Catic et al. BMC Medical Genetics(2020) 21:202
https://doi.org/10.1186/s12881-020-01143-6

RESEARCH ARTICLE

Open Access

KANK1-NTRK3 fusions define a subset of
BRAF mutation negative renal metanephricadenomas
Aida Catic1,2* , Amina Kurtovic-Kozaric2,3, Ardis Sophian1, Lech Mazur1, Faruk Skenderi3, Ondrej Hes4,
Stephen Rohan5, Dinesh Rakheja6,7, Jillene Kogan1,8,9,10 and Michael R. Pins8,9

Abstract
Background: Metanephric adenoma (MA) is a rare benign renal neoplasm. On occasion, MA can be difficult todifferentiate from renal malignancies such as papillary renal cell carcinoma in adults and Wilms̕ tumor in children.
Despite recent advancements in tumor genomics, there is limited data available regarding the genetic alterations
characteristic of MA. The purpose of this study is to determine the frequency of metanephric adenoma cases
exhibiting cytogenetic aberration t (9;15)(p24;q24), and to investigate the association between t (9,15) and BRAF
mutation in metanephric adenoma.
Methods: This study was conducted on 28 archival formalin fixed paraffin-embedded (FFPE) specimens frompatients with pathologically confirmed MA. Tissue blocks were selected for BRAF sequencing and fluorescent in situ
hybridization (FISH) analysis for chromosomal rearrangement between KANK1 on chromosome 9 (9p24.3) andNTRK3 on chromosome 15 (15q25.3), which was previously characterized and described in two MA cases.
Results: BRAFV600E mutation was identified in 62% of our cases, 9 (38%) cases were BRAFWT, and 4 cases were
uninformative. Of the 20 tumors with FISH results, two (10%) were positive for KANK1-NTRK3 fusion. Both cases were
BRAFWT suggesting mutual exclusivity of BRAFV600E and KANK1-NTRK3 fusion, the first such observation in the literature.
Conclusions: Our data shows that BRAF mutation in MA may not be as frequent as suggested in the literature and
KANK-NTRK3 fusions may account for a subset of BRAFWT cases in younger patients. FISH analysis for KANK1-NTRK3
fusion or conventional cytogenetic analysis may be warranted to establish the diagnosis of MA in morphologically andimmunohistochemically ambiguous MA cases lacking BRAF mutations.
Keywords: Metanephric adenoma, Cytogenetics, Chromosomal translocations, KANK1-NTRK3 fusion, BRAFV600E

Background
Metanephric adenoma (MA) is a rare benign renal
tumor classified under the rubric of metanephric tumors, which also include metanephric stromal tumor
and metanephric adenofibroma [1]. BRAF mutations* Correspondence: aida.catic.00@gmail.com
1
Department of Cytogenetics, ACL Laboratories, Rosemont, IL, USA
2
Department of Genetics and Bioengineering, International Burch University,
Francuske revolucije bb, Ilidza, 71000 Sarajevo, Bosnia and Herzegovina
Full list of author information is available at the end of the article

have been identified in metanephric stromal tumor andmetanephric adenofibroma in addition to metanephricadenoma, which justifies their grouping as family of
metanephric tumors by the World Health Organization(WHO) [2]. MA is uncommon and generally occurs in
adults between the fourth and sixth decades of life andoccasionally in children [3]. The male-to-female ratio is
between 1:2 to 1:3 with a mean age of approximately 41
years [3, 4]. In adults, MA accounts for approximately
0.2% of adult renal epithelial neoplasms [5]. Despite the© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Catic et al. BMC Medical Genetics(2020) 21:202

fact that fewer than 25 cases have been reported in children, it is considered to be the most common benign
pediatric renal epithelial tumor [6, 7]. Including both
pediatric and adult cases, fewer than 200 cases of MA
have been reported in the literature, thus, illustrating its
rarity and the scarcity of available data [5–8].
The majority of MA cases can be diagnosed on routine
hematoxylin and eosin (H&E) stained slides. However, insome challenging cases MA can be difficult to morphologically differentiate from malignant renal neoplasms [9]; particularly the solid variant of papillary renal cell carcinoma(PRCC) and epithelial-predominant Wilms’ tumor (WT)[10]. The distinction between these renal tumor subtypes
can be aided by the use of diagnostic modalities such as immunohistochemistry, cytogenetic studies, and advanced molecular analyses [3]. The correct classification of a renal
tumor is not only critical from a diagnostic standpoint, butalso from a prognostic and therapeutic standpoint [11, 12].
Immunohistochemistry may be helpful in distinguishing metanephric adenoma from solid variant of papillary
renal cell carcinoma (s-pRCC) and epithelial predominant Wilm’s tumor (e-WT). Specifically, MAs are generally diffusely CD57 and WT1 positive, only focally CK7
positive, and CD56 and AMACR negative (rarely weakly
positive), s-pRCCs are generally diffusely CK7 andAMACR positive, often CD57 positive (in contrast toconventional [non solid-variant] pRCC, which is usually
CD57 negative) and WT1 and CD56 negative, and eWTs are generally diffusely CD56 and WT1 positive andvariably CD57, CK7, and AMACR positive.
The genetic alterations underlying MA tumorigenesis have
only been defined relatively recently [11]. Previous cytogenetic studies have revealed a paucity of genetic alterations in
MA. The molecular and cytogenetics data reported in the literature in regard to MA is sparse and often consists of literature reviews of previously published cases or isolated casereports. We and another group each reported a case of MA
showing a t (9;15) [11–13]. A study by Choueiri et al. demonstrated that approximately 90% of MAs harbor BRAFV600E
mutations; the genetics of the remaining 10% in their study
is unclear [14]. BRAFV600E gene mutations are frequently detected in a wide range of benign and malignant human tumors, however, BRAF mutations in renal tumors such as
renal cell carcinoma (RCC), oncocytoma, and WT are essentially absent [4, 15–21]. This data coupled with Choueiri’s
data suggests that BRAF mutations are specific for MA
amongst renal tumors.
The present study was undertaken to determine thefrequency of MA cases exhibiting cytogenetic aberrationt (9;15)(p24;q24) as previously reported in the literature,and to investigate the association between t (9;15) and
BRAF mutation in MAs [11–13]. Two cases included inthis study have previously been reported in literature(case # 1 and #12-Table 1.). Catic et al. have

Page 2 of 9demonstrated the specific gene fusion that results fromthe chromosomal translocation t (9,15)(p24;q24) [11].
Rakheja et al. reported chromosomal translocation mentioned above with only a karyotype [13]. We examined
28 cases of MA at the genetic and molecular level, using
a combination of BRAF sequencing and fluorescent insitu hybridization (FISH) to detect chromosomal rearrangement between KANK1 on chromosome 9(9p24.3) and NTRK3 on chromosome 15 (15q25.3).

Methods
Patients and samples

This study was conducted on 28 archival formalin fixedparaffin-embedded (FFPE) specimens from renal metanephric adenomas. FFPE blocks and H&E stained slides
were obtained from the departments of pathology at four
participating institutions, including Advocate Lutheran
General Hospital (Park Ridge, IL, USA), Northwestern
Memorial Hospital (Chicago, IL, USA), Children’s Medical Center of Dallas (Dallas, TX, USA), and Charles
University Hospital (Plzen, Czech Republic). Fourteen
cases were of American origin and 14 of European origin. All samples received for this study and data reported
have been de-identified. Because this was a retrospective
study, ethics committee ruled that no formal ethics approval was required in this particular study.
The MA specimens and hematoxylin and eosinstained slides were retrieved and reviewed by expert pathologists at each institution. All pathologic specimens
were acquired after partial or complete nephrectomy,
and none were diagnosed by needle biopsy. The diagnosis of MA was then re-confirmed by two genitourinary
pathologists (Fig. 1) and de-identified representative tissue blocks were further selected for BRAFV600E exon 15
sequencing and FISH analysis. Histologically, the tumors
are composed of epithelial cells arranged in tubules andpapillary configurations. The relatively small tumor cells
have a high nuclear: cytoplasmic ratio, ovoid nuclei, uniformly dispersed chromatin, inconspicuous nucleoli,
scant eosinophilic cytoplasm, and ill-defined cell borders
with nuclear overlap, Fig. 1, a and b. Mitoses are notconspicuous. Occasional psammomatoid calcifications
are seen, Fig. 2. Patient demographics and clinicopathologic characteristics such as: age, gender, tumor size, laterality, and chromosomal analysis results were provided
by pathologists at each institution (Table 1).
Fluorescence in situ hybridization (FISH)

Metaphase chromosome spreads and interphase nuclei
were prepared on a glass microscope slide in accordancewith standard cytogenetic procedure and according to
the manufacturer’s instructions. Paraffin embedded tissue slides were cut at 2-μm thickness using a microtome
and floated in a protein free waterbath at 40 °C. A

Catic et al. BMC Medical Genetics(2020) 21:202

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Table 1 Clinical, pathological, cytogenetic, and molecular findingsa
Case no. Age range years Gender Tumor Size (cm) Laterality Karyotype

BRAF Statust(9;15)(p24;q24) FISH

1.

21–30

F

2

Right

46,XX,t(9;15)(p24;q24)

Wild type

Fusion Present

2.

21–30

F

3

Left

46,XX,t(6;22)(q26;q11.2)

Wild type

Normal

3.

41–50

M

12

NA

46,XY

V600E Mutation Normal

4.

51–60

F

1.4

Left

46,XX

V600E Mutation Normal

5.

51–60

M

3

Right

46,XY

V600E Mutation Normal

6.

31–40

F

6.7

Left

NA

V600E Mutation Normal

7.

61–70

F

NA

Right

NA

Uninformative

8.

51–60

M

NA

NA

NA

V600E Mutation Uninformative

9.

51–60

F

0.5

Right

NA

Uninformative

10.

61–70

F

5.1

Right

NA

V600E Mutation Normal

11.

31–40

F

3.4

Left

NA

V600E Mutation Normal

12.

1–10

M

1.7

Right

46,XY,t(9;15)(p24;q24),inv.(12)(q13q15) Wild type

13.

1–10

M

1.5

Right

NA

V600E Mutation Normal

14.

31–40

M

4.3

NA

NA

V600E Mutation Normal

15.

11–20

M

7

Right

NA

Wild type

Normal

16.

NA

NA

NA

NA

NA

Wild type

Normal

17.

51–60

F

NA

Left

NA

Wild type

Normal

18.

51–60

F

5

NA

NA

Uninformative

Uninformative

19.

71–80

M

1.8

Right

NA

Wild type

Normal

20

21–30

F

2

NA

NA

Wild type

Uninformative

21.

61–70

F

4

NA

NA

Wild type

Uninformative

22.

71–80

F

2.5

NA

NA

V600E Mutation Normal

23.

51–60

F

5

NA

NA

Uninformative

24.

51–60

F

3

NA

NA

V600E Mutation Normal

25.

11–20

F

3

Right

NA

V600E Mutation Normal

26.

41–50

F

1.5

NA

NA

V600E Mutation Uninformative

27.

61–70

M

8

NA

NA

V600E Mutation Normal

28.

61–70

M

3

NA

NA

V600E Mutation Normal

Uninformative

Uninformative

Fusion Present

UninformativeaAbbreviations: NA indicates not available, FISH Fluorescent in situ hybridization

concurrent H&E slide was stained and marked by apathologist to delineate the area of tumor for analysis.
Fluorescent in situ hybridization probes were purchasedfrom BlueGnome (Illumina, Cambridge, United Kingdom) and Empire Genomics (Buffalo, NY, USA). Briefly,
prepared slides from the tumor were placed in a Coplin
jar with 40 mL of 2XSSC pH 7.0 at 37 °C for 15 min.
Treatment of slides in 2XSSC was used to artificially age
the chromosomes, making them less sensitive to overdenaturation. Next, the slides were dehydrated in 70, 85,
and 100% ethanol at room temperature for 2 min each,followed by drying on a 50 °C warmer for 15 min. Paraffin embedded tissue slides were baked in a 60 °C ovenfor a minimum of 1 h. These slides were then placed on
the VP processor (Abbott Molecular, Des Plaines, IL,
USA) for de-paraffinization, pretreatment, and protease
digestion. A mixture of 3.5 μl of locus specific identifier(LSI) hybridization buffer (Abbott Molecular, Des
Plaines, IL, USA), 1 μl of sterile water, and 0.5 μl ofprobe was prepared for the BlueGnome probes and 4 μlof Empire Genomics buffer was used with 1 μl of probe
for the Empire probes for each hybridization area. 5 μl ofprobe mixture was applied to each hybridization area ofpatient and control slides. Prepared slides and probes
were then co-denatured using the ThermoBrite machineat a denature temperature of 76 °C for 5 min and thenhybridized overnight. After hybridization, slides were
washed using 40 ml of 0.4X SSC/0.3%NP40 for 2 min,followed by 40 ml of 2X SSC/0.1%NP40 for 1 min to remove any excess or unbound probe. After slides were
air-dried, 10 μl of DAPI II counterstain on a 22 × 22
coverslip was applied to the targeted area of the slide.
FISH analysis was performed following standard techniques using a fluorescent microscope with appropriate

Catic et al. BMC Medical Genetics(2020) 21:202

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Fig. 1 Histologic, immunohistochemical, fluorescence in situ hybridization and molecular genetic analyses findings of metanephric adenoma. a
Hematoxylin and eosin (H&E) of translocation negative metanephric adenoma case, 200x total magnification b H&E slide of translocation positive
metanephric adenoma case, 200x total magnification c Fluorescence in situ hybridization with probes for chromosomes 9 (green signal) and 15(orange/red signal) show normal nuclei with two red and two green signals, (original magnifications 100x) d Abnormal fluorescence in situ
hybridization utilizing 2 color probes for the t(9;15) showing one green (9p24) and one orange/red (15q24) signal on the normal homologues.
The yellow signals represent the fusion of the probes on the abnormal homologues of chromosome 9p24 and 15q24 (original magnifications
100x) e Melting curve analysis of BRAF mutations in metanephric adenoma sample illustrating a sample without a BRAF GTG > GAC (V600E)
mutation of codon 600 (nucleotide 1799) f. Melting curve profiles illustrating the detection of a BRAFV600E mutation of the metanephric adenoma
sample with a GTG > GAC (V600E) mutation of codon 600 (nucleotide 1799). Green melting curve represents quality control (QC) wild-type (WT),
red melting curve represents QC positive for V600E, black curve represents patient samples (E-non-mutated; F-BRAFV600E mutated) are compared,
additional melting peaks or changes in peak-area ratios indicate a sequence alteration under the probe (E and F)

filters (Olympus, Tokyo, Japan). The number ofhybridization signals for each probe was assessed on aminimum of twenty metaphases from fresh tumor
slides and from 200 nuclei on the paraffin embedded
tissue slides with strong and well-delineated signals,
and were further selected for thorough examination
using Applied Spectral Imaging (ASI) software (Carlsbad, CA, USA). Karyotypes were described and reported in accordance with the International System
Committee for Human Cytogenomic Nomenclature(ISCN) 2016 [22].

Probes used to interrogate the 9q24 region were:RP11-143 M15 (9p24.3-green), RP11-59O6 (9p24.3-orange), RP11-130C19 (9p24.3-green), and RP11-1107A23(9p24.3-green). Probes used to interrogate the 15q24 region were: RP11-62D2 (15q25.3-orange) and RP11608H9 (15q25.3-orange).
BRAF mutation analysis

FFPE sections were evaluated for the BRAF mutation ona Roche LightCycler 2.0 instrument (Mannheim,
Germany) utilizing the allelic discrimination by real time(2020) 21:202

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40 °C for 30 s. The method of BRAF exon 15-mutation
analysis interpretation has been previously described [4,11, 14]. Specifically, our assay detects 28 nucleotidechanges involving the following codons: V600E, M, L, R,
Q, D, K, A and G, L597V, S, Q, R and L, K601E, del, andN, A598V, A598_T599insV, T599I, T599_V600insT,
T599_V600insTT, and V600_K601 > E.
Fisher’s exact analysis was performed for comparison
between the differential prevalence of BRAFV600E mutation in patients under the age of 30 and those patientsover 30 years of age. A p value <.05 was used to indicate
statistical significance.

Fig. 2 Hematoxylin and eosin photomicrograph of BRAF wild typecase, showing classic features of renal metanephric adenoma. Smallcells with a high nuclear to cytoplasmic ratio form small tubules,
rudimentary intraluminal buds and sheets

polymerase chain reaction (PCR), which was performedat the Clinical Laboratory Improvements Amendments(CLIA) certified ACL Laboratories (Rosemont, IL, USA).
In short, the appropriate FFPE tissue block was selectedby the pathologist. Four-micrometer-thick sections fromFFPE tissue blocks were enriched by manual microdissection and DNA was isolated by the ZymoPinPiont
method (Irvine, CA, USA), according to the manufacturer’s protocol. Exon 15 of the BRAF gene was amplified from 50 ng of genomic DNA by real time PCR using
sequence specific primers ordered from Invitrogen-Life
Technologies (Carlsbad, CA, USA) (forward primer: 5′CTCTTCATAATGCTTGCTCTGATAGG-3′, and reverse primer: 5′-TAGTAACTCAGCAGCATCTCAGG3′). Melting curve analysis was performed by optimized
fluorescent probes 5′-FL-TGGAGTGGGTCCCATCAG
TTTGAACAGTTGTCTGGATCCATT
SpacerC35’TGGTCTAGCTACAGTGAAATCTC-LC640. The PCR
products were amplified in the following conditions: initial denaturation at 95 °C for 10 min; amplification 45 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 20 s;
melting curves 1 cycle of 95 °C for 2 min, 40 °C for 2
min, and 85 °C at 5 s; and cooling period of 1 cycle atResults
We analyzed 28 MAs. Among those, there were 17
women, 10 men and 1 unknown (F:M, 1.7:1). Patient age
ranged from 9.8 to 73 years with median age of 52.5
years (52 among women; and 53.5 among men). Twenty
patients were over the age of 30, while 7 were under theage of 30 and 1 patient was of unknown age (Table 1
and Table 2). Tumor size ranged from 0.5 cm to 12 cm
with a median size of 3 cm. Patient cohort and clinicalcharacteristics are summarized in Table 1.
Out of 28 cases studied, cytogenetic analysis was available in only six (21%) cases, Table 2. Three (50%) cases
out of 6 exhibited chromosomal aberration. All three patients were under the age of 30. Two cases exhibited
translocation involving chromosome 9 (9p24) andchromosome 15 (15q24), or t (9;15)(p24;q24) [11, 13].
To rule out constitutional abnormality in these two patients, peripheral blood samples revealed normal karyotypes [11, 13]. One additional case that wastranslocation positive exhibited a translocation involving
chromosome 6 (6q26) and chromosome 22 (q11.2) witha karyotype of 46,XX,t (6;22)(q26;q11.2). For this case, it
is unknown if any genes are affected and if constitutional
chromosome analysis was performed to rule out constitutional abnormality. The remaining three tumor cases
demonstrated normal karyotypes of 46,XX or 46,XY andwere over the age of 30. There were no differences notedamong genders. Cytogenetic results are further summarized in Table 2.

Table 2 Summary of cytogenetic and molecular findings
Patients

Karyotype n = 28

BRAFV600E n = 24 (%) Mutation Wild TypeFISH for t(9;15) n = 20 (%)

28

6 (21%)

15 (62%)2 (10%)> 30 years

20

3

13

3

0

< 30 years

7

3

2

5

2 (100%)Total # of cases

9 (38%)

Unknown

1

0

0

1

Female (median age in years)

17 (52)

3

8

5

1

Male (median age in years)

10 (53.5)

3

7

3

1

Unknown

1

0

0

1

1

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Fig. 3 Schematic diagram of genomic location of KANK1-NTRK3 fusion. Exons 1–7 of KANK1 are shown in blue and exons 14–18 of NTRK3 areshown in red

The focus of the study was to determine the frequencyof t (9;15) or KANK1-NTRK3 gene fusion using FISH,
Fig. 1, C and D. Cases 1 through 9 were tested using
FISH probes RP11-130C19 on 9p24.3 (green signals) andRP11-62D2 on 15q25.3 (orange signals) from

BlueGnome (Illumina, Cambridge, United Kingdom).
Due to unavailability of previously used probes from
BlueGnome, the following replacement probes were purchased from Empire Genomics (Buffalo, NY, USA):
RP11-1107A23 on 9p24.3 (green signals) and RP11-

Fig. 4 a Schematic representation of the BRAF status wild type (WT) is colored green and mutation is colored red. Translocation presence ofeither t(9;15) or t (6;22) is colored red and no translocation is colored green. Gray represents uninformative results. b Schematic representation of
the results demonstrating the presence and the absence of translocation, and BRAFV600E mutation or wild type, suggesting mutual exclusivity
between BRAFV600E and KANK1-NTRK3 fusion

Catic et al. BMC Medical Genetics(2020) 21:202

608H9 on 15q25.3 (orange signals). A clinical cytogeneticist picked the best replacement probes based on
available data from the vendor as well as University ofCalifornia Santa Cruz (UCSC) Genome Browser (Genome Build 38), making sure probes covered the genes ofinterest. FISH analysis of the remaining cases (10through 28) was carried out using the Empire Genomics
probes. Clear signals and reportable FISH results wereobtained in 20 (71%) cases. Of the 20 tumors with FISH
results, two tumors (10%) were positive for KANK1NTRK3 fusion (Fig. 1, D). These two cases were also
found to harbor the same translocation t (9;15)(p24;q24)
by conventional cytogenetics analysis [11, 13]. Thus,
concordance between FISH testing and chromosome
analysis evaluation for the detection of the rearrangement and involvement of KANK1-NTRK3 genes was
100% (2/2 cases). The remaining 18 (90%) tumors were
negative for KANK1-NTRK3 fusion, Fig. 1d. FISH ineight tumors (29%) was technically unsuccessful due tolack of hybridization signals and therefore uninformative. This was most likely due to DNA degradation inarchival material, a conclusion supported by the fact thatmany of the same cases were uninformative for BRAF by
PCR. FISH results are summarized in Table 2.
Of the 28 cases studied, the BRAF mutational analysis
was informative in 24 cases (86%), Fig. 1e and f. BRAF
exon 15 analysis was wild type (WT) in 9/24 (38%) cases(Fig. 1e) and mutated in 15/24 (62%) of cases, (Fig. 1f).
Patient cases exhibiting BRAFV600E mutation 13 of 15(87%) were over the age of 30. The patients exhibiting
BRAF wild type results were predominantly (5 of 9; 56%)
under the age of 30. The Fisher exact test statistic value
is 0.026. The result is significant at p < .05.
In the present study, the t (9;15) resulted in a KANK1NTRK3 fusion transcript in which the first seven exons of
KANK1 are fused to exon fourteen of the NTRK3, Fig. 3.
Out of the 9 cases lacking BRAF mutation, 3 cases(33%) exhibited chromosomal translocation. Four out of
six BRAFWT cases did not have cytogenetic results available, but showed normal FISH pattern for t (9;15). The
remaining 2 cases were uninformative by FISH. Overall,
13 of 20 (65%) cases lacking t (9;15) harbored BRAFV600E
mutations. There were no cases demonstrating both thetranslocation and BRAFV600E mutation, suggesting mutual exclusivity between BRAFV600E and KANK1-NTRK3
fusion, Fig. 4 (a and b).

Discussion
Typically, straightforward cases of MA can be diagnosed
solely based on histologic features. Challenging cases require additional diagnostic testing for distinction frommalignant renal tumors. Cytogenetic analysis is laborintensive, expensive, and generally not performed as partof routine clinical practice in classification of MAs;

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therefore, only a handful of previous studies have reported chromosomal aberrations in MAs. Catic et al.
and Rakheja et al., observed balanced translocation t(9;15)(p24;q24) [11, 13], while Lerut et al., reported thepresence of the dual balanced translocation, t(1,22)(q22;q13), and t(15;16)(q21;p13) [23]. However, several studies that have reported cytogenetic findings have reported
normal karyotypes [23–30].
In this study, we confirm that a subset of biologicallydistinct MAs in younger patients (< 30 yrs. of age) exhibit aberrant chromosomal translocations and do notharbor BRAF mutations. The novel findings of our study
are that the typical MAs which do not harbor BRAF mutations can demonstrate cytogenetic aberrations. Additional larger cohort studies are necessary to confirm
and further elucidate the frequency of the cytogenetic
aberrations found in this subset of MAs.
Unlike chromosome analysis data, FISH analysis andimmunohistochemistry analyses on MA are more frequently performed and reported in the literature. Most reported FISH studies have been primarily focused on
testing for trisomies of chromosome 7 and 17, and loss of
Y chromosome. Brown et al., reported trisomies of chromosomes 7 and 17 and loss of Y chromosome in eight of
11 cases classified as MA [31]. However, their eight cases
almost certainly represented examples of solid variant of
PRCC, an entity that was described in the literature subsequent to their publication [32–34]. Recent studies strongly
recommend utilization of FISH analysis to aid in differentiating those cases that deviate from the expected immunohistochemical staining pattern [3, 4].
Recent studies of MA have emphasized the importanceof advanced molecular testing. Previous studies have demonstrated that the vast majority of MAs harbor BRAF
V600E
mutations, and that epithelial WTs lack BRAF V600E
mutations [19, 20, 35]. Choueiri et al. published the largest
series of MA demonstrating that 90% of MAs harbor a
BRAF V600E mutation [14]. This was the first large study toshed light on the molecular underpinnings of MA and toinvestigate BRAFV600E mutation in this indolent tumor.
Additionally, they tested 129 non-MA renal neoplasms
and detected BRAF V600E mutation in only one PRCC.
Cytogenetic analysis of this PRCC case revealed the presence of trisomy of chromosomes 7 and 17. Padilha et al.
and Choueiri et al. suggest that molecular BRAFV600E mutation analysis is a valuable diagnostic tool in the differential diagnosis of this rare kidney tumor that may bediagnostically challenging [8, 14].
More recently, Chami et al. studied pediatric MA cases for
BRAFV600E mutations and found three out of four cases to bepositive for BRAFV600E mutation; 10 cases of pediatric renal
cell carcinoma and 10 cases of Wilms’ tumor did not exhibit
BRAFV600E mutation [6]. To date, there are no cases reported
in literature of Wilms’ tumor exhibiting BRAF mutations.

Catic et al. BMC Medical Genetics(2020) 21:202

Similarly, Udager et al. evaluated eleven MA cases for
BRAFV600E mutations and found eight out of elevencases to be positive for BRAFV600E mutation. Of thethree cases negative for BRAFV600E mutation, two exhibited a novel BRAFV600D mutation, of which one had acompound BRAFV600D and BRAFK601L mutation [4].
Overall, 90% of all published MA cases evaluated for
BRAF mutations harbor BRAF exon 15 mutations. Most
recent published study by Chan et al. examined the genetic profiles using next-generation sequencing on elevenconventional MAs and revelated all eleven cases harboring BRAF V600E mutations [35].
Our assay, which is capable of detecting 28 different
BRAF mutations including BRAFV600D and BRAFK601L
only detected BRAFV600E mutations. Our results differ
significantly from those in the literature in the overalllower frequency of BRAF mutations (62% vs. 90%). Thismay be due to the fact that in our study 25% of our patients were under the age of 30. We observed a possible
trend towards patients younger than 30 years-old showing BRAFWT tumors. This data suggests that MA inyounger patients may be genetically distinct from its
counterpart in older patients, but a larger patient cohort
is needed to confirm this observation.
Lastly, we compared BRAFV600E mutation analysis results with FISH for KANK1-NTRK3 gene fusion. Of thenine cases that exhibited BRAFWT, 3 cases were thosethat demonstrated chromosomal aberrations by conventional karyotyping. Of the three cases that showedchromosomal translocations, two were positive for t (9;15) and KANK1-NTRK3 gene fusion by FISH. The
remaining six BRAFWT cases did not have conventional
cytogenetic analyses. Four of these six cases were negative for KANK1-NTRK3 fusion by FISH, and 2 cases
were uninformative. Overall, 13 of 20 (65%) cases lacking t (9;15) harbored BRAF mutations. There were nocases with both t (9;15) and BRAFV600E mutation suggesting exclusivity between BRAFV600E and t (9;15) andthat the latter may be the genetic event behind a subsetof BRAFWT MAs.
A significant limitation of our study is the retrospective nature of case series with inability to test the BRAFwild type cohort for additional mutations. Unfortunately,
many cases had very little or no additional sample material to perform NTRK3, pan-Trk and BRAF immunohistochemistry testing.

Conclusion
In conclusion, we report KANK1-NTRK3 fusion without
BRAFV600E mutation in two MA cases. This finding supports the initial suggestion that KANK1-NTRK3 is thepathogenetically significant fusion transcript in tumors
carrying a t (9,15)(p24;q24) and lacking BRAFV600E mutation in younger patient cohort. In this study, we have

Page 8 of 9

provided additional evidence that metanephric adenomas have relatively noncomplex karyotypes and havedistinctive cytogenetic profiles. The cytogenetic profile
can be useful in resolving a differential diagnosis ofmetanephric adenoma.
Classic histopathological features of MA coupled witha documented BRAFV600E mutation are diagnostic of
MA; however, we and others have demonstrated that theabsence of BRAFV600E mutation does not exclude a diagnosis of MA. For those case lacking BRAF mutations, alternative testing such as FISH analysis for KANK1NTRK3 fusion and/or cytogenetic chromosome analysis
to look for t(9;15)(p24;q24) may be warranted.
Abbreviations
MA: Metanephric adenoma; FFPE: Formalin fixed paraffin-embedded;
H&E: Hematoxylin and eosin; WHO: World Health Organization;
PRCC: Papillary renal cell carcinoma; WT: Wilms’ tumor; ASI: Applied Spectral
Imaging; ISCN: International System Committee for Human Cytogenomic
Nomenclature; PCR: Real time polymerase chain reaction; CLIA: ClinicalLaboratory Improvements Amendments
Acknowledgements
We would like to thank Peter J. Socki, BS, HTL, ASCP and the technical staff
at ACL Laboratories Cytogenetics Department (Rosemont, IL) for their
excellent technical assistance, and the Advocate Lutheran General HospitalJames R. & Helen D. Russell Institute for Research and Innovation, Park Ridge,
IL for their assistance in study design.
Authors’ contributions
A.C. selected, directed, and reported testing, wrote manuscript. A.S.
performed FISH analysis. L.M. performed BRAF mutation analysis. F.S., O. H,
S.R., and D.R. provided us with FFPE samples, patient demographics andcontributed their pathology expertise. A.K. designed figures and tables, andassisted in writing manuscript. J.K. and M.R.P. provided guidance andexpertise in developing this study. All authors read and approved the final
manuscript.
Funding
Not applicable.
Availability of data and materials
The data generated and/or analyzed during the current study is available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
No administrative permissions were required to access the raw data andspecimens used in this study. All samples were de-identified and only relevant information pertaining to this study was shared. The need for consent
to participate was also waived by the ethics committee at the Advocate Lutheran General Hospital James R. & Helen D. Russell Institute for Researchand Innovation. The ethics committee at the Advocate Lutheran General
Hospital James R. & Helen D. Russell Institute for Research and Innovation
ruled that no formal ethics approval was required for this study.
Consent for publication
Not applicable.
Competing interests
All authors declare that they have no competing interests.
Author details
1
Department of Cytogenetics, ACL Laboratories, Rosemont, IL, USA.
2
Department of Genetics and Bioengineering, International Burch University,
Francuske revolucije bb, Ilidza, 71000 Sarajevo, Bosnia and Herzegovina.
3
Department of Clinical Pathology, Cytology and Human Genetics, ClinicalCenter of the University of Sarajevo, Sarajevo, Bosnia and Herzegovina.

Catic et al. BMC Medical Genetics(2020) 21:202

4
Department of Pathology, Charles University Hospital Pilsen, Pilsen, Czech
Republic. 5Department of Pathology, Saint Joseph Hospital, Denver, CO, USA.
6
Department of Pathology and Pediatrics, University of Texas Southwestern
Medical Center, Dallas, TX, USA. 7Departments of Pathology and Laboratory
Medicine, Children’s Health, Dallas, TX, USA. 8Department of Pathology,
Advocate Lutheran General Hospital, Park Ridge, IL, USA. 9Department of
Pathology, Chicago Medical School of Rosalind Franklin University of
Medicine and Science, North Chicago, IL, USA. 10Advocate Medical Group
Genetics, Park Ridge, IL, USA.

Received: 6 May 2020 Accepted: 9 October 2020

References
1. Grignon DJ, Eble J, Argani P, Hartmann A. Metanephric Tumours. In: Moch
H, Humphry PA, Ulbright TM, Reuter VE, eds. World Health Organization
classification of Tumours of the urinary system and male genital Organs, 4thed. Lyon, France: IARC Press; 2016:45–47.
2. Argani P, Lee J, Netto GJ, Zheng G, Tseh-Lin M, Park BH. Frequent BRAF
V600E mutations in Metanephric stromal tumor. Am J Surg Pathol. 2016;40:
719–22.
3. Kinney SN, Eble JN, Hes O, et al. Metanephric adenoma: the utility of
immunohistochemical and cytogenetic analyses in differential diagnosis,
including solid variant papillary renal cell carcinoma and epithelialpredominant nephroblastoma. Mod Pathol. 2015;28:1236–48.
4. Udager AM, Pan J, Magers MJ, et al. Molecular and immunohistochemical
characterization reveals novel BRAF mutations in metanephric adenoma. Am
J Surg Pathol. 2015;39:549–57.
5. Li G, Tang Y, Zhang R, Song H, Zhang S, Niu Y. Adult metanephric adenoma
presumed to be all benign? A clinic perspective. BMC Cancer. 2015;15:310.
6. Chami R, Yin M, Marrano P, Teerapakpinyo C, Shuangshoti S, Thorner PS.
BRAF mutations in pediatric metanephric tumors. Hum Pathol. 2015;46:
1153–61.
7. Bruder E, Passera O, Harms D, et al. Morphologic and molecularcharacterization of renal cell carcinoma in children and young adults. Am J
Surg Pathol. 2004;28:1117–32.
8. Mantoan Padilha M, Billis A, Allende D, Zhou M, Magi-Galluzzi C.
Metanephric adenoma and solid variant of papillary renal cell carcinoma:common and distinctive features. Histopathology. 2013;62:941–53.
9. Pins MR, Jones EC, Martul EV, Kamat BR, Umlas J, Renshaw AA. Metanephricadenoma–like tumors of the kidney: report of 3 malignancies withemphasis on discriminating features. Arch Pathol Lab Med. 1999;123:415–20.
10. Blanco LZ Jr, Schein CO, Patel T, et al. Fine-needle aspiration of metanephricadenoma of the kidney with clinical, radiographic and histopathologic
correlation: a review. Diagn Cytopathol. 2013;41:742–51.
11. Catic A, Kurtovic-Kozaric A, Johnson SH, Vasmatzis G, Pins MR, Kogan J. A
novel cytogenetic and molecular characterization of renal metanephricadenoma: Identification of partner genes involved in translocation t (9;15)(p24; q24). Cancer Genet. 2017:214–5 9–15.
12. Catic A, Kurtovic-Kozaric A, Sophian A, et al. Prevalence of KANK1-NTRK3
fusion in renal metanephric adenomas that lack BRAF mutations. Cancer
Genet. 2018:224–5 59.
13. Rakheja D, Lian F, Tomlinson GE, Ewalt DH, Schultz RA, Margraf LR. Renal
metanephric adenoma with previously unreported cytogenetic
abnormalities: case report and review of the literature. Pediatr Dev Pathol.
2005;8:218–23.
14. Choueiri TK, Cheville J, Palescandolo E, et al. BRAF mutations in metanephricadenoma of the kidney. Eur Urol. 2012;62:917–22.
15. Chappé C, Padovani L, Scavarda D, et al. Dysembryoplastic neuroepithelial
tumors share with pleomorphic xanthoastrocytomas and gangliogliomas
BRAF(V600E) mutation and expression. Brain Pathol. 2013;23:574–83.
16. Brastianos PK, Taylor-Weiner A, Manley PE, et al. Exome sequencing
identifies BRAF mutations in papillary craniopharyngiomas. Nat Gene. 2014;46:161–5.
17. Taube JM, Begum S, Shi C, Eshleman JR, Westra WH. Benign nodal nevi
frequently harbor the activating V600E BRAF mutation. Am J Surg Pathol.
2009;33:568–71.
18. Olgac S, Hutchinson B, Tickoo SK, Reuter VE. Alphamethylacyl-CoA racemase
as a marker in the differential diagnosis of metanephric adenoma. Mod
Pathol. 2006;19:218–24.

Page 9 of 9

19. Miao J, Kusafuka T, Fukuzawa M. Hotspot mutations of BRAF gene are notassociated with pediatric solid neoplasms. Oncol Rep. 2004;12:1269–72.
20. Caliò A, Eble JN, Hes O, et al. Distinct clinicopathological features inmetanephric adenoma harboring BRAF mutation. Oncotarget. 2016;8:54096–105.
21. Gattenlöhner S, Etschmann B, Riedmiller H, Müller-Hermelink HK. Lack of
KRAS and BRAF mutation in renal cell carcinoma. Eur Urol. 2009;55:1490–1.
22. McGowan-Jordan J, Simons A, Schmid M, eds. ISCN 2016: an international
system for human Cytogenomic nomenclature (2016). Basel, Switzerland: S.
Karger; 2016.
23. Lerut E, Roskams T, Joniau S, et al. Metanephric adenoma during pregnancy:
clinical presentation, histology, and cytogenetics. Hum Pathol. 2006;37:
1227–32.
24. Jones EC, Pins M, Dickersin GR, Young RH. Metanephric adenoma of thekidney. A clinicopathological, immunohistochemical, flow cytometric,
cytogenetic, and electron microscopic study of seven cases. Am J Surg
Pathol. 1995;19:615–26.
25. Brisigotti M, Cozzutto C, Fabbretti G, Sergi C, Callea F. Metanephricadenoma. Histol Histopathol. 1992;7:689–92.
26. Granter SR, Fletcher JA, Renshaw AA. Cytologic and cytogenetic analysis ofmetanephric adenoma of the kidney: a report of two cases. Am J Clin
Pathol. 1997;108:544–9.
27. Gatalica Z, Grujic S, Kovatich A, Petersen RO. Metanephric adenoma:
histology, immunophenotype, cytogenetics, ultrastructure. Mod Pathol.
1996;9:329–33.
28. Tsuji M, Murakami Y, Kanayama HO, Sano T, Kagawa S. A case of renal
metanephric adenoma: histologic, immunohistochemical and cytogenetic
analyses. Int J Urol. 1999;6:203–7.
29. Renshaw AA, Freyer DR, Hammers YA. Metastatic metanephric adenoma ina child. Am J Surg Pathol. 2000;24:570–4.
30. Kohashi K, Oda Y, Nakamori M, et al. Multifocal metanephric adenoma inchildhood. Pathol Int. 2009;59:49–52.
31. Brown JA, Sebo TJ, Segura JW. Metaphase analysis of metanephricadenoma reveals chromosome Y loss with chromosome 7 and 17 gain.
Urology. 1996;48:473–5.
32. Brunelli M, Eble JN, Zhang S, Martignoni G, Cheng L. Metanephric adenoma
lacks the gains of chromosomes 7 and 17 and loss of Y that are typical ofpapillary renal cell carcinoma and papillary adenoma. Mod Pathol. 2003;16:
1060–3.
33. Pan CC, Epstein JI. Detection of chromosome copy number alterations inmetanephric adenomas by array comparative genomic hybridization. Mod
Pathol. 2010;23(12):1634–40.
34. Kuroda K, Hiroi M, Toi M, Enzan H. Review of metanephric adenoma of thekidney with focus on clinical and pathobiological aspects. Histol
Histopathol. 2003;18(1):253–7.
35. Chan E, Stohr B, Croom N, et al. Molecular characterization of metanephricadenomas beyond BRAF: genetic evidence for potential malignant
evolution. Histopathol. 2020;76:1084–90.

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