Oncogenic Mutation Screening in Solitary Fibrous Tumors

Elizabeth G. Demicco, Khalida Wani, Kenneth Aldape, Alexander J. Lazar, and Wei-Lien Wang

Solitary Fibrous Tumor (SFT)Background Phenotypic spectrum comprising classic solitary fibrous tumor

(hypocellular variant) and hemangiopericytoma (hypercellular variant)

Rarely metastasizing– Behavior can be difficult to predict

Limited data on cytogenetic abnormalities. Relatively simple genomic alterations Few recurrent features

Limited data on genetic abnormalities

PDGFRB mutations, 2/88 pleural SFT

No mutations in DDR1 (n=8), ERBB2 (n=10), FGFR1 (n=15), PDGFRB (n=39)

TP53 one case, dedifferentiated SFT of nasal cavity

Solitary Fibrous Tumor (SFT)Background

Examine a large series of solitary fibrous tumors for common oncogenic mutations.

Purpose

Screening Approach to Mutational Analysis (Sequenom MassArray)

Detection method for single nucleotide polymorphisms (SNP)

Step 1. PCR-based Allele (WT vs. mutant) specific probes Single nucleotide extension across site of SNP Different probe sizes allow for allele differentiation and multiplexed reaction

Step 2. MALDI-TOF mass spectrometry Analyze primer extension product Time of flight mass spectrometry differentiates based on probe size

Gene # Sites Alleles

AKT1 1 G49A

AKT2 1 G49A

AKT3 1 G49A

BCOR 1 A4220GCT

CC2D1A 1 C3036G

EPHA3 1 G2283TC

FOXL2 1 C402G

GRM3 1 G2608A

JAK2 1 G1849T

MGA 1 C5421A

PPP2R1A 1 T769G

RET 1 T2753C

RPL22 1 A44CGT

SFRS9 1 C722A

SMO 1 G970A

SRC 1 C1591T

TGM2 1 T734C

CDK4 2 C70T; G71A

CSMD1 2 G1225T; C9013T

FBXO4 2 T68A; C226A

FGFR1 2 C754A; C374T

FGFR2 2 T1647GA; C755G

GNA11 2 A626TC; C547T

GNAQ 2 A627T; A626TCG

Gene # Sites Alleles

CTNNB1 12 A95CGT; G94CAT, G101ATC; A107CG; T104AGC; T97GCA; T109GCA; C110GTA; T133GCA; C134GTA; A121GCT; C122TAG

KIT 12 A2447GTC; G2446CAT; A1924G; T1727C; A1696G; T2466GCA; A2464TCG; C1900T; T1676CAG ; T1679AGC ; T2474C ; T1657A

BRAF 12 A1781GT; G1756A; G1391ATC; G1397ATC; G1396CA; G1406ATC; G1405CA; A1801G; T1790GA; G1800ATC; G1800ATC; T1799ACG_F; T1799ACG_R; G1798TA

GNAS 2 G602A; C601AT

PIK3CA 28 C3137T; T1258C; G328A; G1252A; G1357A; G1624AC; A1625TG; A1634CGT; G1635CT; G1633AC; C2727G; G353A; A3140GT_F; A3140GT_R; C3139T; A2102C; G333C; G3129ATC; A3127G; T1035A; C1616G; C178A; C1636GA; A1637TCG; G263A; C1214T; A3073TG; A3062G; T3061CA

IDH1 3 G209A; C394TGA; G395AT

IDH2 4 G419TA; A514GT; G515TA; G516T

MAP2K7 4 C870T; G485A; T811A; C932T

PDGFRA 5 A2525T; G2524TA; C1977A; A1975T ; T1682A

FBXW7 6 C1393T; G1394AT; G1436AT; C1513TA; G1514ATC; C1745T

RAF1 6 G955T; C1837G; A344G; G1005C; T775G; T1018G

FGFR3 7 G1108T; G1138A; G2089T; A1949TC ; C742T ; C746G ; A1118G

KRAS 7 G436CA; G28A; G35ACT; G34ACT; G38ACT; G37ACT; C181GAT

MET 7 A3335GT; C3334T; T3803C; A1124G ; C2962T ; C3029T ; T3742CG

ALK 8 T3521GC; C3522AG; T3520CAG; T3734G; C3735AG; T3733GA; T3512A; G3824AT

EGFR 8 G2155TA; A2579T; T2573G; T2582AG; T2158C ; C2369T ; C2561T ; A2438G

NRAS 8 G436A; G35ACT; G34ACT; G38ACT; G37ACT; C181GAT; A183TCG; A182GCT

Tumor Characteristics

122 tumors (105 patients) 72 Primary 9 Local recurrence 41 Metastasis

13 Patients with multiple tumors tested 6 primary and metastasis(es) 5 multiple metastases 2 local recurrence(s) +/- metastasis

Site Number of Patients

Gender (% F)

Age at first diagnosis, years (median, range)

All 105 48.5% 53, 16-89

Meningeal 24 38.9% 45, 16-89

Pleural 22 36.4% 63, 35-81

Intra-abdominal

27 51.9% 53, 27-71

Extremity 15 70.0% 57, 31-69

Trunk 10 53.3% 51, 29-78

Head and neck

7 57.1% 56, 39-72

Patient Demographics

Variable Run 1 Run 2 Total

Number of cases (each in duplicate)

31 96 127

Total number of polymorphism sites tested

175 (41 genes) 174 (41 genes) 174/175

% Successful reads 96.5% 91.9% 93.0%

Duplicate concordance success rate (out of all possible reads) 94.4% 86.5% 88.4%

Duplicate failure rate due to no result one or both duplicates (out of all possible reads)

5.3% 13.2% 11.3%

Duplicate discordance rate (WT/mut read) (out of all possible reads) 0.3% 0.3% 0.3%

General Quality Control Indicators

Run 1 Run 2 Total

Number SNPs detected (% of all useable reads)

12 (0.23%)

21 (0.15%)

33 (0.17%)

Site

ALK T3733G (p.F1245V) - 1 1BRAF T1799A* (p.V600E) 2 1 3KRAS G35A (p.G12D)** 1 10 11KRAS G34A (p.G12S)** - 1 1KRAS C181A (p.Q61K) - 1 1MET C2962T (p.R988C) 1 1 2MET C3029T (p.T1010I) - 3 3NRAS G35A (p.G12D)** 7 3 10NRAS G38A (p.G13D)** 1 - 1

* Mutation identified on forward probe, WT on reverse** Problems with probes later reported – Determined to be WT by CAST-PCR

Potential Mutations Identified by Sequenom

Characteristics of Tumors with Identified SNPs

* Presence of SNP did not correlate with c-MET expression by IHC

Patient (phenotypic variant)

Mutation Tumor status Primary Site Site this metastasis

Outcome

SFT118 (hypercellular)

MET C3029T (p.T1010I)*

Metastasis (10 yr)

Intra-abdominal

Peritoneum DoD 18 yr

SFT118 (hypercellular)

MET C3029T (p.T1010I)*

Metastasis(14 yr)

Intra-abdominal

Peritoneum DoD 18 yr

SFT055 (hypocellular)

MET C3029T (p.T1010I)*

Primary Intra-abdominal

Dead other causes 30 months

SFT048 (hypercellular)

MET C2962T (p.R988C)*

Primary Intra-abdominal

Metastasized at 40 mo.DoD 8.5 yr

SFT092 (hypocellular)

MET C2962T (p.R988C)*

Primary Pleura LTFU 1 month

SFT067 (hypercellular)

ALK T3733G (p.F1245V)

Primary Intra-abdominal

NED 69 mo

SFT112 (hypercellular)

KRAS C181A (p.Q61K)

Metastasis (13 yr)

Intra-abdominal

Peritoneum Dod 16 yrs

MET C3029T (p.T1010I) and C2962T (p.R988C)

Also designated as T992I and R970CJuxtamembrane domain

Initially reported as rare somatic oncogenic mutations Identified in a wide range of tumors Frequency in involved cancers ~1-10%

Later proposed to represent germline polymorphisms Seen in ~1% of individuals without cancer (1/96) Germline in several patients with cancer No evidence of transformation in cell models

T1010I as cooperative germline oncogenic mutation? Identified in ~4.5% familial CRC Mutation proposed to indirectly activate c-MET via inhibition of inhibitor Not initiator, may promote progression

ALK T3733G (p.F1245V)

Kinase-activating mutation Rare mutation Reported in neuroblastoma

KRAS C181A (p.Q61K)

Activating mutation reported rarely in colonic and lung adenocarcinoma, misc. other carcinomas various sites Much more rare than the equivalent NRAS mutation

Summary

We performed screening SNP analysis in a large series of SFT

Confirmed that SFT have low frequency of mutations in oncogenes commonly reported in other malignancies

7/122 cases (5.7%)Abdominal location predominantInsufficient data on relevance to status, prognosis

Conclusions Mutations in commonly identified oncogenes do not appear to function in pathogenesis of SFT.

Alternative mechanisms? Mutations in uncommon genes

Imprinting

miRNA regulation

Studies are ongoing

Thank You.