ANTICANCER RESEARCH 34: 5105-5110 (2014) Sorafenib Efficacy in Thymic Carcinomas Seems Not to Require c-KIT or PDGFR-alpha Mutations MARIA PAGANO1, NURIA MARIA ASENSIO SIERRA1, MICHELE PANEBIANCO1, GIULIO ROSSI2, ROBERTA GNONI1, GIANCARLO BISAGNI1 and CORRADO BONI1 Department of Oncology and Advanced Technologies, 1Oncology Unit and 2Operative Unit of Pathology, Azienda Ospedaliera S.Maria Nuova/IRCCS of Reggio Emilia, Reggio Emilia, Italy Abstract. Purpose: To retrospectively evaluate sorafenib in the United States) (1), they are among the most common activity and safety in patients with metastatic thymic mediastinal primary tumours with up to 50% of anterior carcinoma (TC) and to correlate outcome with c-KIT and mediastinal masses proving to be of thymic origin. Males PDGFR-alpha mutational status. Patients and Methods: have a slightly higher risk of developing thymic carcinomas Patients with metastatic thymic carcinoma treated with than females and this risk rises with age, reaching a peak in sorafenib after at least one prior line of chemotherapy were the seventh decade of life. included. Objective response rate (ORR) and toxicity were Thymic carcinomas (TC) are aggressive neoplasms with evaluated. Analysis of c-KIT and PDGFR-alpha mutational poor prognosis and limited effective therapeutic options status was performed retrospectively. Results: From (2-5). October 2007 to August 2011, 5 patients with metastatic In early stages, surgery is the standard treatment, while thymic carcinoma were evaluated. A median of 8 cycles of systemic chemotherapy is mainly reserved for patients with sorafenib (range=3–29) were administered. Two patients an unresectable or recurrent disease (1, 2). (40%) displayed a partial response (PR), two patients Current regimens consist of a combination of drugs almost presented stable disease (SD), while one patient had always including a platinum compound; other effective drugs progression. The median progression-free (PFS) and overall are doxorubicin, etoposide, cyclophosphamide, epirubicin survival were 28 weeks and 92 weeks, respectively. At and vincristine. The response rate with this regime is 50- mutational analysis, only one patient with PR had c-KIT 90%, with a median survival of 12-48 months (6-8). mutation in exon 17 and was successfully treated with Expression of the protein c-KIT, the product of the proto- sunitinib for 12 months after progression to sorafenib. No oncogene c-KIT immunohistochemically-recognized by the PDGFR-alpha mutations were found. Conclusion: antibody CD117, has been reported in a wide variety of Sorafenib activity seems independent from the c-KIT and human solid tumours, including TC type C, and is thought PDGFR-alpha mutational status. After progression, to play an important role in multiple cellular functions like sequence treatment with a different tyrosine kinase inhibitor survival, adhesion, differentiation, proliferation and can be considered. These results are promising and need functional maturation (9). further confirmation on larger, possibly prospective, series Several studies have analyzed the presence of c-KIT of patients. mutations in TC, evidencing that mutational alterations, although uncommon, are present in about 10% of cases (10- Although thymic malignancies are relatively rare (0.2% to 13). Promising results have been previously published in 1.5% of all malignancies or 0.13 per 100,000 person-years patients with TC harboring c-KIT mutations and treated with selective tyrosine kinases inhibitors (TKI), namely sunitinib (14, 15), imatinib (16) and sorafenib (17, 18). Based on the aforementioned data and our own clinical Correspondence to: Dr. Pagano Maria, Department of Oncology experience with one patient treated with sorafenib (17), we and Advanced Technologies, Oncology Unit, Azienda Ospedaliera decided to treat with sorafenib a series of patients with TC S. Maria Nuova/IRCCS , Viale Risorgimento, 80 42123- Reggio type C. Emilia, Italy. Tel: +39 0522296628, Fax: +39 0522296854, e-mail: In this brief manuscript we report the efficacy and toxicity [email protected] results obtained with sorafenib in this oncological setting, Key Words: Thymic carcinoma, c-KIT, tyrosine kinase, PDGFR also investigating the correlation between clinical response alpha, sorafenib. and mutational status of c-KIT and PDGFR-alpha genes. 0250-7005/2014 $2.00+.40 5105 ANTICANCER RESEARCH 34: 5105-5110 (2014) Table I. Patients’ characteristics and outcome. Patient Gender Age Previous Stage Histology Status PS Response Toxicity TTP Following Survival No. yr therapy c-Kit/PDGFR (weeks) therapy (weeks) 1 M 50 Debulking; PEI; IVB liver, Νeuro- c-Kit 1 RP Ipertension, 116 Sunitinib; 180 metastasectomy; PEI; lung, endocrine Εxon 17 skin toxicity PEI metastasectomy; brain G2 IFO-ADR; longostatina; RT 2 M 61 Debulking; IVB lung B2 Wild-type 0 PD Diarrhea G2; 12 None 60 RT; PEI. HFS G2 3 F 75 ADOC; RT; IVB lung Epidermoid Wild-type 0 RP HFS G3; 28 None 76 ADOC Diarrhea G3 4 M 70 CHOP; DDP-VP; IVB lung, Thymus Wild-type 2 SD None 32 Novantrone 92 Debulking; adrenal carcinoma Mitoxantrone; PEI. 5 F 66 Debulking; IVB pleura,B2 Wild-type 1 SD None 16 Adriamicina- 116 PEI; RT; soft tissue 5FU; Metastesectomy; Imatinib; PEI; RT. Paclitaxel w PR, Partial response; PD, progressive disease; SD, stable disease; RT, radiotherapy; PEI, cisplatin, etoposide, ifosfamide; IFO-ADR, ifosfamide, adriamicina; CHOP, adriablastina, cisplatin, vincristine, prednisone; DDP-VP, cisplatin, etoposide; ADOC, cisplatin, doxorubicin, vincristine, cyclophosphamide; TTP, time-to-progression. Patients and Methods embedded block were applied on noncover-slipped slides for microdissection and DNA extraction. Briefly, microdissection was Patients. From October 2007 to August 2011 5 patients with performed under direct observation with an inverted microscope metastatic TC refractory to conventional treatment were treated with using a sterile needle. Each microdissected sample was directly sorafenib in our Institution. transferred to an Eppendorf tube containing digestion buffer (2 All patients had a diagnosis of type C TC according to the 2004- mg/ml proteinase K in 50 mM Tris (pH 8.5), 1 mM EDTA, 0.5% World Health Organization (WHO) classification of thymic tumors. Tween 20). The tubes were then incubated overnight at 56˚C and In all cases, surgical specimens were fixed in 10% buffered-formalin followed by 10 min of incubation at 95˚C to eliminate any remaining and then routinely paraffin-embedded. Immunohistochemistry was proteinase K activity. Polymerase chain reaction) (PCR) was performed using an automated immunostainer (BenchMark, Ventana, performed in 20 μl reactions containing 2.0 μl DNA, 2 μl of Tucson, AZ, USA). The primary antibodies used in all cases were commercial PCR buffer (Applied Biosystems, Foster City, CA, the following: CD117 (clone A4502, Dakopatts, Glostrup, Denmark; USA), 1.0 to 2.0 mM of MgCl2, 200 mM of each dNTP, 20 pmol of 1:200 dilution without antigen retrieval), CD5 (clone SP19, Ventana; each primer, and 3 units of AmpliTaq Gold polymerase (Applied prediluted with microwave antigen retrieval), p63 (clone 4A4, Biosystems). The PCR reaction was carried out on Uno II NeoMarkers, Fremont, CA, USA; 1:400 dilution with microwave Thermoblock (Biometra, Gottingen, Germany). Initial denaturation antigen retrieval), chromogranin (clone LK2H10, Ventana; prediluted at 95˚C for 10 minutes was followed by 41 cycles and a final with microwave antigen retrieval), synaptophysin (polyclonal, extension step (10 minu at 72˚C). The cycles included denaturation Ventana; prediluted with microwave antigen retrieval). at 95˚C for 1 minute, annealing at 55˚C to 66˚C for 1 minute, and The institutional Ethical Committee of our Hospital (Comitato extension at 72˚C for 2 min. The amplified DNA was Etico Provinciale – IRCCS Arcispedale S. Maria Nuova – Reggio electrophoresed on 2% agarose gel for 1 hour at 110 V. The Emilia) approved the treatment and patients gave written informed amplification products were then purified by using the MinElute consent. PCR purification Kit (Qiagen, Valencia, CA, USA) as indicated by All clinicopathological features are reported in Table I. the manufacturer’s instructions. PCR products were then sequenced in both directions with ABI Prism BigDye Terminator v1.1 Cycle Treatment. Treatment consisted in 400 mg of sorafenib orally twice Sequencing kit (Applied Biosystems), using the same primers as daily continuously until progression or unacceptable toxicity. The those employed for PCR. The PCR products were finally purified by response to treatment was evaluated according to Response Centri-Sep Spin Columns (Applied Biosystems) and subsequently Evaluation Criteria In Solid Tumors (RECIST) 1.1 criteria and the ran on the ABI Prism 310 automatic sequencer (Applied toxicity was monitored as clinical practice. Biosystems). The data were analyzed with the Sequencing Analysis 5.2 Software (Applied Biosystems). The forward and reverse DNA extraction and mutational analysis. Five-μm thick, oligonucleotide primers used to amplify c-KIT exons 9, 11, 13, 14 hematoxylin-eosin-stained sections from a representative paraffin- and 17, and PDGFRα exons 12, 14 and 18 are listed in Table II. 5106 Pagano et al: Sorafenib Efficacy in Thymic Carcinomas Table II. Oligonucleotide primers used in the study. Primer Name Primer sequence Annealing temperature Product size (bp) Exon 9 c-KIT Forward 5’- ATG CTC TGC TTC TGT ACT GCC -3’ Reverse 5’- CAG AGC CTA AAC ATC CCC TTA-3’ 58˚C 238 bp Exon 11 c-KIT