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The Journal of Molecular Diagnostics, Vol. -, No. -, - 2015
jmd.amjpathol.org
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.
Q2
Q3
Q4
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
FLA 5.2.0 DTD JMDI412_proof 1 July 2015 3:22 pm EO: JMD14_0221
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
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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,
2
jmd.amjpathol.org
Materials and Methods Patients and Tissues
-
The Journal of Molecular Diagnostics
FLA 5.2.0 DTD JMDI412_proof 1 July 2015 3:22 pm EO: JMD14_0221
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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 (
The Journal of Molecular Diagnostics, Vol. -, No. -, - 2015
jmd.amjpathol.org
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.
Q2
Q3
Q4
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
FLA 5.2.0 DTD JMDI412_proof 1 July 2015 3:22 pm EO: JMD14_0221
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
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,
2
jmd.amjpathol.org
Materials and Methods Patients and Tissues
-
The Journal of Molecular Diagnostics
FLA 5.2.0 DTD JMDI412_proof 1 July 2015 3:22 pm EO: JMD14_0221
187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
print & web 4C=FPO
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
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 (
