype 3/20 (15%) 3/16 (19%)
WGD 2/20 (10%) 0
Class 2 N = 25 N = 24
Loss of 1 23 (92%) 23 (96%)
Del (1p) 2 (8.0%) 1 (4%)
-1,-Y 12/17 (71%) 16/16 (100%)
-1,-X 3/8 (38%) 5/8 (63%)
+7 6 (24%) 3/24 (13%)
-14 6(24%) 10/24 (42%)
-21 5 (20%) 6/24 (25%)
-22 5 (20%) 5/24 (21%)
Simple karyotype 22 (88%) 4/24 (17%)
Complex karyotype 3 (12%) 5/24 (21%)
WGD 8(32%) 5/24 (21%)
Class 3 N = 18 N = 51
+7 5/18 (28%) 9/51 (18%)
-Y or -X 7/18 (39%) 10/51 (20%)
Simple karyotype 13 (72%) 32/51 (63%)
Complex karyotype 5 (28%) 10/51 (20%)
WGD 0 0
Abbreviation: WGD, whole genome doubling.
ANDERSON ET AL. 3
monosomy 1 in 84% cells and loss of Y in 3% cells in a case that
showed only loss of Y by karyotype (case no. 10-048, Table S1),
suggesting that the loss of chromosome 1 clone could not be identi-
fied by karyotype analysis due likely only normal cells grew. Loss Y in
this case may be age-related chromosome alteration.
To test further the possibility of using FISH alone as a diagnostic
test, we have examined randomly selected karyotypically normal and
failure RO cases by FISH using probes for chromosomes X, Y, 1, and
CCND1. We identified 6 of 22 (27%) with CCND1 rearrangement,
6 of 15 (40%) with loss of chromosome 1, 5 of 18 (28%) cases with
loss of Y. These data therefore suggest that FISH using a panel of pro-
bes targeting the most recurrent chromosome abnormalities may
serve as diagnostic test for identifying classes 1 and 2 ROs.
3.3 | Clinical correlations
No clinical and histological correlations were identified between vari-
ous classes of ROs, except age and WGD. Ages between the RO
classes were statistically significant, where class 1 ROs occur in youn-
ger patients (class 1, 55.07 years; class 2, 64.6 years; class 3, 69.5 years)
(P = .0014). Frequencies of WGD between RO classes are also statisti-
cally significant with class 2 exhibiting a higher frequency (P = .01).
3.4 | Validation of the present data with Mitelman
database
A total of 102 karyotypically abnormal ROs were reported in
Mitelman database. To identify the type and frequency of chromo-
some abnormalities characteristic of each class of RO, we chose
91 cases after excluding 11 cases reported by us before. We com-
pared frequencies of chromosome abnormalities with our series of
63 cases (Tables 1 and 2). Overall this analysis showed similar types
and frequencies of chromosome aberrations in all three classes of
ROs. Interestingly, partial deletion of chromosome arm 1p in class
1 RO was present in two tumors in the present series and in one case
FIGURE 1 Patterns of chromosome
abnormalities in various classes of renal
oncocytomas. Red, class 1; blue, class 2; light
green, class 3. Light blue in class 2 indicates
partial deletion of chromosome arm 1p
FIGURE 2 Cytogenetic and FISH analyses of class 1 RO. Partial karyotypes showing 11q13 rearrangements with (A) 4q and (B) 9p (arrows
indicate rearranged chromosomes). FISH analysis using the CCND1 (orange)/CEP 11 (green) probe (C, partial metaphase) and the two-color
CCND1 break apart probe (D, interphase). Rearranged CCND1 is shown by white arrows. E, The frequency of 11q13 translocation rearrangement
partners
4 ANDERSON ET AL.
in the Mitelman database. WGD in class 2 RO was found in eight
cases in our series and five in the Mitelman database.
This analysis further confirms that 11q13 rearrangements occur as
a sole abnormality in the majority of the class 1 RO. The chromosome
1 and sex chromosome losses occur together in most cases of class
2 RO. Loss of Y was seen as a sole abnormality in class 3 RO in 4 of
16 (25%) in our series and 5 of 51 (10%) in the Mitelman database.
Loss of Y can be an age-associated abnormality. Trisomy 7 was found
to be a sole abnormality in class 3 RO in three of 16 (19%) in our
series and 5 of 51 (10%) in the Mitelman database. Trisomy 7 is a non-
specific change that can be seen in proliferative kidney epithelial cells.
Among the structural abnormalities, t(6;9)(p12;p23-24) and 19p13
rearrangement was seen in four cases each of the class 3 RO in the
Mitelman database and one each in our series.
4 | DISCUSSION
Karyotypic characterization of chromosome aberrations has greatly
contributed in our understanding of the biological events underlying
the origin and progression of major histologic subtypes clear cell and
papillary renal cell cancer.
6-11
Various recurrent nonrandom chromo-
some changes reflecting copy number alterations and mutations
have been identified in clear cell, papillary, and chromophobe
RCCs.
12-14
However, the genetic and molecular basis of sporadic RO
is relatively poorly understood. Only a small number of cases with
karyotypic aberrations have been reported in the literature.
5,15-18
These and other molecular studies have identified two major groups
of RO, one with monosomy 1 accompanied by losses of Y in males
or X chromosome in females, and a second one with 11q13
rearrangement.
19
Here we report the largest series of karyotypically
characterized RO that revealed three distinctly different and mutu-
ally exclusive classes of chromosome abnormalities. We have previ-
ously reported a small series of RO cases where we showed 11q13
rearrangements and loss of chromosome 1 as mutually exclusive
cytogenetic aberrations.
5
The rearrangement of 11q13 with multiple translocation partners
in the genome is a distinct feature of class 1 RO. Others and we have
previously shown that CCND1 at 11q13 is the target of rearrangement
with the breakpoints outside of the coding region resulting in
increased expression of cyclin D1.
5,18-20
CCND1 chromosomal
rearrangement and amplification results in over expression of cyclin
D1 in many tumor types.
21
CCND1 translocation due to juxtaposition
to immunoglobulin genes acts as a driver mutation in several forms of
malignant lymphomas.
22,23
CCND1 also participates in translocations
with other genes than immunoglobulin genes in other tumors resulting
in alternative mechanisms of cyclin D1 overexpression.
24,25
However,
the gene(s) or genomic sequences that drive the deregulated expres-
sion of cyclin D1 in RO are not known. As 11q13 rearrangements
occur with multiple chromosomal partners in class 1 RO, it is likely that
enhancers similar to immunoglobulin-type genes may be involved in
these translocations. The transcriptional activation mechanisms of
CCND1 deregulation in RO remains to be examined. In hematologic
malignancies, there are number of genes (eg, KMT2A and BCL6) known
to be involved in translocations with multiple partners in the genome.
As CCND1/11q13 translocations with multiple genomic regions occur
as a sole abnormality in RO, they are likely to act as driver mutations.
Loss of chromosome 1 in association with sex chromosome loss is
characteristic feature of class 2 RO. This class of RO shows a male
predominance (68% males vs 32% females) with more frequent loss of
Y in males (71% cases) compared to loss of X in females (38%). The
biological consequence of chromosome 1 loss is not well understood.
However, no significant differences in expression of genes located on
chromosome 1 have been found in a small series of RO cases reported
recently.
19
Interestingly, we found two cases showing a common dele-
tion at 1p22-p34 (one with deletion distal to 1p21 and other with an
interstitial deletion at 1p22-p34). This limited number of cases with
deletion at 1p22-p34 potentially points to a critical region of deletion
that might harbor important genes. However, larger series of class
2 RO cases need to be studied in order to identify if a critical region
of deletion on chromosome 1 exists. The biological consequence of
chromosome losses of Y or X, 14, 21, and 22 also remains unknown.
Although it is well established that abnormalities involving chromo-
some 1 are frequent in a wide-variety of tumors, the biologic conse-
quences of these karyotypic aberrations remain poorly understood.
Class 3 RO comprising 18 tumors showed simple (≤3 changes) or
complex karyotypes (≥4 changes) with gains, losses, and structural
rearrangements. No class 3 tumors had 11q13 translocations or chro-
mosome 1 abnormalities. Thirteen tumors showed simple karyotypes
and five had complex karyotypes. The only recurrent changes identi-
fied were loss of Y or X in seven cases, gain of chromosome 7 in five
cases, and structural rearrangements at 2q37 in three cases. Loss of
the Y chromosome and/or trisomy 7 was seen in seven cases. These
abnormalities are nonspecific and can occur in benign proliferative
epithelial cells. Interestingly, telomeric associations (tas) were seen in
three tumors. It is unclear what the significance of these seemingly
nonrecurrent chromosome abnormalities is in class 3 RO. Subjecting
these tumors to extensive genomic characterization may shed some
light on the genetic basis of this class of RO.
In the present series of RO, we observed WGD in both classes
1 and 2, but not in class 3 tumors. It was most frequent in class 2 with
32% tumors exhibiting WGD. Tetraploidy resulting from genome dou-
bling is a common phenomenon in many cancer types. It has been
hypothesized that tetraploidization in cancer is an intermediate step
before aneuploidy onset, early event in tumor progression, and occurs
in transition from premalignant state to malignant state.
14,26,27
In 9 of
10 RO tumors with evidence for WGD it was only present in sub-
clones, suggesting that duplication occurred as a later event. Consis-
tent with this, tetraploid clones have been reported to occur later in
tumor development,
28
as well as both before and after other copy
number alterations in different tumor types.
29
It has also been shown
that tetraploid cells exhibit tolerance of chromosomal instability com-
pared to diploid clones and associate with poor prognosis in colorectal
tumors.
26
As ROs are benign, whether the presence of WGD fuels
further genomic changes accelerating tumor progression is unknown.
ANDERSON ET AL. 5
