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Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma
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Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma Q12
Qiushi Wang,* Xin Yang,* Yong He,y Qiang Ma,* Li Lin,* Ping Fu,* and Hualiang Xiao* From the Departments of Pathology* and Respiration,y Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, China Accepted for publication April 14, 2015.
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Address correspondence to Hualiang Xiao, Department of Pathology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, 10#, Changjiangzhilu, Daping, Yuzhong District, Chongqing 400042, China. E-mail: [email protected].
Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive nonesmall-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients. (J Mol Diagn 2015, -: 1e6; http://dx.doi.org/10.1016/j.jmoldx.2015.04.002)
Lung cancer is the leading cause of cancer-related death worldwide. Recently, successful strategies to improve personalized lung cancer therapy have focused on the epidermal growth factor receptor (EGFR)etargeted therapies, such as tyrosine kinase inhibitors (TKIs) (gefitinib or erlotinib).1 A recent study suggested that a gene rearrangement comprising portions of the EML4 gene and the ALK gene in nonesmall-cell lung cancer (NSCLC) cells might be a promising therapeutic target and a diagnostic molecular marker.2 When compared with chemotherapy, crizotinib (a targeted inhibitor of ALK and C-met) used in second-line treatment significantly prolonged progression-free survival (PFS), increased response rates, and improved the quality of life in patients with advanced and ALK-positive NSCLC.3 ALK-rearranged lung cancer can be identified by fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and RT-PCR. FISH is recommended by the National
Comprehensive Cancer Network Clinical Practice Guidelines in Oncology for NSCLC (version 3, 2014) for the clinical diagnosis of ALK-positive lung cancer. However, FISH requires specialized technical resources and expertise.4 FISH diagnosis is very subjective, which limits accurate quantification. IHC is relatively inexpensive and can be routinely performed on formalin-fixed, paraffin-embedded (FFPE) tissues. However, IHC is a rapid screening method but associated with relatively low sensitivity and specificity, except when the ALK monoclonal antibody D5F3 and the Ventana Diagnostic System (F. Hoffmann-La Roche AG, Basel, Switzerland) were Q5 used.5 RT-PCR is commonly used to identify known fusion Supported by grant 81472189 from the National Natural Science Foundation of China (Y.H.) and grant 2012AA02A201 from 863 Major Project of China (Y.H.). Q1 Disclosures: None declared.
Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2015.04.002
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Wang et al Table 1 Clinical Profile of Genetically Screened Patients with Lung Adenocarcinoma Characteristic Sex Male Female Smoking status Never-smokers Smokers Specimen type Surgical Biopsy Total
No. (%) of patients, N Z 103
No. (%) ALK (FISH) positive
51 (49.5) 52 (50.5)
8 (15.7) 6 (11.5)
66 (64.1) 37 (35.9)
9 (13.6) 5 (13.5)
57 (55.3) 46 (44.7) 103
9 (15.8) 5 (10.9) 14 (13.6)
FISH, fluorescence in situ hybridization.
variants of ALK gene rearrangements with high sensitivity but semiquantitatively.6 Treatment with EGFR TKIs was effective in advanced NSCLC because of the abundance of EGFR mutations.7 The median PFS and OS rates of patients with low abundance of EGFR mutations were shorter than those with high abundance but significantly longer than those with wildtype tumors. It is still unclear if the abundance of ALK gene rearrangements was predictive of treatment efficacy with crizotinib. Unfortunately, the current methods are not adequate to detect the abundance of ALK gene rearrangements accurately. Droplet Digital PCR (ddPCR), a recently developed technique, involves emulsification and PCR amplification inside thousands of small droplets, each droplet containing one or no molecules of target DNA or RNA.8 Precise and absolute quantification of the number of target DNA or RNA molecules in the reaction is simply achieved by counting the number of positive and negative droplets.9 The strategy reduces competitive amplification, allowing detection of 0.001% mutant fractions, which is 1000 times lower than real-time PCR. In this study, we used ddPCR to reveal a highly sensitive and specific detection of EML4-ALK gene rearrangements in mRNA derived from clinical lung adenocarcinoma FFPE samples. We further discuss the potential benefit of crizotinib in the treatment of patients with low copy numbers of ALK rearrangements.
and smoking history of the patients were obtained from their medical records.
RNA Isolation and cDNA Synthesis Total RNA was extracted and purified from freshly cut FFPE tissue sections using the RNeasy kit (Qiagen, Valencia, CA), after ascertaining the tumor percentage in each specimen. The RNA quality was verified by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA), and not more than 1 mg of RNA was reverse transcribed to cDNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA). The total volume of cDNA obtained from the reverse Q6 transcription reaction was 20 mL.
FISH FISH was performed on unstained 4-mm-thick FFPE specimens using the Vysis LSI ALK dual-color, break-apart FISH probes (Abbott Molecular, Des Plaines, IL), according to the Q7 manufacturer’s instructions. Slides were read on a fluorescence microscope (BX51; Olympus, Tokyo, Japan) and independently evaluated by two experienced pathologists. Q8 Tumor cells containing at least one pair of green and orange signals, split apart by 2 signal diameters, or a single orange signal without the corresponding green signal were diagnosed as positive for ALK gene rearrangement. The criteria of 15% break-apart signals or orange single signals isolated in 50 tumor cells were used as the ALK-positive cutoff levels.10
ddPCR Workflow
This study was approved by the Clinical Ethics Committee of Daping Hospital and Research Institute of Surgery, Third Military Medical University. Signed informed consent was obtained from all patients. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma patients between January 2013 and February 2014. They were randomly selected after diagnosis of lung adenocarcinoma, and none of them had received ALK inhibitor treatment or chemotherapy before surgery or biopsy. The clinical data
The ddPCR was performed as previously described.11,12 TaqMan reaction mixture was assembled from a 2 ddPCR master mix (BioRad Laboratories, Hercules, CA), 20 primers and probes (final concentrations of 900 and 250 nmol/L, respectively), and 1 mL of cDNA in a final volume of 20 mL. Each reaction mixture was loaded into a sample well of an eight-channel disposable droplet generator cartridge (BioRad Laboratories). The emulsified samples were generated from a droplet generator (QX100) and then transferred onto a 96-well plate. After heat-sealing with a foil seal, the emulsified samples underwent a 2-step thermal cycling protocol in a C-1000 touch thermal cycler (BioRad Laboratories) as follows: 95 C 10 minutes, 40 cycles of 95 C 30 seconds and 60 C 60 seconds (ramp rate set to 2 C per second), and 98 C 10 minutes. The 96well droplet PCR plates were loaded into a droplet reader, which automatically reads the droplets from each well of the plate. Analysis of the ddPCR data were performed with QuantaSoft analysis software (BioRad Laboratories). Three Q9 main variants of EML4-ALK were analyzed in this study, and forward primers of these variants were as follows: 50 AGCCCACACCTGGGAAAGGAC-30 for variant 1, 50 CAGCTACATCACACACCTTGACTGGT-30 for variant 2,
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Materials and Methods Patients and Tissues
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ddPCR Quantification of EML4-ALK
and 50 -CTTGGGAAAATTCAGATGATAGCCG-30 for variant 3. These variants shared a unique reverse primer 50 AGCTTGCTCAGCTTGTACTCAGGG-30 and probe 50 FAM-CATGGCTTGCAGCTCCTGGTGCTTCC-TAMRA-30 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The sequence of the different primers is as follows: forward primer 50 -CATCTTCCAGGAGCGAGATCCC-30 , reverse primer 50 -ATGGTTCACACCCATGACGAACA-30 , and probe 50 -FAM-GAGTACGTCGTGGAGTCCACTGGCGTC-TAMRA-30 .
Evaluation and Statistical Analysis Two patients carried low copy numbers (<15%, based on FISH test) of ALK gene rearrangement after crizotinib treatment. Their responses were evaluated according to the Response Evaluation Criteria in Solid Tumors. PFS was assessed from the date of the TKI treatment to the date of disease progression or death. Overall survival was not measured as the end point because these two patients were still alive at the completion of the study.
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311 312 313 314 315 316 317 318 319 Figure 1 ALK gene rearrangement results for 320 103 clinical formalin-fixed, paraffin-embedded 321 samples detected by fluorescent in situ hybridiza322 tion (FISH) and Droplet Digital PCR (ddPCR). For 323 easy identification, some of the negative samples 324 (colored gray) were truncated. A: ALK status 325 detected by FISH (15% was used as the cutoff 326 point for ALK positivity). B: Normalized variant 1 (v1 nor) results of EML4-ALK. C: Normalized variant 327 2 (v2 nor) results of EML4-ALK. D: Normalized 328 variant 3 (v3 nor) results of EML4-ALK; 14 ALK329 positive samples detected by FISH (black, red, 330 green, and blue; A) were also assessed via ddPCR 331 for EML4-ALK copy number alterations. The results 332 are highlighted by the red (B), green (C), and blue 333 (D) colors. The specimen numbered 1318991 (30% 334 ALK break-apart signals by FISH test, colored 335 black; A) failed the ddPCR test and finally identi336 fied as KIF5B-ALK gene rearrangement by PCRbased direct sequencing. 337 338 339 340 341 342 343 344 345 346 347 348 We analyzed the association of ALK gene rearrangement 349 rates with clinical characteristics and smoking history, using 2 350 Pearson’s c test. P < 0.05 was considered statistically 351 significant. All analyses were performed using SPSS sta352 tistical software version 18.0 (SPSS Inc, Chicago, IL). 353 354 355 Results 356 357 Clinical Features of Tumors 358 359 The study population consisted of 51 men (49.5%) and 52 360 women (50.5%), with a mean age of 60.52 years (range, 39 to 361 77 years). On the basis of their smoking history, 66 patients 362 were classified as never-smokers (64.1%) and 37 as smokers 363 (35.9%). Among the tumor specimens, 57 were obtained by 364 surgical resection (55.3%) and 46 by biopsy (44.7%) 365 (Table 1). ½T1 366 367 368 ALK Rearrangement Assessed by FISH 369 370 Of the 103 patients, 14 (13.6%, eight men and six women) 371 were FISH positive with the dual-color, break-apart probe 372
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Wang et al 373 Table 2 Patients’ Clinical and ALK Gene Profile 374 ddPCR copy number Smoking 375 No. Age, years Sex ALK rearrangements FISH (%) (normalized) history, years Disease stage 376 1317769 72 M Variant 1 19 0.010082 30 IIIa 377 1314926 68 M Variant 1 27 0.015359 17 IIa 378 1303203 48 M Variant 1 27.6 0.053933 Non IIIa 379 1305735 77 M Variant 1 42 0.182692 30 Ia 380 c140457 66 F Variant 2 16 0.018750 Non IV 381 1309153 67 F Variant 2 26 0.044444 Non Ib 382 1318736 60 M Variant 3 15 0.003200 Non IIIb 383 1314415 76 F Variant 3 17 0.003750 Non IIIa 384 1307896 63 F Variant 3 16 0.004061 Non IIIa 385 1303308 64 M Variant 3 15 0.004384 30 IIb 386 1309028 57 M Variant 3 20 0.004663 30 IIIa 387 1315381 56 M Variant 3 33 0.089189 40 IV 388 1310046 59 F Variant 3 60 0.372222 Non Ia 389 1318991 67 M KIF5B-ALK 30 50 IV 390 1406350 58 F Variant 1 6.5 0.000103 Non IV 391 1308361 69 F Variant 3 7.1 0.000734 Non IIa 392 1303465 58 F Variant 3 7.6 0.001078 Non IIa 393 1315036* 63 M Variant 1 8 0.009386 Non IV 394 1309931 62 F Variant 1 8.2 0.000687 Non IV 395 1319454 64 F Variant 1 8.4 0.000616 Non IIIb 396 1308639 59 F Variant 1 8.6 0.001802 Non IIIb 397 1314673 55 M Variant 1 9.2 0.002336 20 IIIb 398 1306902 63 M Variant 3 9.4 0.001023 Non IIIa 399 1301868 39 M Variant 3 9.7 0.002258 Non IV 400 1306691 38 M Variant 3 9.8 0.001980 Non IV 401 1319396 70 M Variant 1 10 0.004294 Non IV 402 1303717* 42 M Variant 3 10 0.010984 30 Iay 403 1407832-1 72 M Variant 1 11.2 0.003717 40 IV 404 1308989 52 F Variant 1 12 0.001910 Non IV 405 1317381 68 M Variant 2 12 0.004918 20 IV 406 407 *These two patients had received crizotinib treatment after unsuccessful chemotherapies and had partial response and stable disease, respectively. y 408 IV when he received crizotinib treatment. 409 F, female; M, male; ddPCR, Droplet Digital PCR; FISH, fluorescence in situ hybridization. 410 411 ½F1 (Table 1 and Figure 1D). The ALK-positive rates in men and the positive cases mentioned above, 16 patients were 412 detected with low copy numbers of EML4-ALK rearrangewomen were 15.7% and 11.5%, respectively, with no sig413 ments (nine with variant 1, one with variant 2, and six with nificant differences. Either tumor heterogeneity or insuffi414 variant 3), which were tested by FISH with break-apart cient tumor cells might have contributed to the lower 415 signals <15%. Considering the limited ALK-positive cases ALK-positive rate in biopsy specimens (10.9%) compared 416 and their crizotinib treatment data, the EML4-ALK positive with surgical specimens (15.8%), with no significant dif417 threshold of ddPCR cannot be safely determined currently. ferences. No significant correlation was seen between the 418 ALK-positive rate and smoking history. 419 420 Patients with Low Copy Numbers of EML4-ALK Gene 421 ddPCR Detection of EML4-ALK Rearrangement Rearrangement Respond Well to Crizotinib 422 423 Three variants of the EML4-ALK rearrangements were Two patients with low copy numbers of EML4-ALK gene 424 detected in this study. GAPDH was used as the reference rearrangements (ALK rearranged cells ratio <15% by FISH and 425 gene. Eight samples of reactive lymph nodes that were used EML4-ALK/GAPDH as 0.011 and 0.009 by ddPCR) (Table 2 ½T2 426 as the false-positive control in ddPCR test finally revealed a and Figure 2) had received crizotinib treatment after unsuc- ½F2 427 negligible false-positive rate (Supplemental Figure S1). The cessful chemotherapy. The patient with 10% ALK rearranged 428 results indicated that 13 of 14 ALK FISH-positive cases tumor cells (EML4-ALK/GAPDH Z 0.011, variant 3) had a 429 were confirmed by ddPCR (four with variant 1, two with partial response to crizotinib (Figure 3). The other patient ½F3 430 431 variant 2, and seven with variant 3) (Figure 1, AeC). The with 8% ALK rearranged tumor cells (EML4-ALK/ 432 case missed by ddPCR was identified as KIF5B-ALK rearGAPDH Z 0.009, variant 1) had stable disease. The PFS of 433 rangement by PCR-based direct sequencing. In addition to these patients was 7 and 5 months, respectively. Therefore, 434
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superior to real-time quantitative PCR for accurate assessment of CCL4 gene copy number variation.8 Our work indicates that ddPCR can be used as a molecular analytical tool of precision in measuring the copy numbers of EML4-ALK fusion at the RNA levels in FFPE samples from patients with lung adenocarcinoma.
Correlation of ALK Gene Translocation with Effectiveness of Crizotinib Wu et al18 suggested that the relative abundance of EGFR mutations predicted the outcome of EGFR TKI treatment. Accurate quantification of EGFR mutations is important not only for better selection of patients but also for developing better treatment strategies for patients with a low abundance of EGFR mutations. We hypothesized that the rearranged ALK gene copy numbers might also predict the degree of benefit from crizotinib.
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ddPCR Quantification of EML4-ALK
Benefit of Crizotinib Oral Treatment in Patients with Low Copy Numbers of ALK Gene Fusion Figure 2
Fluorescent in situ hybridization (FISH) and Droplet Digital PCR (ddPCR) tests reveal low copy numbers of EML4-ALK fusion in a patient successfully treated with crizotinib. A: FISH test revealed 10% tumor cells harbored break-apart ALK signals, which failed the positive cutoff point of 15% (arrows indicate break-apart ALK signals). B: ddPCR test revealed low copy numbers of EML4-ALK translocation (normalized variant 3 Z 0.010984). Nor., normalized; V, version.
patients with low copy numbers of ALK gene rearrangements might benefit from crizotinib treatment.
Discussion
Median PFS and overall survival rates of patients with low abundance of EGFR mutations were significantly shorter than those with high abundance but were significantly longer than those with wild-type tumors. The objective response rates were 62.7%, 44.4%, and 16.1% for patients with high abundance of EGFR mutations, low abundance of EGFR mutations, and wild-type EGFR, respectively.18 Of the 16 patients with low abundance of EML4-ALK fusion identified in this study, 2 had received crizotinib treatment after unsuccessful chemotherapy. The PFS rates of the patients with 0.011 (10% by FISH) and 0.009 (8% by FISH) EML4-ALK/GAPDH rates were 7 (partial response) and 5 (stable disease) months, respectively. These
High Sensitivity of ddPCR with Absolute Quantification for ALK Gene Rearrangement Detection
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Patients with lung adenocarcinoma and ALK gene rearrangements benefited from crizotinib treatment.13 However, the limited rearrangement rate requires sensitive and reliable screening for ALK gene rearrangements. FISH, although well standardized, failed to distinguish the fusion variants of ALK. Interpretation of the FISH results is subjective, and substantial variation in interpretation has been reported.10,14 Furthermore, interpretation of the ALK FISH analysis is more complex than for other FISH assays. ddPCR is a reliable choice for ALK gene rearrangement detection, in addition to commonly used FISH, IHC, and RT-PCR. Compared with FISH and real-time quantitative PCR, ddPCR is known to be more precise and sensitive,15 better at detecting rare genetic variants,11 more objective17 and most importantly associated with absolute quantification. These advantages have gradually promoted the use of ddPCR in clinical molecular diagnostics.18 ddPCR offers a unique approach to distinguishing HER2þ from HER2 samples due to its absolute quantitative nature, thereby eliminating the need for calibration curves.12 ddPCR was also
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Figure 3 Chest computed tomography (CT) revealing a decrease in the primary tumor in a patient with 10% (EML4-ALK/GAPDH Z 0.010984, variant 3) ALK rearranged tumor cells after 7 months of crizotinib treatment (partial response). A: CT scan before crizotinib treatment. B: CT scan after 7 months of crizotinib treatment. Arrows indicate decreased tumor and nonreduced pleural effusion.
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Wang et al results revealed the partial benefit of crizotinib therapy in patients with low copy numbers of ALK translocation. The proposed ALK FISH-positive cut point for nonesmallcell lung cancer is 15% tumor cells with split ALK signals19 but without substantial case load accompanying crizotinib treatment. The abundance of EGFR mutations predicted the degree of benefit from EGFR TKI treatment,18 indicating that patients with lower abundance of EGFR mutation benefited from EGFR TKI treatment but relatively less than those with higher ones. Our studies indicated that patients with ALK rearrangement copy numbers lower than 15% still benefited from crizotinib treatment. Therefore, the ALK-positive cutoff point of FISH test may be too high in some cases. A more appropriate positive threshold for ALK rearrangement of patients with nonesmallcell lung cancer should be determined using large numbers of clinical cases along with data evaluating crizotinib treatment and ALK rearrangement copy numbers.
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Uncited Reference 16. 11.
Acknowledgments We thank Shirong Wei for assistance on RNA purification, He Xiao for assistance with statistical analysis, and Yun Zhao (BioRad Laboratories) for assistance using the ddPCR protocol.
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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2015.04.002. 14.
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Supplemental Figure S1 Raw data of Droplet Digital PCR (ddPCR) for false-positive control. Eight samples of reactive lymph nodes were used as the false-positive control of the ddPCR test and finally revealed negligible positive signals.
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Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma Q12
Qiushi Wang,* Xin Yang,* Yong He,y Qiang Ma,* Li Lin,* Ping Fu,* and Hualiang Xiao* From the Departments of Pathology* and Respiration,y Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, China Accepted for publication April 14, 2015.
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Address correspondence to Hualiang Xiao, Department of Pathology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, 10#, Changjiangzhilu, Daping, Yuzhong District, Chongqing 400042, China. E-mail: [email protected].
Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive nonesmall-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients. (J Mol Diagn 2015, -: 1e6; http://dx.doi.org/10.1016/j.jmoldx.2015.04.002)
Lung cancer is the leading cause of cancer-related death worldwide. Recently, successful strategies to improve personalized lung cancer therapy have focused on the epidermal growth factor receptor (EGFR)etargeted therapies, such as tyrosine kinase inhibitors (TKIs) (gefitinib or erlotinib).1 A recent study suggested that a gene rearrangement comprising portions of the EML4 gene and the ALK gene in nonesmall-cell lung cancer (NSCLC) cells might be a promising therapeutic target and a diagnostic molecular marker.2 When compared with chemotherapy, crizotinib (a targeted inhibitor of ALK and C-met) used in second-line treatment significantly prolonged progression-free survival (PFS), increased response rates, and improved the quality of life in patients with advanced and ALK-positive NSCLC.3 ALK-rearranged lung cancer can be identified by fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and RT-PCR. FISH is recommended by the National
Comprehensive Cancer Network Clinical Practice Guidelines in Oncology for NSCLC (version 3, 2014) for the clinical diagnosis of ALK-positive lung cancer. However, FISH requires specialized technical resources and expertise.4 FISH diagnosis is very subjective, which limits accurate quantification. IHC is relatively inexpensive and can be routinely performed on formalin-fixed, paraffin-embedded (FFPE) tissues. However, IHC is a rapid screening method but associated with relatively low sensitivity and specificity, except when the ALK monoclonal antibody D5F3 and the Ventana Diagnostic System (F. Hoffmann-La Roche AG, Basel, Switzerland) were Q5 used.5 RT-PCR is commonly used to identify known fusion Supported by grant 81472189 from the National Natural Science Foundation of China (Y.H.) and grant 2012AA02A201 from 863 Major Project of China (Y.H.). Q1 Disclosures: None declared.
Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2015.04.002
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Wang et al Table 1 Clinical Profile of Genetically Screened Patients with Lung Adenocarcinoma Characteristic Sex Male Female Smoking status Never-smokers Smokers Specimen type Surgical Biopsy Total
No. (%) of patients, N Z 103
No. (%) ALK (FISH) positive
51 (49.5) 52 (50.5)
8 (15.7) 6 (11.5)
66 (64.1) 37 (35.9)
9 (13.6) 5 (13.5)
57 (55.3) 46 (44.7) 103
9 (15.8) 5 (10.9) 14 (13.6)
FISH, fluorescence in situ hybridization.
variants of ALK gene rearrangements with high sensitivity but semiquantitatively.6 Treatment with EGFR TKIs was effective in advanced NSCLC because of the abundance of EGFR mutations.7 The median PFS and OS rates of patients with low abundance of EGFR mutations were shorter than those with high abundance but significantly longer than those with wildtype tumors. It is still unclear if the abundance of ALK gene rearrangements was predictive of treatment efficacy with crizotinib. Unfortunately, the current methods are not adequate to detect the abundance of ALK gene rearrangements accurately. Droplet Digital PCR (ddPCR), a recently developed technique, involves emulsification and PCR amplification inside thousands of small droplets, each droplet containing one or no molecules of target DNA or RNA.8 Precise and absolute quantification of the number of target DNA or RNA molecules in the reaction is simply achieved by counting the number of positive and negative droplets.9 The strategy reduces competitive amplification, allowing detection of 0.001% mutant fractions, which is 1000 times lower than real-time PCR. In this study, we used ddPCR to reveal a highly sensitive and specific detection of EML4-ALK gene rearrangements in mRNA derived from clinical lung adenocarcinoma FFPE samples. We further discuss the potential benefit of crizotinib in the treatment of patients with low copy numbers of ALK rearrangements.
and smoking history of the patients were obtained from their medical records.
RNA Isolation and cDNA Synthesis Total RNA was extracted and purified from freshly cut FFPE tissue sections using the RNeasy kit (Qiagen, Valencia, CA), after ascertaining the tumor percentage in each specimen. The RNA quality was verified by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA), and not more than 1 mg of RNA was reverse transcribed to cDNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA). The total volume of cDNA obtained from the reverse Q6 transcription reaction was 20 mL.
FISH FISH was performed on unstained 4-mm-thick FFPE specimens using the Vysis LSI ALK dual-color, break-apart FISH probes (Abbott Molecular, Des Plaines, IL), according to the Q7 manufacturer’s instructions. Slides were read on a fluorescence microscope (BX51; Olympus, Tokyo, Japan) and independently evaluated by two experienced pathologists. Q8 Tumor cells containing at least one pair of green and orange signals, split apart by 2 signal diameters, or a single orange signal without the corresponding green signal were diagnosed as positive for ALK gene rearrangement. The criteria of 15% break-apart signals or orange single signals isolated in 50 tumor cells were used as the ALK-positive cutoff levels.10
ddPCR Workflow
This study was approved by the Clinical Ethics Committee of Daping Hospital and Research Institute of Surgery, Third Military Medical University. Signed informed consent was obtained from all patients. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma patients between January 2013 and February 2014. They were randomly selected after diagnosis of lung adenocarcinoma, and none of them had received ALK inhibitor treatment or chemotherapy before surgery or biopsy. The clinical data
The ddPCR was performed as previously described.11,12 TaqMan reaction mixture was assembled from a 2 ddPCR master mix (BioRad Laboratories, Hercules, CA), 20 primers and probes (final concentrations of 900 and 250 nmol/L, respectively), and 1 mL of cDNA in a final volume of 20 mL. Each reaction mixture was loaded into a sample well of an eight-channel disposable droplet generator cartridge (BioRad Laboratories). The emulsified samples were generated from a droplet generator (QX100) and then transferred onto a 96-well plate. After heat-sealing with a foil seal, the emulsified samples underwent a 2-step thermal cycling protocol in a C-1000 touch thermal cycler (BioRad Laboratories) as follows: 95 C 10 minutes, 40 cycles of 95 C 30 seconds and 60 C 60 seconds (ramp rate set to 2 C per second), and 98 C 10 minutes. The 96well droplet PCR plates were loaded into a droplet reader, which automatically reads the droplets from each well of the plate. Analysis of the ddPCR data were performed with QuantaSoft analysis software (BioRad Laboratories). Three Q9 main variants of EML4-ALK were analyzed in this study, and forward primers of these variants were as follows: 50 AGCCCACACCTGGGAAAGGAC-30 for variant 1, 50 CAGCTACATCACACACCTTGACTGGT-30 for variant 2,
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Materials and Methods Patients and Tissues
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ddPCR Quantification of EML4-ALK
and 50 -CTTGGGAAAATTCAGATGATAGCCG-30 for variant 3. These variants shared a unique reverse primer 50 AGCTTGCTCAGCTTGTACTCAGGG-30 and probe 50 FAM-CATGGCTTGCAGCTCCTGGTGCTTCC-TAMRA-30 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The sequence of the different primers is as follows: forward primer 50 -CATCTTCCAGGAGCGAGATCCC-30 , reverse primer 50 -ATGGTTCACACCCATGACGAACA-30 , and probe 50 -FAM-GAGTACGTCGTGGAGTCCACTGGCGTC-TAMRA-30 .
Evaluation and Statistical Analysis Two patients carried low copy numbers (<15%, based on FISH test) of ALK gene rearrangement after crizotinib treatment. Their responses were evaluated according to the Response Evaluation Criteria in Solid Tumors. PFS was assessed from the date of the TKI treatment to the date of disease progression or death. Overall survival was not measured as the end point because these two patients were still alive at the completion of the study.
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311 312 313 314 315 316 317 318 319 Figure 1 ALK gene rearrangement results for 320 103 clinical formalin-fixed, paraffin-embedded 321 samples detected by fluorescent in situ hybridiza322 tion (FISH) and Droplet Digital PCR (ddPCR). For 323 easy identification, some of the negative samples 324 (colored gray) were truncated. A: ALK status 325 detected by FISH (15% was used as the cutoff 326 point for ALK positivity). B: Normalized variant 1 (v1 nor) results of EML4-ALK. C: Normalized variant 327 2 (v2 nor) results of EML4-ALK. D: Normalized 328 variant 3 (v3 nor) results of EML4-ALK; 14 ALK329 positive samples detected by FISH (black, red, 330 green, and blue; A) were also assessed via ddPCR 331 for EML4-ALK copy number alterations. The results 332 are highlighted by the red (B), green (C), and blue 333 (D) colors. The specimen numbered 1318991 (30% 334 ALK break-apart signals by FISH test, colored 335 black; A) failed the ddPCR test and finally identi336 fied as KIF5B-ALK gene rearrangement by PCRbased direct sequencing. 337 338 339 340 341 342 343 344 345 346 347 348 We analyzed the association of ALK gene rearrangement 349 rates with clinical characteristics and smoking history, using 2 350 Pearson’s c test. P < 0.05 was considered statistically 351 significant. All analyses were performed using SPSS sta352 tistical software version 18.0 (SPSS Inc, Chicago, IL). 353 354 355 Results 356 357 Clinical Features of Tumors 358 359 The study population consisted of 51 men (49.5%) and 52 360 women (50.5%), with a mean age of 60.52 years (range, 39 to 361 77 years). On the basis of their smoking history, 66 patients 362 were classified as never-smokers (64.1%) and 37 as smokers 363 (35.9%). Among the tumor specimens, 57 were obtained by 364 surgical resection (55.3%) and 46 by biopsy (44.7%) 365 (Table 1). ½T1 366 367 368 ALK Rearrangement Assessed by FISH 369 370 Of the 103 patients, 14 (13.6%, eight men and six women) 371 were FISH positive with the dual-color, break-apart probe 372
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Wang et al 373 Table 2 Patients’ Clinical and ALK Gene Profile 374 ddPCR copy number Smoking 375 No. Age, years Sex ALK rearrangements FISH (%) (normalized) history, years Disease stage 376 1317769 72 M Variant 1 19 0.010082 30 IIIa 377 1314926 68 M Variant 1 27 0.015359 17 IIa 378 1303203 48 M Variant 1 27.6 0.053933 Non IIIa 379 1305735 77 M Variant 1 42 0.182692 30 Ia 380 c140457 66 F Variant 2 16 0.018750 Non IV 381 1309153 67 F Variant 2 26 0.044444 Non Ib 382 1318736 60 M Variant 3 15 0.003200 Non IIIb 383 1314415 76 F Variant 3 17 0.003750 Non IIIa 384 1307896 63 F Variant 3 16 0.004061 Non IIIa 385 1303308 64 M Variant 3 15 0.004384 30 IIb 386 1309028 57 M Variant 3 20 0.004663 30 IIIa 387 1315381 56 M Variant 3 33 0.089189 40 IV 388 1310046 59 F Variant 3 60 0.372222 Non Ia 389 1318991 67 M KIF5B-ALK 30 50 IV 390 1406350 58 F Variant 1 6.5 0.000103 Non IV 391 1308361 69 F Variant 3 7.1 0.000734 Non IIa 392 1303465 58 F Variant 3 7.6 0.001078 Non IIa 393 1315036* 63 M Variant 1 8 0.009386 Non IV 394 1309931 62 F Variant 1 8.2 0.000687 Non IV 395 1319454 64 F Variant 1 8.4 0.000616 Non IIIb 396 1308639 59 F Variant 1 8.6 0.001802 Non IIIb 397 1314673 55 M Variant 1 9.2 0.002336 20 IIIb 398 1306902 63 M Variant 3 9.4 0.001023 Non IIIa 399 1301868 39 M Variant 3 9.7 0.002258 Non IV 400 1306691 38 M Variant 3 9.8 0.001980 Non IV 401 1319396 70 M Variant 1 10 0.004294 Non IV 402 1303717* 42 M Variant 3 10 0.010984 30 Iay 403 1407832-1 72 M Variant 1 11.2 0.003717 40 IV 404 1308989 52 F Variant 1 12 0.001910 Non IV 405 1317381 68 M Variant 2 12 0.004918 20 IV 406 407 *These two patients had received crizotinib treatment after unsuccessful chemotherapies and had partial response and stable disease, respectively. y 408 IV when he received crizotinib treatment. 409 F, female; M, male; ddPCR, Droplet Digital PCR; FISH, fluorescence in situ hybridization. 410 411 ½F1 (Table 1 and Figure 1D). The ALK-positive rates in men and the positive cases mentioned above, 16 patients were 412 detected with low copy numbers of EML4-ALK rearrangewomen were 15.7% and 11.5%, respectively, with no sig413 ments (nine with variant 1, one with variant 2, and six with nificant differences. Either tumor heterogeneity or insuffi414 variant 3), which were tested by FISH with break-apart cient tumor cells might have contributed to the lower 415 signals <15%. Considering the limited ALK-positive cases ALK-positive rate in biopsy specimens (10.9%) compared 416 and their crizotinib treatment data, the EML4-ALK positive with surgical specimens (15.8%), with no significant dif417 threshold of ddPCR cannot be safely determined currently. ferences. No significant correlation was seen between the 418 ALK-positive rate and smoking history. 419 420 Patients with Low Copy Numbers of EML4-ALK Gene 421 ddPCR Detection of EML4-ALK Rearrangement Rearrangement Respond Well to Crizotinib 422 423 Three variants of the EML4-ALK rearrangements were Two patients with low copy numbers of EML4-ALK gene 424 detected in this study. GAPDH was used as the reference rearrangements (ALK rearranged cells ratio <15% by FISH and 425 gene. Eight samples of reactive lymph nodes that were used EML4-ALK/GAPDH as 0.011 and 0.009 by ddPCR) (Table 2 ½T2 426 as the false-positive control in ddPCR test finally revealed a and Figure 2) had received crizotinib treatment after unsuc- ½F2 427 negligible false-positive rate (Supplemental Figure S1). The cessful chemotherapy. The patient with 10% ALK rearranged 428 results indicated that 13 of 14 ALK FISH-positive cases tumor cells (EML4-ALK/GAPDH Z 0.011, variant 3) had a 429 were confirmed by ddPCR (four with variant 1, two with partial response to crizotinib (Figure 3). The other patient ½F3 430 431 variant 2, and seven with variant 3) (Figure 1, AeC). The with 8% ALK rearranged tumor cells (EML4-ALK/ 432 case missed by ddPCR was identified as KIF5B-ALK rearGAPDH Z 0.009, variant 1) had stable disease. The PFS of 433 rangement by PCR-based direct sequencing. In addition to these patients was 7 and 5 months, respectively. Therefore, 434
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superior to real-time quantitative PCR for accurate assessment of CCL4 gene copy number variation.8 Our work indicates that ddPCR can be used as a molecular analytical tool of precision in measuring the copy numbers of EML4-ALK fusion at the RNA levels in FFPE samples from patients with lung adenocarcinoma.
Correlation of ALK Gene Translocation with Effectiveness of Crizotinib Wu et al18 suggested that the relative abundance of EGFR mutations predicted the outcome of EGFR TKI treatment. Accurate quantification of EGFR mutations is important not only for better selection of patients but also for developing better treatment strategies for patients with a low abundance of EGFR mutations. We hypothesized that the rearranged ALK gene copy numbers might also predict the degree of benefit from crizotinib.
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ddPCR Quantification of EML4-ALK
Benefit of Crizotinib Oral Treatment in Patients with Low Copy Numbers of ALK Gene Fusion Figure 2
Fluorescent in situ hybridization (FISH) and Droplet Digital PCR (ddPCR) tests reveal low copy numbers of EML4-ALK fusion in a patient successfully treated with crizotinib. A: FISH test revealed 10% tumor cells harbored break-apart ALK signals, which failed the positive cutoff point of 15% (arrows indicate break-apart ALK signals). B: ddPCR test revealed low copy numbers of EML4-ALK translocation (normalized variant 3 Z 0.010984). Nor., normalized; V, version.
patients with low copy numbers of ALK gene rearrangements might benefit from crizotinib treatment.
Discussion
Median PFS and overall survival rates of patients with low abundance of EGFR mutations were significantly shorter than those with high abundance but were significantly longer than those with wild-type tumors. The objective response rates were 62.7%, 44.4%, and 16.1% for patients with high abundance of EGFR mutations, low abundance of EGFR mutations, and wild-type EGFR, respectively.18 Of the 16 patients with low abundance of EML4-ALK fusion identified in this study, 2 had received crizotinib treatment after unsuccessful chemotherapy. The PFS rates of the patients with 0.011 (10% by FISH) and 0.009 (8% by FISH) EML4-ALK/GAPDH rates were 7 (partial response) and 5 (stable disease) months, respectively. These
High Sensitivity of ddPCR with Absolute Quantification for ALK Gene Rearrangement Detection
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Patients with lung adenocarcinoma and ALK gene rearrangements benefited from crizotinib treatment.13 However, the limited rearrangement rate requires sensitive and reliable screening for ALK gene rearrangements. FISH, although well standardized, failed to distinguish the fusion variants of ALK. Interpretation of the FISH results is subjective, and substantial variation in interpretation has been reported.10,14 Furthermore, interpretation of the ALK FISH analysis is more complex than for other FISH assays. ddPCR is a reliable choice for ALK gene rearrangement detection, in addition to commonly used FISH, IHC, and RT-PCR. Compared with FISH and real-time quantitative PCR, ddPCR is known to be more precise and sensitive,15 better at detecting rare genetic variants,11 more objective17 and most importantly associated with absolute quantification. These advantages have gradually promoted the use of ddPCR in clinical molecular diagnostics.18 ddPCR offers a unique approach to distinguishing HER2þ from HER2 samples due to its absolute quantitative nature, thereby eliminating the need for calibration curves.12 ddPCR was also
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Figure 3 Chest computed tomography (CT) revealing a decrease in the primary tumor in a patient with 10% (EML4-ALK/GAPDH Z 0.010984, variant 3) ALK rearranged tumor cells after 7 months of crizotinib treatment (partial response). A: CT scan before crizotinib treatment. B: CT scan after 7 months of crizotinib treatment. Arrows indicate decreased tumor and nonreduced pleural effusion.
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Wang et al results revealed the partial benefit of crizotinib therapy in patients with low copy numbers of ALK translocation. The proposed ALK FISH-positive cut point for nonesmallcell lung cancer is 15% tumor cells with split ALK signals19 but without substantial case load accompanying crizotinib treatment. The abundance of EGFR mutations predicted the degree of benefit from EGFR TKI treatment,18 indicating that patients with lower abundance of EGFR mutation benefited from EGFR TKI treatment but relatively less than those with higher ones. Our studies indicated that patients with ALK rearrangement copy numbers lower than 15% still benefited from crizotinib treatment. Therefore, the ALK-positive cutoff point of FISH test may be too high in some cases. A more appropriate positive threshold for ALK rearrangement of patients with nonesmallcell lung cancer should be determined using large numbers of clinical cases along with data evaluating crizotinib treatment and ALK rearrangement copy numbers.
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Uncited Reference 16. 11.
Acknowledgments We thank Shirong Wei for assistance on RNA purification, He Xiao for assistance with statistical analysis, and Yun Zhao (BioRad Laboratories) for assistance using the ddPCR protocol.
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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2015.04.002. 14.
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Supplemental Figure S1 Raw data of Droplet Digital PCR (ddPCR) for false-positive control. Eight samples of reactive lymph nodes were used as the false-positive control of the ddPCR test and finally revealed negligible positive signals.
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