Primary Renal Paragangliomas and Renal Neoplasia Associated with Pheochromocytoma/Paraganglioma:

Analysis of von Hippel-Lindau (VHL), Succinate Dehydrogenase (SDHX)

and Transmembrane Protein 127 (TMEM127)

Sounak Gupta, M.B.B.S., Ph.D.1, Jun Zhang, M.D. Ph.D.2, Dragana Milosevic1, John R. Mills, Ph.D1,

Stefan K. Grebe, M.D., Ph.D.1, Steven C. Smith, M.D., Ph.D.3, Lori A. Erickson, M.D.1

1Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN;

2Department of Laboratory Medicine and Pathology, Mayo Clinic, Phoenix, AZ;

3Departments of Pathology and Urology, VCU Health, Richmond, VA.

Submitted to: Endocrine Pathology

Address for correspondence and requests for reprints:

Lori A. Erickson, M.D.

Department of Laboratory Medicine and Pathology

Mayo Clinic

200 First Street SW

Rochester, MN 55905

Phone: 507-284-1199

Fax: 507-284-1599

Email:

Supplementary Table 1. Primer Sequences: TMEM127

Amplification
TMEM127 (exon 1) Forward / 5'- GGGAAAGCATCGCGGGA -3'
TMEM127 (exon 1) Reverse / 5'- TCAGGGTCTCAGCCAGAGGA -3'
TMEM127 (exon 2) Forward / 5'- CGGAGCGTTGCTATCCTCCA -3'
TMEM127 (exon 2) Reverse / 5'- TCTGGTCCCTGGCTATCTCT -3'
TMEM127 (exon 3) Forward / 5'- GTGGAATTTGCTCCAGCTACC -3'
TMEM127 (exon 3) Reverse / 5'- CCTGACACTCTGGGAAAGAC -3'
TMEM127 (exon 4) Forward / 5'- GAGAATGGGGTTTGGGTAGAGA -3'
TMEM127 (exon 4) Reverse / 5'- CAGAGTCTCTCCTGGGGAAGG -3'
Sequencing
TMEM127 (exon 1) Forward / 5'- GGACCGCCTCGAACTACCA -3'
TMEM127 (exon 1) Reverse / 5'- TCTGGGACCGGCGGTTTG -3'
TMEM127 (exon 2) Forward / 5'-GTTGCTATCCTCCACCGGAC-3'
TMEM127 (exon 2) Reverse / 5'-CCCTGGCTATCTCTGCTTCAG-3'
TMEM127 (exon 3) Forward / 5'- CTCCCAGGACTGTGGTGGA -3'
TMEM127 (exon 3) Reverse / 5'- TGGGAAAGACTGGAGCTTGG -3'
TMEM127 (exon 4) Forward / 5'- GGGTAGAGAGGCTGTGATTGG -3'
TMEM127 (exon 4) Reverse / 5'- CTGGGGAAGGTTTGGAGAAGAT -3'

Supplementary Table 1. Primer Sequences: TMEM127

Supplementary Table 2. Grading system for Adrenal Pheochromocytomas and Paragangliomas (GAPP)

Case No. / Architectural Pattern / Cellularity / Comedo Necrosis / Vascular/ Capsular Invasion / Mitosis/ 10 hpf / Ki67 Labeling Score / Ki67 % / Functional Status / SDHB IHC (PC /PGL) / Total Score / Metastasis (PC /PGL)
1 / 0 / 1 / 0 / 1 / 0 / 2 / 10.4 / NF / 0 / 4 / +
2 / 1 / 1 / 0 / 1 / 2 / 1 / 1.6 / NA / 2+ / NS* / +
3 / 0 / 1 / 0 / 0 / 0 / 0 / 0.3 / NA / 1+ / NS* / 0
4 / 0 / 1 / 0 / 0 / 2 / NA / NA / NA / NA / NS* / 0
5 / 1 / 1 / 0 / 1 / 2 / 1 / 1.4 / NE/NM / 3+ / 5 / 0
6 / 0 / 2 / 0 / 0 / 0 / 1 / 1.3 / NE/NM / 3+ / 4 / 0
7 / 0 / 1 / 0 / 1 / 0 / 1 / 1.3 / NF / 2+ / 3 / 0
8 / 1 / 2 / 0 / 1 / 1 / 1 / 1.8 / NE/NM / 3+ / 6 / +
9 / 1 / 2 / 0 / 0 / 0 / 0 / 0.7 / NF / 2+ / 3 / 0
10 / 0 / 1 / 2 / 0 / 0 / 1 / 2.4 / NF / 0-1+ / 4 / 0
11 / 0 / 1 / 0 / 0 / 4 / 1 / 2.0 / NF / 2+ / 2 / 0
12 / 0 / 2 / 0 / 0 / 0 / 0 / 0.6 / NE/NM / 3+ / 3 / 0
13 / 0 / 1 / 0 / 0 / 0 / 1 / 1.0 / NE/NM / 1+ / 3 / 0
14 / 2 / 1 / 0 / 1 / 4 / 1 / 1.5 / NE/NM / 2+ / 6 / 0
Summarized Data (Total Score)
Non-Metastatic: 3.7 (3-6) (n=9, 2 excluded); Metastatic: 5 (4 & 6) (n=2, 1 excluded); p=0.2

Supplementary Table 2. Grading system for Adrenal Pheochromocytomas and Paragangliomas (GAPP)

hpf= high power field; NA: (Data) Not Available; IHC: Immunohistochemistry; PC =Pheochromocytoma; PGL=Paraganglioma; NS*= Not Scored (due to lack of data on functional status).

Mitosis/10 hpf & SDHB IHC scores are depicted here, but are not a component of the GAPP scoring scheme.

Architectural pattern: Zellballen (Score0); Large & Irregular Cell Nest (Score1); Pseudorosette (even focal) (Score1).

Cellularity: Low cellularity (<150cells/400x field) (Score0); Moderate cellularity (150-250 cells/400x field) (Score1); High cellularity (>250cells/400x field) (Score2)

ComedoNecrosis: Absent (Score0); Present (Score2)

Vascular/ Capsular Invasion: Absent (Score0); Present (Score1)

Ki67 Labeling Score: Ki67 <1% (Score 0); Ki67 1-3% (Score 1); Ki67 >3% (Score 2)

Functional Status: E: Epinephrine NE: Norepinephrine, DA: Dopamine, NF: Nonfunctional. E or E+NE (Score 0); NE or NE+DA (Score 1); Nonfunctional (Score 0)

Total Score: Range 0 to 10