quantiﬁcation of anaplastic lymphoma kinase protein … · 2020. 3. 11. · quantiﬁcation of...

Quantification of Anaplastic Lymphoma KinaseProtein Expression in Non–Small Cell Lung Cancer

Tissues from Patients Treated with CrizotinibTodd Hembrough,1,2 Wei-Li Liao,1,2 Christopher P. Hartley,3,9 Patrick C. Ma,4,5,10 Vamsidhar Velcheti,5

Christopher Lanigan,6 Sheeno Thyparambil,1,2 Eunkyung An,1,2 Manish Monga,7 David Krizman,1,2

Jon Burrows,1 and Laura J. Tafe3,8*

BACKGROUND: Crizotinib has antitumor activity inALK (anaplastic lymphoma receptor tyrosine kinase)-rearranged non–small cell lung cancer (NSCLC). Thecurrent diagnostic test for ALK rearrangement isbreakapart fluorescence in situ hybridization (FISH),but FISH has low throughput and is not always reflec-tive of protein concentrations. The emergence of mul-tiple clinically relevant biomarkers in NSCLC neces-sitates efficient testing of scarce tissue samples. Wedeveloped an anaplastic lymphoma kinase (ALK) pro-tein assay that uses multiplexed selected reaction mon-itoring (SRM) to quantify absolute amounts of ALKin formalin-fixed paraffin-embedded (FFPE) tumortissue.

METHODS: After validation in formalin-fixed cell lines,the SRM assay was used to quantify concentrations ofALK in 18 FFPE NSCLC samples that had been testedfor ALK by FISH and immunohistochemistry. Resultswere correlated with patient response to crizotinib.

RESULTS: We detected ALK in 11 of 14 NSCLC sampleswith known ALK rearrangements by FISH. AbsoluteALK concentrations correlated with clinical response in 5of 8 patients treated with crizotinib. The SRM assay didnot detect ALK in 3 FISH-positive patients who had notresponded to crizotinib. In 1 of these cases, DNA se-quencing revealed a point mutation that predicts a non-functional ALK fusion protein. The SRM assay did notdetect ALK in any tumor tissue with a negative ALKstatus by FISH or immunohistochemistry.

CONCLUSIONS: ALK concentrations measured by SRMcorrelate with crizotinib response in NSCLC patients.The ALK SRM proteomic assay, which may be multi-plexed with other clinically relevant proteins, allows forrapid identification of patients potentially eligible for tar-geted therapies.© 2015 American Association for Clinical Chemistry

Targeted cancer therapies designed to disrupt proteins inoncogenic signaling pathways are the current focus ofcancer drug development. Several targeted therapy regi-mens have been approved by the US Food and DrugAdministration for the treatment of advanced lung can-cer. The first targeted therapy regimens are the epidermalgrowth factor receptor (EGFR)11 tyrosine kinase inhibi-tors (TKIs), such as erlotinib and afatinib, which showefficacy against tumors harboring activating mutations inEGFR (epidermal growth factor receptor)12 kinase do-main. In 2013, ALK (anaplastic lymphoma receptor ty-rosine kinase) 2p23 rearrangements were validated asadditional molecular targets in TKI-based therapy forlate-stage non–small cell lung cancer (NSCLC) with USFood and Drug Administration approval of the anaplas-tic lymphoma kinase (ALK) inhibitor crizotinib. Thiswas followed by the recent approval of the second-generation ALK TKI ceritinib, which is active in the ma-jority of patients who have acquired crizotinib resistance(1, 2 ). Other therapeutic agents for patients with ALK-rearranged disease are currently in clinical trials (3 ).

1 OncoPlex Diagnostics, Rockville, MD; 2 NantOmics, LLC, Rockville, MD; 3 Department ofPathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH; 4 Department of Trans-lational Hematology and Oncology Research, Taussig Cancer Institute, 5 Department ofHematology and Medical Oncology, Taussig Cancer Institute, and 6 Robert J. TomsichPathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH; 7 MaryBabb Randolph Cancer Center, West Virginia University, Morgantown, WV; 8 GeiselSchool of Medicine at Dartmouth, Hanover, NH; 9 current affiliation: Department of Pa-thology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI; 10 cur-rent affiliation: Mary Babb Randolph Cancer Center, West Virginia University, Morgan-town, WV.

* Address correspondence to this author at: Department of Pathology, Dartmouth-Hitchcock Medical Center, One Medical Center Dr, Lebanon, NH 03756. [email protected].

Received July 2, 2015; accepted October 15, 2015.Previously published online at DOI: 10.1373/clinchem.2015.245860© 2015 American Association for Clinical Chemistry11 Nonstandard abbreviations: EGFR, epidermal growth factor receptor; TKI, tyrosine ki-

nase inhibitor; NSCLC, non–small cell lung cancer; ALK, anaplastic lymphoma kinase;FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; FFPE, formalin-fixed paraffin-embedded; SRM, selected reaction monitoring; MS, mass spectrometry;BLAST, Basic Local Alignment Search Tool; Pfu, Pyrococcus furiosus complex matrix;LOD, limit of detection; LOQ, limit of quantification.

12 Human genes: ALK, anaplastic lymphoma receptor tyrosine kinase; EML4, echinodermmicrotubule associated protein-like 4; EGFR, epidermal growth factor receptor; KRAS,Kirsten rat sarcoma viral oncogene homolog; BRAF, B-Raf proto-oncogene, serine/threonine kinase.

Clinical Chemistry 62:1252–261 (2016)

Cancer Diagnostics

252

ALK rearrangements are found in 2%–5% ofNSCLC patients; most patients are relatively young, witha history of never or light smoking, and have tumors withnonsquamous histology. Current evidence-based guide-lines recommend testing all patients with advanced,nonsquamous NSCLC for ALK rearrangements (4 ). Inpatients with ALK-rearranged NSCLC, crizotinib yieldsbetter response rates (65% vs 20%) and higherprogression-free survival (7.7 vs 3 months) than standardcytotoxic chemotherapy (5 ).

Under typical physiological conditions, ALK is notexpressed in the adult lung, whereas it is embryonicallyexpressed in early developmental regulation. ALK rear-rangement leads to aberrant expression of the ALK fusionprotein and constitutive activation of the ALK kinasedomain (5, 6 ). Oncogenic activation of ALK occurs ow-ing to intrachromosomal inversion in chromosome 2,leading to fusion of the tyrosine kinase domain of ALKwith a 5� end partner such as EML4 (echinoderm micro-tubule associated protein-like 4). EML4 is the most com-mon ALK fusion partner in NSCLC; however, �20EML4-ALK transcript variants have been described, and�6 other partner genes have been identified (7–11). Theclinical significance of different types of ALK rearrange-ments is under investigation (12 ).

Presently, fluorescence in situ hybridization (FISH)is the standard test to detect ALK rearrangements. TheALK Break-Apart FISH Probe Kit (Abbott Molecular)was used in clinical trials of crizotinib and approved bythe Food and Drug Administration as a companion di-agnostic in 2013. However, the presence of ALK generearrangement is not always reflective of ALK proteinconcentrations. In patients with advanced disease, a smalltissue biopsy is often the only material available; hence,extracting as much phenotypic and molecular informa-tion as possible from a limited tissue sample is desirableand warranted.

Although ALK FISH detects the breakpoint of theALK gene, ALK immunohistochemistry (IHC) detectsALK protein overexpression. Multiple studies have dem-onstrated ALK IHC to be tightly correlated with ALKFISH, suggesting that IHC could be used as the routinescreening method to identify pathologic ALK rearrange-ment in NSCLC (13–16). Consequently, many groupshave attempted to standardize and validate IHC methodsto supplement or replace ALK FISH (17–19). Othershave reported discrepancies between IHC and FISH(20–22). A study of 3244 consecutive NSCLC casesfound that FISH and IHC were discordant in 46.6% ofALK-rearranged tumors; both types of discordant cases(FISH�/IHC� and FISH�/IHC�) responded to crizo-tinib (22 ). IHC testing of a single protein requires 1–2formalin-fixed paraffin-embedded (FFPE) sections, andtesting of multiple proteins can rapidly deplete small tis-sue samples.

Both IHC and FISH have technical limitations.IHC is sensitive to preanalytical variables such as delayedfixation, fixation time, and the age of cut tissue sections(23 ). FISH is a DNA-based assay, and many preanalyti-cal tissue handling steps may lead to DNA degradationand test failure (24 ). Both FISH and IHC are semiquan-titative, and their interpretation can be somewhat subjec-tive and challenging. McLeer-Florin et al. reported that,in their hands, 19 of 100 lung adenocarcinomas analyzedby FISH were uninterpretable (16 ).

To overcome these limitations, we have developeda proteomic assay to detect and quantify ALK expres-sion in FFPE tumor tissue. Liquid Tissue® selectedreaction monitoring (SRM) is a mass spectrometry(MS)-based multiplexed assay that allows simultane-ous measurement of the absolute amount of dozens oftargeted proteins in FFPE tissues, including ALK (25–27 ). The assay results were compared with ALK test-ing results by FISH and IHC and correlated with re-sponse to crizotinib therapy.

Materials and Methods

PATIENTS AND TISSUE SAMPLES

After institutional review board approval at the medicalcenter sites, we identified FFPE tissue sections (n � 18)from 17 NSCLC patients who had been tested for ALKrearrangement in 2006–2013 (14 ALK FISH�; 4 ALKFISH�). The samples were 14 lung resection samples, 2lymph node metastases, and 2 brain metastases. Thetissues had been archived at Dartmouth-HitchcockMedical Center, Lebanon, NH (n � 12; 11 patients);Cleveland Clinic, Cleveland, OH (n � 5); and WestVirginia University (n � 1). All tissue samples andclinical annotations (extracted from medical records)were anonymized.

SAMPLE PREPARATION

Tissue sections (10 �m) were cut from FFPE blocks,placed onto the energy transfer coating of Director® mi-crodissection slides, and deparaffinized. We used lasermicrodissection to isolate tumor cells from FFPE sectionsand prepare Liquid Tissue lysates, as previously described(25–27). Total protein concentration for each lysate wasmeasured with a bicinchoninic acid protein assay (MicroBCA™, Thermo Fisher Scientific).

SRM ASSAY DEVELOPMENT

We used trypsin digestion mapping of recombinant ALKprotein (UniProtKB acc. no. Q9UM73) to identify can-didate peptides from the intracellular domain for assaydevelopment. The resulting peptides were analyzed witha TSQ Vantage triple quadrupole mass spectrometer(Thermo Scientific) equipped with a nanoAcquityLCsystem (Waters). We used Pinpoint1.0 and Xcalibur2.1

ALK Protein Quantification in NSCLC

Clinical Chemistry 62:1 (2016) 253

(Thermo Scientific) software to identify the optimaltryptic peptides for SRM analysis. Peptides containingmethionine or cysteine residues were excluded because ofongoing unpredictable oxidation. Candidate peptideswere then screened in FFPE tumor samples and in theALK-rearranged H3122 cell line to assess the capability ofthe assay for detecting ALK protein.

The selected optimal peptide, DPEGVPPLL-VSQQAK, encoded by exon 29 of the ALK gene, wasC-terminal to the ALK kinase domain and found to beunique to ALK compared to the entire human proteomewith the Basic Local Alignment Search Tool (BLAST), aspreviously described (25–27). Unlabeled (DPEGVP-PLLVSQQAK) and isotopically labeled [DPEGVPPLL-VSQQAK (13C6

15N2)] versions of this peptide weresynthesized to develop and perform the assay (Aqua Ul-timate grade peptide; Thermo Scientific). SRM transi-tions used for quantification of the unlabeled DPEGVP-PLLVSQQAK peptide were m/z 789.43/498.22 (b5

�1),540.82 (y10

�2), and 1080.64 (y10�1) (Q1/Q3), and the

transitions used for the isotopically labeled internal stan-dard were m/z 793.44/498.22 (b5

�1), 544.83 (y10�2),

and 1088.65 (y10�1) (Q1/Q3). The optimized collision

energy was 24V for all 3 transitions. We used the sameliquid chromatography gradient and MS parameters asthose previously described (25–27), except that Pronto-SIL 200-5-C18AQ reversed-phase particle (C18, 5 �m,200-Å pore size; Bischoff Chromatography) was used forcolumn packing in this study.

A calibration curve was generated in a Pyrococcusfuriosus complex matrix (Pfu; Agilent Technologies) todetermine the assay’s limits of detection (LOD) andquantification (LOQ). Briefly, we added unlabeled pep-tide to 11 separate tubes containing Pfu and a constantconcentration of isotopically labeled internal standardpeptide (5000 amol) to provide a final concentrationranging from 75 to 25 000 amol unlabeled peptide perconcentration point, including a zero sample (matrixsample processed without unlabeled peptide but with in-ternal standard). Each concentration point was run inquintuplicate on the LC-MS system.

We identified optimal quantification peptides for 11other proteins by trypsin digestion mapping of recombi-nant proteins specific for each target and similarly devel-oped an assay for each protein.

MEASUREMENT OF ALK IN FFPE TISSUE SAMPLES AND DATA

ANALYSIS

A known amount of isotopically labeled internal stan-dard peptide was added to each lysate prepared from themicrodissected tissue. We calculated the amount of ALKin each sample (attomoles per microgram of total pro-tein) from the ratio of unlabeled to labeled peak areasmultiplied by the known amount of spiked isotopicallylabeled internal standard, normalized to the amount of

total protein injected. Unlabeled and labeled peptidepeak areas were exported from Pinpoint 1.0. Lysates wereanalyzed in triplicate except for samples DH3 and DH5,for which measurements were from a single SRM analysisbecause of sample limitations.

IHC, FISH, AND DNA SEQUENCING

We selected tumor samples on which ALK IHC and/orALK FISH had been previously performed. IHC was per-formed on 4- to 5-�m-thick FFPE tumor samples withthe D5F3 ALK antibody (1:100, Cell Signaling Technol-ogy) at the respective institutions. Any ALK immunore-activity within the tumor cells was interpreted as a posi-tive result. ALK FISH results had been obtained with theALK Break Apart FISH Probe Kit (Abbott Laboratories).A minimum of 50 cells were counted by 2 observers. Theorange-green split signals and the single orange signal(with loss of the 5� green signal) were counted as rear-ranged patterns; a rearrangement rate of �15% was con-sidered positive for ALK rearrangement. DNA sequenc-ing for the region encoding the optimal ALK peptide wasperformed by Macrogen (Rockville, MD). Briefly, a210-bp DNA fragment generated by PCR amplificationwas analyzed by Sanger sequencing with a 3730XL DNAanalyzer (Life Technologies). DNA concentrations intumor tissue lysates were determined with a QubitssDNA Assay Kit (Life Technologies). Primer sequencesused for PCR amplification were ALK-F (5�-ATCTCTGCAGCTGTGGGTTT-3�) and ALK-R(5�-TGTAATCAACACCGCTTTGC-3�).

Results

TISSUE AND PATIENT CHARACTERISTICS

Eighteen tissues were identified from 17 patients withadenocarcinomas (n � 15) and adenosquamous carcino-mas (n � 2). All patients had been tested with both ALKFISH and ALK IHC except 1 with only FISH results.There were 14 ALK-rearranged cases (by FISH) and 12cases with ALK protein expression (by IHC). EightFISH-positive patients had received crizotinib treatment(Table 1).

SRM ASSAY DEVELOPMENT

For development of the ALK SRM assay, multiple pep-tides obtained from a tryptic digest of recombinant ALKwere measured with MS. When ALK translocation oc-curs, breakpoints in the N-terminal fusion partner canvary; however, the usual ALK breakpoint occurs at exon20 of the kinase domain. Consequently, we focused onpeptides within the intracellular domain including pep-tides in or C-terminal to the kinase domain. The re-sulting 2 intracellular candidate peptides, SNQEV-LEFVTSGGR and DPEGVPPLLVSQQAK, were thenextensively screened in formalin-fixed cell lines and FFPE

254 Clinical Chemistry 62:1 (2016)

Tabl

e1.

Char

acte

ristic

sofA

LKpr

otei

nco

ncen

tratio

ns(b

ySR

M),

ALK

sequ

encin

gre

sults

,ALK

FISH

,and

ALK

IHC

in18

non–

smal

lcel

llun

gca

ncer

FFPE

tissu

es.

Sam

ple

IDTu

mo

rhi

sto

log

y

Mea

nA

LKSR

Mp

rote

inco

ncen

trat

ion,

amo

l/μg

(SD

)A

LKse

que

ncin

gA

LKFI

SHA

LKIH

CC

rizo

tini

btr

eatm

ent

Ove

rall

pat

ient

resp

ons

e

Dar

tmo

uth

Med

ical

Cen

ter

DH

1A

DC

a15

3.9

(13.

5)W

TPo

sitiv

ePo

sitiv

eY

esPF

S7

mo

nths

;NED

insp

ring

2012

;lo

stto

follo

w-u

p

DH

2A

DC

261.

0(2

6.3)

NA

Posi

tive

Posi

tive

Yes

PFS

14m

ont

hs;t

hen

NED

DH

3A

DC

223.

3bN

APo

sitiv

ePo

sitiv

eY

esPF

S22

mo

nths

;the

nN

ED;p

oss

ibly

ind

epen

den

tofc

rizo

tinib

bec

ause

pat

ient

was

on

criz

otin

ibo

nly

5m

ont

hs

DH

4A

DC

(LN

)45

3.0

(28.

1)W

TPo

sitiv

ePo

sitiv

eY

esPF

S12

mo

nths

;the

nA

WD

DH

5cA

DSC

C21

6.4b

NA

Posi

tive

Posi

tive

No

Aliv

e,N

ED

DH

6cA

DSC

C(L

N)

126.

3(1

2.7)

WT

Posi

tive

Posi

tive

No

Aliv

e,N

ED

DH

7A

DSC

CN

DW

TN

egat

ive

Neg

ativ

eN

o

DH

8A

DC

(bra

in)

ND

WT

Neg

ativ

eN

egat

ive

No

DH

9A

DC

ND

HT

G->

APo

sitiv

eN

egat

ive

Yes

PD;t

hen

DO

D

DH

10A

DC

ND

WT

Neg

ativ

eN

egat

ive

No

DH

11A

DC

ND

WT

Neg

ativ

eN

egat

ive

No

DH

12A

DC

(bra

in)

ND

WT

Posi

tive

Posi

tive

Yes

PRo

fliv

erm

etas

tase

sfo

r10

mo

nths

;the

nD

OD

Cle

vela

ndC

linic

ALK

002

AD

C33

6.6

(2.5

)W

TPo

sitiv

ePo

sitiv

eY

esPF

S29

mo

nths

;ini

tialP

R,t

hen

CR

and

curr

ently

NED

ALK

081

AD

C13

8.8

(20.

7)W

TPo

sitiv

ePo

sitiv

eN

oA

live

ALK

083

AD

C15

3.9

(12.

9)W

TPo

sitiv

ePo

sitiv

eN

oA

live

ALK

516

AD

C10

6.4

(6.4

)W

TPo

sitiv

ePo

sitiv

eN

oA

live,

NED

ALK

634

AD

C13

6.3

(24.

6)W

TPo

sitiv

ePo

sitiv

eN

oO

S14

mo

nths

;DO

D

Wes

tVir

gin

iaU

nive

rsity

CD

0370

AD

CN

DN

APo

sitiv

eN

AY

esPF

S<

2m

ont

hs;D

OD

aAD

C,ad

enoc

arcin

oma;

WT,

wild

type

;PFS

,pro

gres

sion-

frees

urviv

al;N

ED,n

oev

iden

ceof

dise

ase;

NA,n

otav

aila

ble;

LN,ly

mph

node

;AW

D,al

ivewi

thdi

seas

e;AD

SCC,

aden

osqu

amou

scel

lcar

cinom

a;HT

G->

A,he

tero

zygo

usG-

>A

poin

tmut

atio

non

DNA

regi

onen

codi

ngfo

rthe

mas

s-spe

ctrom

etry

-targ

eted

pept

ide;

ND,n

otde

tecte

d;PD

,pro

gres

sive

dise

ase;

DOD,

died

ofdi

seas

e;PR

,par

tialr

espo

nse;

CR,c

ompl

ete

resp

onse

;OS,

over

alls

urviv

al.

bM

easu

rem

ents

inDH

3an

dDH

5we

refro

mas

ingl

eSR

Man

alys

is.c

Sam

ples

DH5

and

DH6

are

from

the

sam

epa

tient

.



clinical samples. The H3122 cell line expressed 396amol/�g ALK protein. Some tumors expressed amountsof ALK that were so low (�150 amol/�g) that only theDPEGVPPLLVSQQAK peptide was detected (LOD 75amol); therefore, this peptide was selected for clinicalassay development. CVs for the various concentrationpoints in the calibration curve ranged from 1.1% to20.1% for samples analyzed in quintuplicate. Theamount of light peptide recovered (attomoles) was plot-ted against the amount of light peptide spiked (atto-moles) to create a calibration curve (see SupplementalTable 1, which accompanies the online version of thisarticle at http://www.clinchem.org/content/vol62/issue1). The LOD was determined by identifying thelowest concentration in the calibration curve where theCV from replicate measurements was �25% and accu-racy from replicate measurements was �80%; the LOQwas determined by identifying the next-highest concen-tration of the calibration curve above the LOD. TheLOD and LOQ were 75 and 100 amol, respectively, witha linear regression value of R2 � 1 for the calibrationcurve with 10 spiking concentration points (Fig. 1A),and R2 � 0.93 when excluding the 3 highest concentra-tions (Fig. 1A inset). The calibration curve showed lin-earity (R2 � 0.9) and low variations over a protein con-centration range of 2 orders of magnitude.

ALK QUANTIFICATION

The SRM assay detected ALK in 11 tissue samples (from10 patients) with concentrations of 106–453 amol/�g oftumor protein (Table 1). ALK was not detected by SRMin any of the FISH�/IHC� samples (n � 4), nor was itdetected in 3 FISH� cases. Of these 3 cases in whichSRM absolute protein concentrations were discordantwith FISH, 1 was IHC�, 1 was IHC�, and 1 had notbeen tested by IHC.

To evaluate the ALK SRM assay reproducibility andprecision, ALK concentrations were measured in 8 of the14 FISH� tissues on 2 different LC-MS systems (systemsR and S) by 2 different operators. Each sample was ana-lyzed in triplicate on each system. The interinstrumentCVs for 6 measurements from the same sample were1.6%–17.7%. Results from systems R and S had an R2

value of 0.9694 (Fig. 1B, online Supplemental Table 2),demonstrating that the SRM assay reproducibly gener-ated a low level of variance between the 2 systems.

RESPONSE TO CRIZOTINIB

Of 8 patients treated with crizotinib, 5 had a positivetumor response (progression-free survival 7–29 months;1 was lost to follow up); all 5 patients were ALK-positiveby FISH, IHC, and SRM. The 3 remaining patients werediscordant between ALK protein concentrations andALK FISH and had nonresponse or partial response tocrizotinib (Table 1; Fig. 2).

VERIFICATION OF ASSAY BY DNA SEQUENCING

Because 3 of 14 ALK rearrangement–positive tissue sam-ples (DH9, DH12, and CD0370) did not have detect-able concentrations of ALK protein, we sought to inves-tigate the DNA sequence of a 210-bp region on exon 29of the ALK gene, where the ALK targeted peptide and itsflanking amino acids sequences are encoded. Thirteen ofthe 14 samples sequenced carried no mutation withinthe 210-bp region tested (Fig. 3A), but sample DH9, theFISH�/IHC�/SRM� case, was found to carry aheterozygous nonsense point mutation (G-�A) withinALK targeted peptide–encoding DNA sequence. Thepoint mutation introduced a stop codon (p.Q1429X);therefore, a nonfunctional (truncated) fusion proteinwould likely be produced, possibly resulting in total lackof any protein product (Fig. 3B).

0

10 000

20 000

30 000

0 10 000 20 000 30 000

Rec

over

ed (a

mol

)

Spiked (amol)

LOD: 75 amolLOQ: 100 amol

R2 = 1

A

0

100

200

300

400

500

0 100 200 300 400 500

Sys

tem

S (a

mol

/µg)

System R (amol/µg)

R2 = 0.9694

B

R² = 0.9315

0

200

400

600

0 200 400 600

Reco

vere

d (a

mol

)

Spiked (amol)

Fig. 1. Development of ALK SRM assay and assessment ofassay precision.(A), Calibration curve generated in Pfu. Inset, Calibration curveomitting the 3 highest concentrations (25 000, 5000, and 1000amol). (B), Assessment of assay precision and reproducibility formeasuring ALK protein concentrations in 8 ALK FISH+ NSCLC FFPEtumor tissues. Each sample was analyzed on 2 different LC-MSsystems.


http://www.clinchem.org/content/vol62/issue1

http://www.clinchem.org/content/vol62/issue1

Discussion

Quantitative mass spectrometry represents an emergingclinical method that is highly specific, reproducible, andquantitative and has decreased sensitivity to preanalyticalvariation. In addition, MS-based proteomic analysis ofFFPE tissue is capable of multiplexing analysis of 100analytes from a small amount of tissue (25–27). The costof reagents, instrument time, and personnel are greaterwith the ALK-SRM assay compared with ALK IHC.However, multiplexing the ALK protein with additionalpredictive/prognostic proteins in a single SRM assaymakes the overall cost comparable to or less than per-forming IHC or FISH for each of these proteins individ-ually. Also, in contrast to IHC, this SRM method quan-tifies proteins on the basis of a unique sequence of aminoacids, and thus does not have the same limitations astraditional antibody-based protein detection methodsand is less susceptible to problems resulting from preana-lytical tissue handling and time elapsed from tissue sec-tioning (27 ).

In this study, we evaluated ALK protein expressionin 18 NSCLC tissues (14 FISH� and 4 FISH�) withALK FISH, ALK IHC, or both. Of the 14 samples withALK rearrangement by FISH, 11 had measurable ALK

0 6 12 18 24 30

DH9

CD0370

DH12

DH1

DH4

DH2

DH3

ALK002

Duration of response (months)

337*223*261*453*154*

**

**

**

AWD

Lost to follow-up

Pa�ent ID

*amol/µg** <LOD

Fig. 2. Duration of response among patients treated withcrizotinib.Arrows indicate patients in complete remission (no evidence ofdisease). Numbers in the bar indicate ALK protein expression de-termined by SRM. Patients in green had positive response(progression-free survival 7–29 months), patient in purple(DH12) had partial response, and patients in orange had progres-sive disease while on crizotinib.

heterozygous G->A point muta�onResulted in p.Q1429X

N-terminal Fusion partner PK domainDH9 DH9

DH9: FISH+, IHC−, SRM−

* Q S V L L P P V G E P D

DH9

B

K A Q Q S V L L P P V G E P D

N-terminal Fusion partner PK domainDH5DH5

DH5: FISH+, IHC+, SRM+

DH1

A

ALK SRMtargeted pep�de

In-frame fusion site(breakpoint)

Fig. 3. Comparison of SRM with ALK FISH, IHC, and DNA sequencing in selective cases.FISH and paired IHC for patient DH5 to represent FISH+/IHC+ cases; DNA sequence within the MS targeted peptide from DH1 to representwild-type sequence. (A), ALK FISH testing shows deletion of the 5` (green) signal with retained 3` (orange) signal, consistent with rearrange-ment. Arrows indicate the rearranged red signal. (B), The FISH+, SRM−, and IHC− case (DH9) showed a nonsense point mutation that resultedin a stop codon (p.Q1429X); therefore, nonfunctional fusion protein would be produced.



protein expression by MS, whereas 3 ALK FISH� sam-ples had undetectable amounts of ALK protein. Amongthe patients who responded to crizotinib treatment, allbut 1 (5 of 6) had measureable ALK protein by massspectrometry.

Interestingly, the 3 FISH�/SRM� instances repre-sent 2 cases in which the ALK-targeted crizotinib therapywas ineffective and 1 in which a modest response was seenfollowed by prompt tumor progression. In 1 such case(DH9), the patient had immediate disease progressionupon crizotinib initiation. We confirmed nonexpressionof ALK protein by IHC in this patient, and with DNAsequencing identified a DNA point mutation in the ALKgene that predicts a truncated ALK protein. In this case,ALK protein quantities appeared to be more predictive ofresponse than FISH testing. A second patient with aFISH�/SRM� ALK profile was treated with crizotinibwithout response for a brief period (progression free sur-vival �2 months). Again, for this patient, the unsatisfac-tory crizotinib response could be predicted by the ab-sence of ALK protein expression. The third FISH�/SRM� case was ALK IHC�; this patient with stage IVdisease had a partial response of liver metastases and sta-bilization of lung disease on crizotinib therapy but died ofprogressive disease after 10 months. Of note, upon re-view of other proteins analyzed by multiplex SRM, this

patient’s tumor expressed high concentrations of METprotein (Fig. 4), a second target of crizotinib activity, anda possible explanation for the partial response in this pa-tient’s liver lesions; similar responses have been previ-ously reported (28–30).

Other studies have reported FISH� cases that werenegative for ALK by IHC (20–22), supporting the con-clusion that in such cases there is an ALK rearrangementthat leads to a nontranslated or nonfunctional fusion pro-tein. There are also reports of FISH� cases that showedgenomically complex ALK rearrangements by next-generation sequencing. In 2 separately reported cases,ALK FISH was negative, ALK IHC was positive, andnext-generation sequencing showed atypical ALK rear-rangements; both patients rapidly responded to crizo-tinib (31, 32 ), again supporting the contention that ALKexpression is necessary for crizotinib activity.

Of note, ALK SRM was evaluated against gold stan-dard FISH testing for ALK rearrangement, which is in-herently limited. In a large study of consecutive NSCLCcases tested with both FISH and IHC, 70 of 150 ALK-altered cases were detected by 1 method but not theother; either test alone would have missed approximately30% of ALK-positive cases. In a small sample of thesepatients (n � 44), both types of discordant cases(FISH�/IHC� and FISH�/IHC�) responded to crizo-

Fig. 4. NSCLC tissue expression for each of the targets in a multiplex SRM analysis, sorted by K7 expression from low to high, leftto right.The 15 samples represent a mixture of 13 ALK rearrangement positive controls and 2 ROS1 rearrangement positive controls. Note that DH12showed an increased expression of MET (highlighted in yellow).


tinib (22 ). Other studies found similar discrepancies be-tween IHC and FISH (20–22). Although we applied thebenchmark of crizotinib response/nonresponse, the re-sults of this study should be considered in light of certainlimitations. First, 1 criticism of SRM technology inFFPE tissue is its inability to identify very low expressionconcentrations (i.e., �LOD). Second, a potential prob-lem with peptide-based measurements is that any devia-tion from the canonical sequence will prevent detection;the expansion of the assay to multiple peptides, even ifthe confirmatory peptides do not have the same level ofdiagnostic sensitivity, would help to address this prob-lem. Finally, this study’s sample size was small; crizotinibresponse data from a larger group of patients will be nec-essary to confirm the clinical sensitivity, specificity andutility of the SRM assay.

To reduce testing costs, some have proposed hierar-chical ALK screening algorithms on the basis of patientcharacteristics (e.g., young age, never or light smoker),histology (adenocarcinoma), and absence of other onco-genic driver mutations (33 ). However, ALK rearrange-ments have been found in older patients and smokers(34 ), squamous cell and adenosquamous carcinoma cases(35, 36 ), and rarely, patients with mutations in EGFR,Kirsten rat sarcoma viral oncogene homolog (KRAS), andB-Raf proto-oncogene, serine/threonine kinase (BRAF)(20–22, 37, 38 ). Although the triage of samples basedon clinicopathological features does increase the successrate of detection of ALK rearrangements (33 ), it is not anoptimal strategy, since a portion of patients who could

benefit from ALK inhibitors would unavoidably be ex-cluded (4, 39, 40 ).

The ALK SRM assay was multiplexed to enable con-current assessment of clinically actionable proteins indic-ative of gene rearrangement (ALK, ROS1, RET, TRK,and others), histology markers (K7, TTF1, K5, p63),receptor tyrosine kinase targets (EGFR, HER2, HER3,MET, IGF1R, FGFR2), proteins involved in immunecheckpoints (PD-L1), and predictive/prognostic proteinmarkers for chemotherapy agents. For example, as dem-onstrated in Fig. 4, multiplex SRM analysis of biomark-ers in FFPE NSCLC tissues identified moderately highMET expression (732 amol/�g) in a brain biopsy tissueof patient who was SRM�/FISH�/IHC� for ALK. Met-astatic diseases tend to have greater tumor heterogeneity;it is possible that there was a higher expression of MET inthe metastatic liver tumor than in the primary lung tu-mor, and this may have contributed to the partial re-sponse to crizotinib (which is active against both ALKand MET).

Proteomics analysis of ALK as part of a multiplexedproteomic screening of patient tissue on initial biopsycould save time, tissue, and the expense of multiple FISHor IHC testing to detect different biomarkers. In theevent that ALK expression is detected by SRM, ALK-positive cases could be reflexed to FISH to confirm eligi-bility for treatment with ALK-targeted therapy (Fig. 5).After critical testing of the assay’s performance, the pro-posed testing protocol would provide clinicians withvaluable diagnostic information and ensure that all pa-

ALK SRM assay(mul�plex proteomic assay)

SRM Nega�ve

Pla�num-based ortargeted therapy

based on SRM data

Lung Cancer (NSCLC)

SRM Posi�ve

Posi�ve

ALK Rearrangement Posi�ve(ALK-targeted therapy)

ALK FISH Test

Nega�ve

ALK Rearrangement Nega�ve

Fig. 5. Potential ALK testing algorithm in NSCLC.The proposed strategy would allow the opportunity for patients with lung cancer to be tested for ALK expression and multiple predictive/prognostic biomarkers.



tients whose lung cancers express ALK and other clini-cally actionable markers have the opportunity to receivetreatment.

Author Contributions: All authors confirmed they have contributed to theintellectual content of this paper and have met the following 3 requirements: (a)significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intel-lectual content; and (c) final approval of the published article.

Authors’ Disclosures or Potential Conflicts of Interest: Upon man-uscript submission, all authors completed the author disclosure form. Dis-closures and/or potential conflicts of interest:

Employment or Leadership: T. Hembrough, NantOmics; W.-L.Liao, NantOmics; S. Thyparambil, NantOmics; E. An, NantOmics;D. Krizman, NantOmics; J. Burrows, OncoPlex Diagnostics.Consultant or Advisory Role: None declared.Stock Ownership: T. Hembrough, NantOmics; W.-L. Liao, Nant-Omics; S. Thyparambil, NantOmics; E. An, NantOmics; D. Krizman,NantOmics.Honoraria: None declared.Research Funding: NantOmics.Expert Testimony: None declared.Patents: T. Hembrough, W.-L. Liao, S. Thyparambil, and D. Krizmanare coinventors of PCT/US2014/031138.

Role of Sponsor: Dartmouth and NantOmics codesigned the study.NantOmics played no role in the choice of enrolled patients. Dart-mouth and NantOmics both reviewed, interpreted data, and approvedthe manuscript.

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