Lung Adenocarcinoma Biomarker Incidence in Hispanic Versus Non-Hispanic White Patients

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1 Lung Adenocrcinom Biomrker Incidence in Hispnic Versus Non-Hispnic White Ptients Elizbeth McQuitty, MD; Wei Zhng, MD; Hether Hendrickson, MB, MBA; Fermin O. Tio, MD; Jishree Jgirdr, MD; Rndll Olsen, MD, PhD; Philip T. Cgle, MD Context. Lung cncer is the leding cuse of cncer deths in the United Sttes nd worldwide. Biomrker testing is criticl to personlized therpy in lung denocrcinom nd hs been extensively investigted in non- Hispnic whites, Asins, nd Africn Americns. However, little informtion ddresses the underlying genetic chnges in lung denocrcinom mong Hispnic ptients in the United Sttes. Objective. To identify trgetble biomrkers other thn EGFR nd EML4-ALK in Hispnic ptients with lung denocrcinom. Design. We tested DNA extrcted from 85 lung denocrcinom specimens collected from 40 Hispnic nd 43 non-hispnic white ptients for previously reported muttions in KRAS, MET, BRAF, mtor, STAT3, JAK2, PIK3CA, AKT1 through AKT3, nd PTEN with custom Sequenom mssarray ssy (Sequenom, Sn Diego, Cliforni). Results. Muttions in KRAS were identified in 11 cses (13%; 6 Hispnic [7%], 5 non-hispnic white [6%]) nd hd no correltion with sex, ge, or smoking history. Muttions in PIK3CA were identified in 2 of the 40 Hispnic ptients (5%), including one ptient (2.5%) with concurrent KRAS muttion. The tumors were wild type for ll other genes tested. Conclusions. Trgetble biomrkers other thn EGFR nd EML4-ALK were identified in 7 of the 40 Hispnic ptients (18%) nd 5 of the 43 non-hispnic white ptients (12%), suggesting similr muttionl frequency. Our highly multiplexed genotyping ssy detected ctionble muttions in 14% (12 of 83) more ptients thn would hve been identified by EGFR nd EML4-ALK testing lone. (Arch Pthol Lb Med. 2014;138: ; doi: / rp oa) Lung cncer is the leding cuse of cncer deths in the United Sttes 1 nd worldwide. 2 Nevertheless, disese burden is uneqully shred mong the predominnt ethnicities of the US popultion. According to the Surveillnce, Epidemiology, nd End Results dtbse, 3 non-hispnic whites nd Africn Americns hve the highest incidence nd mortlity due to lung cncer, followed by Asin/Pcific Islnders. Intriguingly, Hispnics hve lower lung cncer incidence nd mortlity thn ny of these groups. 4 Accepted for publiction My 15, Published s n Erly Online Relese June 26, From the Deprtment of Pthology, University of Texs Helth Sciences Center, Sn Antonio (Drs Zhng nd Jgirdr); the Deprtment of Antomic nd Clinicl Pthology, South Texs Veterns Helth Cre System, Sn Antonio (Dr Tio); nd the Deprtment of Pthology nd Genomic Medicine, Houston Methodist Hospitl, Houston, Texs (Drs McQuitty, Olsen, nd Cgle nd Ms Hendrickson). Dr McQuitty is now with the Deprtment of Pthology nd Immunology, Bylor College of Medicine, Houston. The uthors hve no relevnt finncil interest in the products or compnies described in this rticle. Presented s n bstrct t the nnul meeting of the College of Americn Pthologists; October 13 16, 2013; Kissimmee, Florid. Reprints: Philip T. Cgle, MD, Deprtment of Pthology nd Genomic Medicine, Houston Methodist Hospitl, 6565 Fnnin St, M227, Houston, TX (e-mil: pcgle@houstonmethodist.org). Incidence nd outcome disprities mong ptients with lung cncer of different ethnicities re likely multifctoril nd compound. However n underlying genetic bsis is likely. 5 Oncogenic muttions in EGFR hve been found in non smll cell lung cncers in only 19% of Africn Americns 6 9 nd 17% of non-hispnic whites, 10,11 compred with 66% of Asins nd 33% of Hispnics. 15,16 Furthermore, EML4-ALK rerrngements re reported in pproximtely 6% to 7% of Asins with lung denocrcinom, compred with only 1% to 2% of non-hispnic white ptients Trgeted therpies re vilble for both biomrkers, nd stndrd of cre requires testing ll lung denocrcinoms for EGFR muttions nd EML4-ALK rerrngements. Mny other driver muttions hve been described in non smll cell lung cncer, including KRAS, MET, BRAF, mtor, STAT3, JAK2, PIK3CA, AKT1 through 3, nd PTEN. Therpies trgeting ech of these biomrkers re currently vilble nd/or in development The incidence of these trgetble muttions hs been unevenly described mong ethnic groups, with prticulr pucity of informtion bout lung cncer genetics in Hispnic ptients living in the United Sttes. In this study, we investigted 85 lung denocrcinoms tken from 83 ptients, 40 Hispnic nd 43 non-hispnic white, for specific, previously reported muttions with therpeutic gents either vilble or in development, using highly multiplexed genotyping ssy. 390 Arch Pthol Lb Med Vol 138, Mrch 2014 Lung Adenocrcinom Biomrker McQuitty et l

2 MATERIALS AND METHODS With pprovl by the institutionl review bord (IRB HSC H), lung denocrcinom tumor tissues from 40 Hispnic nd 43 non-hispnic white ptients were retrieved from the surgicl pthology rchives t the University of Texs Helth Sciences Center (Sn Antonio) nd the Audie L. Murphy Veterns Affirs Hospitl (Sn Antonio). Demogrphic informtion ws collected by chrt review nd included ge t dignosis, sex, stge t dignosis, nd smoking sttus. Formlin-fixed, prffin-embedded tumor tissue ws cut in 10- lm sections onto unchrged glss slides. One slide from ech cse ws reviewed by bord-certified pthologist nd mrked for mcrodissection to enrich the smple for tumor cells compred with benign cells. The number of sections used for genomic DNA extrction rnged from 5 to 10 depending on tumor volume. Tissue ws deprffinized using Citrisolv (Fisher Scientific Irelnd, Dublin) followed by tretment in 100% ethnol. Puregene cell lysis buffer (Qigen, Almed, Cliforni) ws pplied, nd tumor tissue ws scrped from the slides nd digested with proteinse K (Qigen). The DNA ws purified using Qigen EZ1 tissue kit on n utomted worksttion ccording to the mnufcturer s instructions. The quntity nd qulity of genomic DNA (gdna) ws ssessed using NnoDrop spectrophotometer (NnoDrop Technologies, Wilmington, Delwre), nd gdna ws stored t 208C. Trget genes were selected bsed on literture serch of lung denocrcinom biomrkers with therpeutic gents either vilble or currently in development. These included KRAS, MET, BRAF, mtor (FRAP1), STAT3, JAK2, PIK3CA, AKT1 through AKT3, nd PTEN (Tble 1). A custom chip to test for these lleles ws designed using the MssARRAY Assy Design 3.0 softwre (Sequenom, Sn Diego, Cliforni). Moleculr testing ws performed t the Methodist Hospitl in Houston, Texs, using Sequenom MssARRAY instrument (Sequenom). Locus-specific polymerse chin rection nd detection primers were selected, nd lung tumor DNA ws mplified in multiplex polymerse chin rection, followed by single-bse extension rection. The resulting nucleotides were deslted nd trnsferred to 384- element SpectroCHIP rry, nd lleles were discriminted by mss spectrometry. A v 2 contingency tble nlysis ws employed to identify significnt ssocitions mong muttion sttus nd sex, ge, ethnicity, smoking history, nd stge t dignosis. Significnce ws bsed on P,.05 for the Fisher exct test. There ws one cse tht hd only PIK3CA muttion, which ws not used in the nlyses becuse of underrepresenttion. RESULTS Lung denocrcinom tumor tissue from 85 specimens representing 83 ptients, 40 Hispnic (48%) nd 43 non- Hispnic white (52%), ws vilble for testing. Of the 40 Hispnic ptients, 5 (13%) were nonsmokers, 13 (33%) were smokers, nd 22 (55%) hd unknown smoking sttus. Of the 43 non-hispnic white ptients, 8 (19%) were nonsmokers, 21 (49%) were smokers, nd 14 (33%) hd unknown smoking sttus. The 14 Hispnic femle ptients (35%) were nonsmokers or hd unknown smoking sttus. Among the 12 non-hispnic white femle ptients (28%), 2 were smokers nd 10 were nonsmokers or hd unknown smoking history. Medin ge t dignosis mong Hispnics ws 63 yers nd ws 62 yers for non-hispnic whites. KRAS muttions were present in 11 (13%) of the 83 lung denocrcinoms tested, including 6 from the 40 Hispnic ptients (15%) nd 5 from the 43 non-hispnic white ptients (12%) (P ¼.65; Tbles 2 nd 3). 24 The KRAS muttions mong Hispnics included chnges to codons 12, 13, nd 59, but only to codons 12 nd 61 mong non- Hispnic whites. Averge ge mong Hispnics with KRAS muttions ws 67 yers; verge ge mong non-hispnic whites with similr muttions ws 65 yers. Seven ptients (12% of the 57 men tested) with KRAS muttions were mle nd 4 were femle (15% of the 26 women tested) (P ¼.70; Tbles 2 nd 3). Among 11 ptients with KRAS-mutted tumors, 4 (36%) were smokers, 2 (18%) were nonsmokers, nd 5 (45%) hd unknown smoking histories (P ¼.74). PIK3CA muttions were identified in 2 Hispnic (5%) nd no (0%) non-hispnic white ptients (Tbles 2 nd 3). One encoded n E545K mino cid chnge in n 84-yer-old mle smoker, nd the other ws n H1047L chnge in 60- yer-old womn with unknown smoking sttus. A concurrent A59T KRAS muttion ws identified in the second ptient. No muttions in MET, BRAF, mtor (FRAP1), STAT3, JAK2, AKT1-3, or PTEN were detected. COMMENT According to the US Census Bureu, in 2012, pproximtely 53.3 million Hispnics live in the United Sttes nd comprise 17% of the popultion (n ¼ 313,933,954). By 2060, the US Hispnic popultion is expected to exceed million. 25 Estimtes from the Surveillnce, Epidemiology, nd End Results dtbse re tht Hispnics living in the United Sttes hve lung cncer incidence of 33.5 new dignoses per Thus, more thn Hispnic ptients in the United Sttes cn be expected to be dignosed with lung cncer this yer. Stndrds of cre mndte testing ll lung denocrcinoms for EGFR muttions nd EML4-ALK rerrngements. Mny dditionl biomrkers with ssocited trgeted therpies hve been described, nd clinicl oncologists incresingly recognize these moleculrly defined subgroups Relible informtion bout biomrker frequencies nd disprities mong ethnic groups is criticl, nd our study provides key biomrker dt bout US Hispnics with lung denocrcinom. Two studies hve previously reported muttion frequency of KRAS codons 12 nd 13 in ptients with non smll cell lung cncer in Ltin Americ. In the first of these, non smll cell lung cncer specimens from Argentin, Colombi, Peru, nd Mexico hd KRAS muttion frequency of 16.6% (n ¼ 108). In the second study, 16 KRAS muttion frequency mong 206 non smll cell lung cncer specimens from Brzilin ptients ws 14.6% (n ¼ 30). Neither study included nlysis of codons 59 nd 61, nd neither nlyzed dditionl biomrkers implicted in non smll cell lung cncer. The Hispnic popultion living in the United Sttes includes considerbly more heterogeneous popultion thn my hve been represented in previous studies, rising the possibility tht KRAS muttion frequency mong US Hispnics my differ from tht reported in Ltin Americ. In ddition to incresed diversity, our study differed from previous reports by inclusion of KRAS codons 12, 13, 59, nd 61 nd limited ptients to those dignosed with lung denocrcinom, rther thn the wider spectrum of non smll cell lung cncer. We discovered tht US Hispnic ptients with lung denocrcinom hd KRAS muttion frequency of 15% (6 of 40), consistent with the results from Ltin Americ. Notbly, 2 of 40 Hispnic ptients (5%) in our study hd KRAS codon 59 muttions, finding tht would not hve been detected in the previous studies. Absent these cses, KRAS muttion rte mong US Hispnics would hve been 10% (4 of 40), suggesting diversity of the US Hispnic Arch Pthol Lb Med Vol 138, Mrch 2014 Lung Adenocrcinom Biomrker McQuitty et l 391

3 Tble 1. Alleles Tested Gene Alleles Tested AKT1 E17K (rs ), F35L AKT2 C574-1G.T, V90L AKT3 Q124L BRAF L597S/R/Q/V V600E/K/R/L G469V/R/S/E/A G466V JAK2 V617F KRAS Composite ssys for codon 12 muttions G13V/D A59T Q61E/K/L/R/P/H MET T1010I MTOR A8S, K42M, L2201L, L888F, M1747L, S1821S PIK3CA E542K, E545K, Q546K, H1047R/L, M1043L, S541F PTEN G251C, R130G, R233* STAT3 D661V STAT4 D661Y STAT5 G402C STAT6 N647I STAT7 Y640F With Custom MssARRAY Chip on Sequenom Pltform, Sequenom, Sn Diego, Cliforni. * Indictes premture stop codon. popultion might slightly dilute the KRAS codon 12 nd 13 muttion frequency. Alterntively, the Ltin Americn popultion my hve n even higher KRAS muttion frequency thn previously reported if chnges to codons 59 nd 61 re included in future nlyses. Furthermore, codon 59 muttions my be more frequent mong Hispnics, either those living in the United Sttes or in US nd Ltin Americn popultions, thn it is mong non- Hispnic whites, n issue tht only lrger follow-up studies cn resolve. No meningful correltion between smoking history nd KRAS muttion sttus ws identified in our study. Thirty-six of 83 ptients (43%) hd unknown smoking histories, including 5 of those 36 ptients (14%) with KRAS muttions, nd the bsence of smoking history in these cses my hve compromised our bility to detect meningful correltion. We identified 2 of the 40 Hispnic ptients (5%) with PIK3CA muttions. PIK3CA is reportedly mutted in 1% to 3% of non smll cell lung cncers, most frequently in squmous cell crcinom of the lung, nd is ssocited with resistnce to EGFR tyrosine kinse inhibitors. 27 One of our 83 ptients (1%) hd concurrent PIK3CA nd KRAS muttions. In recent study, 30 mong 23 ptients with Tble 2. Cse No. Smoking Sttus Age, y/sex Lung Adenocrcinom Muttions in 40 Hispnic Ptients Stge Grouping t Dignosis Mutted Gene Muttion Additionl Mutted Gene Muttion 1 Nonsmoker 69/M IIA WT 2 Nonsmoker 75/M IIB KRAS A59T 3 Nonsmoker 53/F N/A WT 4 Nonsmoker 61/F IA WT 5 Nonsmoker 83/M IB WT 6 Smoker 71/M IIIA KRAS G12V 7 Smoker 54/M IA WT 8 Smoker 84/M IB PIK3CA E545K 9 Smoker 72/M IA WT 10 Smoker 73/M IIIA KRAS G12D 11 Smoker 68/M IIIA WT 12 Smoker 80/M IB WT 13 Smoker 79/M IIIB WT 14 Smoker 66/M IA WT 15 Smoker 55/M IV WT 16 Smoker 63/M IV WT 17 Smoker 89/M IA WT 18 Smoker 62/M IA WT 19 Unknown 63/F IIA WT 20 Unknown 55/F N/A WT 21 Unknown 53/F N/A WT 22 Unknown 62/F IA WT 23 Unknown 56/M N/A WT 24 Unknown 55/F N/A WT 25 Unknown 54/F N/A WT 26 Unknown 67/F N/A WT 27 Unknown 49/M N/A WT 28 Unknown 60/F N/A KRAS A59T PIK3CA H1047L 29 Unknown 61/M N/A WT 30 Unknown 60/M N/A KRAS G12D 31 Unknown 59/M N/A WT 32 Unknown 58/M IA WT 33 Unknown 66/F IA WT 34 Unknown 60/F IIIB KRAS G13D 35 Unknown 66/F IIB WT 36 Unknown 63/M IIB WT 37 Unknown 75/M IA WT 38 Unknown 48/F N/A WT 39 Unknown 65/M N/A WT 40 Unknown 69/M N/A WT Abbrevitions: N/A, biopsy only, stging informtion unknown; WT, wild type. Stging informtion per Americn Joint Committee on Cncer, Arch Pthol Lb Med Vol 138, Mrch 2014 Lung Adenocrcinom Biomrker McQuitty et l

4 Tble 3. Lung Adenocrcinom Muttions in 43 Non-Hispnic White Ptients Cse No. Smoking Sttus Age, y/sex Stge t Dignosis Mutted Gene Muttion 41 Nonsmoker 46/F IA WT 42 Nonsmoker 65/F N/A WT 43 Nonsmoker 60/F IIIB KRAS G12V 44 Nonsmoker 60/F IB WT 45 Nonsmoker 62/F IIB WT 46 Nonsmoker 43/F N/A WT 47 Nonsmoker 79/M IA WT 48 Nonsmoker 88/M IA WT 49 Smoker 48/M N/A KRAS Q61L 50 Smoker 62/F IA WT 51 Smoker 70/M IA WT 52 Smoker 59/M IIIA WT 53 Smoker 49/F IA WT 54 Smoker 50/M IA WT 55 Smoker 61/M IIB WT 56 Smoker 72/M IA WT 57 Smoker 80/M IA WT 58 Smoker 65/M IIIB WT 59 Smoker 64/M IB WT 60 Smoker 53/M IIIA WT 61 Smoker 78/M IB WT 62 Smoker 78/M IB WT 63 Smoker 71/M IA WT 64 Smoker 64/M IB WT 65 Smoker 83/M IB KRAS G12V 66 Smoker 63/M IA WT 67 Smoker 64/M IB WT 68 Smoker 58/M IIIA WT 69 Smoker 63/M IA WT 70 Unknown 35/M N/A WT 71 Unknown 68/M IB WT 72 Unknown 70/M IA WT 73 Unknown 62/M IB WT 74 Unknown 60/M IB WT 75 Unknown 71/F IA WT 76 Unknown 60/M IA WT 77 Unknown 60/F IV WT 78 Unknown 63/F IIA WT 79 Unknown 60/M IA WT 80 Unknown 38/M IIB WT 81 Unknown 61/M IB WT 82 Unknown 55/F IIIA KRAS G12A 83 Unknown 77/M N/A KRAS G12V Abbrevitions: N/A, biopsy only, stging informtion unknown; WT, wild type. Stging informtion per Americn Joint Committee on Cncer, Additionl Mutted Gene Muttion PIK3CA-mutted lung denocrcinom, 16 (70%) hd coexisting muttion, nd 10 of the 16 (63%) with coexisting KRAS muttion. The reminder (6 of 16; 38%) hd other concurrent muttions previously documented to occur t low frequency in lung denocrcinom. Ptients with coexisting muttions hd shorter medin survivl thn did those with isolted PIK3CA muttions. Cliniclly, identifiction of coexisting muttions in one ptient in our study my hve led to therpy directed t the mtor pthwy nd to more-ggressive therpeutic regimen. The current stndrds of cre do not require tht testing for lung denocrcinom include either of PIK3CA or KRAS testing, nd both muttions would hve been missed in directed testing for EGFR muttions nd EML4-ALK trnsloction. This cse highlights the importnce of highly multiplexed testing pltforms nd the mndte for pthologists to pursue ssys tht detect n rry of genetic chnges in wht re often smll tumor smples, including biopsies nd fine-needle spirtions. In conclusion, this study is the first, to our knowledge, to compre the genotype of lung denocrcinoms in US Hispnics compred with non-hispnic whites. Our custom, highly multiplexed, genotyping ssy found no sttisticlly significnt difference in the frequency of trgetble biomrkers in lung denocrcinoms in Hispnics compred with non-hispnic whites. These findings do not explin the lower frequency nd better survivl of US Hispnic ptients with lung cncer compred with other ethnicities. However, our findings do underscore the need to test ll ptients newly dignosed with lung denocrcinom, regrdless of ethnicity. We thnk Leif Peterson, PhD, for ssistnce with sttisticl nlysis nd Keith Newton, BS, for technicl ssistnce. References 1. Siegel R, Nishdhm D, Jeml A. Cncer sttistics, CA Cncer J Clin. 2013;63(1): Lozno R, Nghvi M, Foremn K, et l. Globl nd regionl mortlity from 235 cuses of deth for 20 ge groups in 1990 nd 2010: systemtic Arch Pthol Lb Med Vol 138, Mrch 2014 Lung Adenocrcinom Biomrker McQuitty et l 393

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