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Duplex Ratio Tests as Diagnostic Biomarkers in Malignant Melanoma
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The Journal of Molecular Diagnostics, Vol. -, No. -, - 2015
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Duplex Ratio Tests as Diagnostic Biomarkers in Malignant Melanoma Q12 Q1
David A. Moore, Gerald Saldanha, Abdlrzag Ehdode, Mohamed Z. Mughal, Linda Potter, Lovesh Dyall, and James H. Pringle
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From the Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom Accepted for publication May 13, 2015.
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Address correspondence to David A. Moore, Department of Cancer Studies and Molecular Medicine, University of Leicester, Level 3 RK-CSB, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom. E-mail: dam18@le. ac.uk.
Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCRebased assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%e91%) and a specificity of 88% (95% CI, 84%e92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas and 20 metastasizing primary melanomas revealed that DRTs had a marginal benefit as prognostic markers. DRTs have early potential to act as molecular biomarkers of melanoma on FFPE specimens pending validation, and DRTs may have applicability as prognostic markers in melanoma or other tumor types if new DRTs to relevant loci are developed. (J Mol Diagn 2015, -: 1e7; http://dx.doi.org/10.1016/ j.jmoldx.2015.05.001)
Malignant melanoma incidence in the United Kingdom has increased markedly during the past 30 years, with >13,000 new cases diagnosed in 2011 within the United Kingdom alone.1 The prognosis for those with disseminated disease is poor, with a 5-year survival rate of <10%.2 Many thousands of melanocytic lesions, benign and malignant, are excised and undergo histopathologic analysis every year to diagnose or exclude malignancy or dysplasia, for cosmetic reasons, or because lesions are causing physical irritation. Histopathologic analysis is the gold standard for diagnosis of melanocytic tumors. In most melanocytic lesions, a confident diagnosis of benign nevus or malignant melanoma can be made on a standard hematoxylin and eosinestained tissue section alone, and the histologic features of melanocytic lesions are well described and recognized.3 Within a small subset of melanocytic lesions, however, it can be very difficult to differentiate between melanoma and nevus based on hematoxylin and eosin staining, and even specialist dermatopathologists cannot agree over the classification of a subset of cases.4e7 As a result, the misdiagnosis of
melanocytic lesions accounts for a significant proportion of the litigation claims against histopathologists.8 Diagnostically difficult and genuinely histologically ambiguous melanocytic lesions have prompted the search for effective diagnostic biomarkers in melanoma.9 Chromosomal instability is a well-described feature of malignant tumors, with typical patterns of DNA copy number (DCN) change seen across the genome in specific tumor types.10,11 DCN is a useful marker because it is well characterized in malignant tissue and can be assayed in formalin-fixed, paraffin-embedded (FFPE) tissue. Using the information generated from comparative genomic hybridization (CGH) studies, Gerami et al12 found that fluorescent in situ hybridization (FISH) can be used to differentiate between nevi and melanoma. The assay is a combination of four FISH probes (MYB, RREB1, CEP6, and This work was supported by The Pathological Society of Great Britain and Ireland grant SGS 2010/04/01 (Small Grant Scheme 2010). 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.2015.05.001
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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
Moore et al Table 1
Clinical and Pathological Characteristics of the Nevi and Melanoma Included in the Discovery Series Type (no.)
Site (no.)
Series Head and neck Trunk Limb Mean age (range, years) Male/female ratio IDN BCN Breslow depth (mm) Ulcerated (%) Mitotic index Nevi 2 P-M 3 PþM 5
11 5 5
7 12 10
37 (19e67) 65 years (34e88) 69 years (46e88)
6:14 8:12 10:10
15 5
3.9 (2.0e7.4) 5.4 (2.5e9.7)
35 60
5.9 (1e19) 9.2 (1e40)
BCN, benign compound nevus; IDN, intradermal nevus; P-M, primary melanoma without metastasis; PþM, primary melanoma with metastasis.
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CCND1). This is the only melanoma diagnostic biomarker to have been translated into clinical practice and to have become commercially available. Our group has previously described a series of assays using real-time quantitative PCR (qPCR), namely, duplex ratio tests (DRTs) and paralogue ratio tests.13,14 These are used to compare the quantity of DNA at one site in the genome with another site, both in the same qPCR assay. The ratio between a pair of targets should be 1:1 in diploid tissue (assuming equal efficiency of the two reactions), although when the relevant DCN changes are present, such as may occur in cancer, the ratio will differ from 1. A panel of five DRTs was previously able to differentiate between histologically unambiguous cases of nevi and melanoma in a
small set of cases. The analytical validity of DRTs in FFPE Q8 has been found in previous work by our laboratory using both clinical sample and cell line DNA.13 The assays benefit from being relatively simple cheap tests for assessing DCN changes, and multiple assays can be tested with each sample, meaning that a wide range of DNA loci are tested. DRTs may therefore represent a potential alternative to FISH and CGH for the assessment of DCN variation. The assays have disadvantages compared with FISH, however, such as relying on a specific quantity of DNA of adequate quality being extracted from small FFPE lesions into solution and the potential for significant DNA contamination, particularly in small lesions and those lesions with a heavy inflammatory infiltrate.
Table 2 The Duplex Ratio Test Targets for the Series of Seven Existing Duplex Ratio Tests, Including Sequences for the Primers and Probes (Target from Region of Gain Shaded) Assay
Locus
Forward primer
Reverse primer
Probe
5 -TCATGAAGACCTCACAGTAAAAA- 5 -ATCCAGACAACTGTTCAAACTG- VIC-50 -AATCTCGATGGAGTGGGTAGGT-30 ATG-30 TC-30 -MGB 0 0 0 0 PTEN 10q23.3 5 -GCGACTGCGCTCAGTTCTCT-3 5 -TCACAGCGGCTCAACTCTCA-3 FAM-50 -CTCTCGGAAGCTGC-30 MGB RREB1 6p25 50 -TGTCCCAATGACGTCAAGTTC-30 50 -CTACACTCATGACCGCCGAC-30 FAM-50 -GTTGATGGAAGATAGGTCT-30 -MGB 0 0 0 MYB 6q22-23 5 -GCTTGTACAGAAATACGGTCC5 -GCCACCTCTCCCTACATTGTT-3 VIC-50 -TTGCCAAGCACTTAAAGA-30 30 -MGB 0 0 SSR1 6p24.3 5 -CCTTAGATGCCTCATTCCGTT5 -CAGTGTTCAGAGGAAGAGCTFAM-50 -CAGGACTACCAGTTTT0 0 AT-3 GTG-3 AT-30 -MGB PERP 6q24 50 -TACTCAGCGCCATCGCCT-30 50 -AGCATTTCCACCACAGCGAG-30 VIC-50 -TTGCAGTCTAGCGACCAC-30 -MGB 0 0 ASAP1 8q24.1-24.2 5 -CAGGCTAAATCTGGAAAGTTCAA- 5 -GTTTGTCATCCAGATCATCAVIC-50 -AATCTTCGACAGGAGGA0 0 TC-3 TCG-3 GATA-30 -MGB LZTS1 8p22 50 -GTGACCACTCTTCTTTAAGCCAT- 50 -TGGAAAGCCACACCCTCTG-30 FAM-50 -CCTGGGCTGGGTGC-30 AGA-30 MGB 0 CCND1 11q13 5 -TGGTGAACAAGCTCAAGTGGAA-30 50 -CGCCTCTGGCATTTTGGA-30 FAM-50 -CCGCACGATTTCATTGA-30 -MGB LDLRAD3 11p13 50 -ACAACGTCAATAATGGCATCCA-30 50 -GCCTACTTCCGACGCATTCT-30 VIC-50 -TTGCCAAGCACTTAAA30 -MGB 0 0 0 TBX2 17q23 5 -GGCCTAGACCGCGTGATAAA-3 5 -GGTCTACACTGACTTCAGTCGT- VIC-50 -GGTTGAGGGATGCTGGAAACTG-30 30 -MGB HIC1 17p13.3 50 -TGTGCGACGTGATCATCGT-30 50 -CACCACCAGGGACTTGAGG FAM-50 -AAGAGGGCGTTCTGCATAG-30 30 -MGB 0 0 0 AKT3 1q44 5 -CTGGACATCACCAGTCCTAGC5 -ACCCTTGGCTGGTCTGGG-3 VIC-50 -ATAGCAGGGGCACCTT0 TC-3 30 -MGB MIB2 1p36.33 50 -CACCAAGCACCACTCCTTCTG-30 50 -CAGCCGCTTCACTGTGTCAA-30 FAM-50 -CCGGGTCATCGGCGA30 -MGB BRAF
2
7q34
0
0
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Duplex Ratio Tests in Malignant Melanoma Table 3 Means SD DDCt for the Diploid Samples for all Seven Duplex Ratio Tests, with Details of the DDCt Versus Ct Correction
Assay
Means SD DDCt
Significant trend Ct versus Means SD DDCt corrected DDCt
MYB.RREB1 BRAF.PTEN SSR1.PERP ASAP1.LZTS1 TBX2.HIC1 LDLRAD3.CCND1 AKT3.MIB2
0.162 0.220 0.183 0.163 0.040 0.016 0.177
Yes Yes No Yes No No Yes
0.285 0.478 0.377 0.263 0.277 0.443 0.268
0.0197 <0.0001 NA 0.0004 NA NA 0.0002
0.269 0.461 0.242 0.245
NA, not applicable.
Our aim was to test DRTs on an opportunity sample of FFPE tissue comprising nevi, primary melanomas that had not metastasized (P-M), primary melanomas that had metastasized (PþM), and the matched metastases. We made three comparisons, nevi versus primary melanoma, P-M versus PþM, and PþM versus the matched metastases, to determine the value of DRTs for assessing diagnosis, prognosis, and progression, respectively. We found DRTs to be most useful to differentiate between nevi and melanoma. Therefore, we subsequently tested the assays on large representative cohorts of melanomas and nevi to develop a diagnostic algorithm.
Materials and Methods Case Selection FFPE tissue blocks were selected from the University Hospitals of Leicester histopathology archive. The discovery series was intended to verify the findings by Gerami et al12 that DCN assessed on FFPE tissue was a marker of malignancy. This discovery series comprised 20 nevi, 40 primary melanomas, and 20 metastatic melanoma samples. The 20 nevi consisted of unambiguous cases of benign compound nevi and benign intradermal nevi. For inclusion, all nevi required a cross-sectional area of >25 mm2, and there were no junctional nevi included because of the difficulty of microdissecting thin melanocytic lesions. Of the 40 primary melanomas, 20 had no evidence of metastasis with at least 5 years of follow-up, and these were designated P-M. The remaining 20 had histologic evidence of metastasis within 5 years of the primary diagnosis and were designated PþM. The inclusion criteria for both classes of melanoma were sufficient thickness of FFPE tissue block to allow for six sections of tissue to be cut and Breslow thickness >2 mm. The PþMs also required matched FFPE metastatic melanoma tissue to be available for DNA extraction. Exclusion criteria for both classes of melanoma were equivocal diagnosis of primary melanoma, tumor representing <5% sectional area, and a sectional area of <25 mm2. The mean
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311 Breslow depth was 3.9 mm for the P-Ms and 5.4 mm for the 312 PþMs. The mean period from primary to metastasis for the 313 PþMs was 16 months. Clinicopathologic details of these 314 nevi and melanoma are summarized in Table 1. Diploid ½T1 315 control samples were selected to develop a reference range of 316 values for the assays. This consisted of 53 benign reactive 317 tonsils, 26 skin samples from wide local excisions for mel318 anoma (none of which contained histologic evidence of 319 tumor), and 23 histologically tumor-free lymph nodes from 320 321 lymph node dissections for melanoma. 322 Retrospective cohorts of sequentially submitted mela323 noma and nevi were also assembled. The same inclusion 324 and exclusion criteria used in the discovery series were also 325 used for the cohort of nevi. The melanoma inclusion and 326 exclusion criteria were as for the discovery series with the 327 exception that all melanomas >1 mm in thickness were 328 included in an attempt to make the cohort as representative 329 as possible. 330 From January 1, 2000, onward, all melanomas and nevi 331 sequentially diagnosed in the Cellular Pathology Depart332 333 ment of the University Hospitals of Leicester NHS Trust 334 that met these criteria were included in the cohort; hema335 toxylin and eosin histologic findings were reviewed and the 336 paraffin-embedded tissue block inspected to establish 337 whether the criteria were met. The cohorts of melanoma and 338 nevi extended up to the point where >100 cases were 339 included in each. The melanoma cohort consisted of 106 340 tumors from 44 men and 62 women. Breslow depth of the 341 lesions ranged from 1.1 to 21.0 mm (mean, 4.2 mm; median, 342 2.6 mm). The age range was 30 to 95 years (mean, 71 years; 343 median, 75 years). The nevus cohort consisted of 102 344 345 benign nevi (intradermal or compound type) from 51 men 346 and 51 women. The age range was 18 to 84 years (mean, 35 347 years; median, 32 years). The tissue samples underwent 348 tissue microdissection and DNA extraction as previously 349 described.13 Table 4 Interclass Correlation, Bias, Limit of Agreement, CV, and t-Test P Value for Each of the Seven Duplex Ratio Tests
Assay
Interclass correlation coefficient Bias
MYB RREB1 BRAF PTEN SSR1 PERP TBX2 HIC1 ASAP1 LZTS1 LDLRAD3 CCND1 AKT3 MIB2
0.95 0.95 0.95 0.93 0.95 0.96 0.93 0.94 0.95 0.95 0.96 0.96 0.98 0.97
Limit of agreement
0.00091 0.21 to 0.21
t-Test CV (%) P value 8.4
0.98
0.0010
0.27 to 0.27 10.2
0.98
0.0022
0.22 to 0.22
8.2
0.95
0.0043
0.31 to 0.30 12.9
0.94
0.0045
0.17 to 0.18
7.3
0.88
0.0072
0.28 to 0.30 11.0
0.89
0.0053
0.18 to 0.19
0.87
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350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
Moore et al 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 Figure 1 Plot of corrected DDCt (cDDCt) values for the seven duplex ratio tests in 397 the comparison of nevi, primary melanomas that had not metastasized (P-M), and 398 primary melanomas that had metastasized (PþM) in the discovery set. 399 400 401 402 403 404 95 C for 10 minutes followed by 40 cycles of 95 C for DRT Selection 405 15 seconds and 60 C for 60 seconds. Fluorescence was 406 Target genes for the DRTs were identified as previously detected by the automated system, and Ct values were 407 described.13 The selected genes were paired such that a gene 408 determined when the fluorescence reached a DRN value 409 from a region of DCN gain and a gene from a region of of 0.05. 410 DCN loss comprised a single DRT. Ten FFPE tonsil DNA samples were selected as plate 411 Duplex PCR primers and Taqman MGB probes were controls and were present on each of the 96 well plates run 412 designed for these regions using the online applications with each of the assays. 413 Muplex15 and NetPrimer and the PCR design software 414 Q9 Primer Express (Applied Biosystems, Carlsbad, CA). Statistical Analysis 415 Specificity of the assays was checked using the National 416 Center for Biotechnology Information BLAST applicaFor each sample, the DCt value was created by calculating 417 16 Q10 tion . Details of the seven DRTs developed and tested are the difference between the Ct values for the two probes (the 418 419 ½T2 given in Table 2. Ct for target in the region of DNA gain minus the Ct from 420 the target in the region of DNA loss). A DDCt value was 421 then created by calculating the difference between the inqPCR 422 dividual DCt values and the mean DCt of the 10 plate 423 controls. qPCR was performed using the ABI Step One Fast Real 424 SPSS statistical software version 18.0. software (SPSS Time PCR System. Each reaction comprised 10 ng of 425 Inc., Chicago, IL) was used for binary logistic regression extracted DNA from the FFPE sample (in 3 mL made to 426 analysis and to calculate the interclass correlation coefficoncentration of 3.33 ng/mL), 5 mL of Genotyping 427 cient. GraphPad Prism software version 5.02 (GraphPad Mastermix (Applied Biosystems), and 0.3 mL of for428 Software, San Diego, CA) was used to perform paired t-test ward and reverse primers for both targets, each at a 429 comparisons and the Bland-Altman agreement analysis, concentration of 20 pmol/L, 0.2 mL each of the Taqman 430 431 which determined the bias and 95% limits of agreement. MGB probes at a concentration of 50 to 200 nmol/L. 432 Local research ethics committee approval was obtained The reactions were all performed in triplicate. qPCR 433 (IRAS reference 2952). was then performed, with the reactions incubated at 434
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497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
Duplex Ratio Tests in Malignant Melanoma Table 5 Correlation Coefficients and Corresponding P Values for Comparison of Primary Melanomas That Had Metastasized and Corresponding Metastases Assay
Correlation coefficient
Correlation coefficient P value
MYB.RREB1 BRAF.PTEN SSR1.PERP ASAP1.LZTS1 TBX2.HIC1 LDLRAD3.CCND1 AKT3.MIB2
0.663 0.520 0.753 0.532 0.675 0.137 0.654
0.002 0.019 0.0001 0.019 0.001 0.565 0.002
Results Determination of Reference Range in Nonneoplastic Diploid Tissues To develop a range of values for the DRTs in nonneoplastic diploid tissue, the assays were tested against a series of 108 FFPE samples of benign skin, lymph node, and tonsil. Although a DRT would be expected to give a DDCt of zero for any benign tissue sample, the reference range was determined to identify whether differences in assay efficiency occurred, which would be expected to generate skewed Ct values. The DDCt values for the diploid samples, using each of the ½T3 seven DRTs, are summarized in Table 3. When the DDCt values for the diploid samples were compared with the corresponding mean Ct values, for four of the seven assays a significant trend was detected (Table 3). This finding indicates that the DDCt result for these assays is dependent on the mean Ct, which may indicate that the DDCt is influenced by DNA quality because a high Ct for the same starting quantity of DNA reflects poor amplification. This finding suggests that as DNA quality changes, there is an unequal change in the reaction efficiency between the two PCR reactions comprising each assay. Because mean Ct values may vary among different types of lesions (with smaller lesions, such as nevi, with lower DNA yields typically having higher mean Ct values), this represents a potential source of bias. In an attempt to eliminate any potential bias, for the four DRTs for which a significant trend was identified, a correction factor was calculated based on the equation of the line when the two values were plotted. This correction factor was applied to all DDCt values for the diploid, nevi, and melanoma samples, producing a corrected DDCt (cDDCt) for these four assays, and these cDDCt values were used to calculate the DRT score (Table 3). In two of the three DRTs without a significant trend, the mean DDCt was close to zero (<0.05). In those with a significant trend, the cDDCt returned the mean value close to zero (Table 3).
Analytical Validity To determine the reliability of the assays, statistical tests were performed on repeat assays on the same samples.
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559 This is a crucial step in ensuring the analytical validity 560 of DRTs. 561 The Ct and DRT values for 10 plate control samples, 562 which were tested every time an assay was performed, were 563 used to determine assay reliability. The Ct values for the 564 plate control samples on the first two occasions the DRTs 565 were performed were used to determine an intraclass cor566 relation coefficient for each DRT to provide a measure of 567 agreement between assays on the same sample run on 568 569 separate occasions. The DRT values for these samples were 570 also used to determine bias, limits of agreement, coefficient 571 of variation, and paired t-test result. The bias is the mean 572 difference in DRT values between the two occasions. The 573 limits of agreement provide a further measure of analytical 574 error expressed as 2 SDs from the mean difference be575 tween repeat assays. A paired t-test is performed to establish 576 that there is no significant difference in DRT values on the 577 separate occasions, as expected with systematic rather than 578 random error. There are good agreement statistics for all 579 seven DRTs, with low levels of bias and nonsignificant P 580 values on paired t-test comparison (Table 4). ½T4 581 582 Both the MYB.RREB1 and SSR1.PERP assays targeted one 583 locus on the gained p arm and one locus on lost q arm of 584 chromosome 6. The 80 nevi and melanoma DRT results were 585 correlated for these two assays, returning an intraclass cor586 relation coefficient of 0.77. This gives a good indication that 587 both assays are detecting the same changes in chromosome 6 588 and supports the validity of the DRTs. 589 In the 288 melanoma and nevi tested, the initial failure 590 rate for the assays ranged from 3.5% (AKT.MIB2) to 11.1% 591 (ASAP1.LZTS1) on the basis of poor agreement among 592 593 replicate values, with an overall failure rate of 6.0%.
Nevi Versus Melanoma Comparison in the Discovery Series This first comparison made with the discovery series was 20 nevi versus 40 melanomas, half of which had metastasized. This was tested to establish the diagnostic potential of the assays. The assays revealed a significant difference between nevi and primary melanoma samples with all the assays, apart from TBX2.HIC1. Plots of the values generated for all seven Table 6 Means SD Values from Seven Duplex Ratio Tests from Cohorts of 145 Melanoma and 123 Nevi Means SD corrected DDCt Assay
Nevi
MYB.RREB1 BRAF.PTEN SSR1.PERP ASAP1.LZTS1 TBX2.HIC1 LDLRAD3.CCND1 AKT3.MIB2
0.11 0.21 0.09 0.14 0.01 0.15 0.10
Melanoma
0.28 0.24 0.39 0.3 0.28 0.28 0.27
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0.52 0.53 0.67 0.05 0.02 0.40 0.27
0.46 0.57 0.59 0.55 0.48 0.80 0.41
5
594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620
Moore et al 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 Figure 2 Receiver operating characteristic (ROC) curve of the speci640 ficity and sensitivity of the full panel of duplex ratio tests to differentiate 641 between melanoma and nevi from the 268 cases using the DDCt scores. 642 Area under the curve Z 0.933. 643 644 645 ½F1 assays are shown in Figure 1. Binary logistic regression 646 analysis using the seven DRT values generated b values as 647 follows for each of the assays. With these b values, the 648 results of the seven assays were able to correctly differen649 tiate between nevi and primary melanoma with 97.5% 650 sensitivity (95% CI, 94%e100%) and 95% specificity (95% 651 CI, 89%e100%). A receiver operating characteristic curve 652 using the predicted probability values derived from DDCt 653 654 values from all seven DRTs in the comparison between nevi 655 and melanomas yielded an area under the curve value of 656 0.995. 657 658 659 Prognostic Value of DRTs in Melanoma in the Discovery 660 Series and Correlation between Primary Tumor and 661 Corresponding Metastasis 662 663 The comparison of those melanomas that did and which did not 664 metastasize in the discovery series allowed the prognostic value 665 of the DRTs to be tested. There was no statistically significant 666 667 difference between the DRT results for the P-Ms and PþMs 668 with any of the seven DRTs on unpaired t-test analysis. Binary 669 logistic regression analysis using the results of all seven DRTs 670 on the 40 primary melanomas was performed to determine the 671 sensitivity and specificity for separation of PþMs and P-Ms and 672 the percentage correctly predicted. The same analysis was 673 performed using Breslow depth, mitotic index, and age. Both 674 clinical information alone and DRT score alone were able to 675 correctly predict the outcome in 28 (70%) of 40 cases. This was 676 marginally improved to 31 and 10 when DRT scores were used 677 in addition to clinical data. No single DRT score was an inde678 679 pendent predictor of prognosis on multivariate analysis when all 680 clinical variables were included. The results indicate that there 681 may be some, limited, discriminating difference in genetic 682
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instability between PþMs and P-Ms at the loci targeted. The discrimination between metastasizing and nonmetastasizing lesions with DRTs is not as successful as the discrimination between nevi and melanoma on this series. This might be expected because the DRT targets were selected from genomic regions of somatic copy number alteration common to all primary melanomas in general. There was significant correlation between the DRT values for the PþM cases and the corresponding metastases with all the DRTs apart from LDLRAD3.CCND1 (Table 5). ½T5 Paired t-test analysis using the same data revealed no significant differences between the set of PþMs and their matched metastases, with BRAF.PTEN coming closest to significance (P Z 0.07).
Diagnostic Value of DRTs in Large Cohorts of Melanomas and Nevi In the discovery series, the diagnostic potential of the DRT panel was the most striking result. To test this further, the panel was applied to large cohorts of >100 nevi and >100 melanomas. The results from these were combined with those of the discovery series to provide 122 nevi and 146 melanomas for comparison. Details of the cDDCt values generated are listed in Table 6. ½T6 With these b values from regression analysis, the results of the seven assays was able to correctly predict lesions as malignant with a sensitivity of 87% (95% CI, 83%e91%) and a specificity of 88% (95% CI, 84%e92%), with 88% of cases correctly predicted as melanoma or nevus. Separation between nevi and melanoma using predicted probability values developed from all seven assays is represented in a receiver operating characteristic curve (Figure 2), with an ½F2 area under the curve value of 0.933.
Discussion The agreement statistics for the DRTs reveal these assays to be reliable on FFPE tissue. The accuracy of the assays is supported by copy number ratios clustering around 1:1 for the diploid samples (although some DRTs required a correction factor) and the good agreement between the two DRT assays targeting 6p.6q. This study reveals that this panel of DRTs can largely distinguish unambiguous melanomas from nevi when using DNA extracted from FFPE tissues. When applied to the four main categories of melanocytic lesions, the comparison between nevi and primary melanomas revealed the most striking difference, with separation in the discovery series for identifying malignancy using logistic regression analysis with 97.5% sensitivity and 95% specificity using all seven DRTs. When applied to representative cohort specimens, the sensitivity and specificity for detecting malignancy were 87% and 88%, respectively. Comparison of P-Ms and PþMs was less discriminatory, although when combined
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683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744
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Duplex Ratio Tests in Malignant Melanoma with clinical stage, the seven DRT scores correctly separated P-Ms and PþMs in 77.5% of cases. This work supports the concept developed by Bastian et al12 that DCN can be used to differentiate nevi from melanomas when FFPE tissue is used as the starting material. Unlike a FISH assay, DRTs are not limited by the number of targets that can be tested simultaneously, provided there is a suitable yield of DNA from the sample, although the reliance on a suitable DNA yield and the possibility of DNA contamination, particularly in thin or inflamed lesions are both potential limitations of this method. An overall failure rate of 6.0% for the assays is a further limitation. The discovery series was an opportunity sample of cases from a diagnostic archive. For the discovery set only, melanomas >2 mm were used, and for the cohort, only those >1-mm thick were used. This is a potential source of selection bias. For four of the seven assays, there was a significant association between Ct and DDCt values for the diploid samples, implying that DNA quality unequally affected the efficiency of the two duplex reactions. A correction factor was applied to the results of the nevus and melanoma samples to eliminate a potential source of bias. The differentiation between nevi and melanoma using a panel of DRTs is clear grounds for testing this method against difficult and genuinely ambiguous lesions that have follow-up data. This approach would determine their diagnostic potential in clinically problematic cases. This DRT panel also needs to be tested on ambiguous and unambiguous melanocytic lesions in direct comparison with the FISH assay and CGH before any claims can be made as to how it may perform as an alternative assay in clinical practice. Although these DRTs reveal some separation between P-Ms and PþMs, it seems that the benefit of a DRT panel as a prognostic marker is marginal at best. Although it is possible that an alternative panel of DRTs would be better suited to development as prognostic markers, there appears to be greater promise for these assays as diagnostic biomarkers in melanoma. Comparison of PþM and the corresponding metastatic tumor suggests that regions of DCN gain and loss are generally conserved as the primary tumor progresses to metastasis and that there is not a many-fold change in DCN at these loci during this progression, making the assays developed on primary tumor candidates for markers of progression.
Conclusions These DRTs are accurate and reliable and have the capability of differentiating between melanomas and nevi with good sensitivity and specificity using DNA derived from FFPE tissue. The next stage of development for these assays will be to increase the sample size to determine a selection algorithm with cutoff values and then apply the prespecified
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algorithm to problematic melanocytic lesions with clinical follow-up to further test their diagnostic potential. DRTs may have a role as markers of disease progression if they can be applied to circulating cell-free tumor DNA in blood, although further information regarding DCN changes that differentiate the classes of melanomas may be required before these markers can be developed.
References 1. Cancer Research UK: Cancer incidence in the UK in 2011. Cancer Stat Rep January 2015:1e8 2. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, Buzaid AC, Cochran AJ, Coit DG, Ding S, Eggermont AM, Flaherty KT, Gimotty PA, Kirkwood JM, McMasters KM, Mihm MC Jr, Morton DL, Ross MI, Sober AJ, Sondak VK: Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009, 27:6199e6206 3. Urso C, Saieva C, Borgognoni L, Tinacci G, Zini E: Sensitivity and specificity of histological criteria in the diagnosis of conventional cutaneous melanoma. Melanoma Res 2008, 18:253e258 4. Shoo BA, Sagebiel RW, Kashani-Sabet M: Discordance in the histopathologic diagnosis of melanoma at a melanoma referral center. J Am Acad Dermatol 2010, 62:751e756 5. Lodha S, Saggar S, Celebi JT, Silvers DN: Discordance in the histopathologic diagnosis of difficult melanocytic neoplasms in the clinical setting. J Cutan Pathol 2008, 35:349e352 6. Barnhill RL, Argenyi ZB, From L, Glass LF, Maize JC, Mihm MC Jr, Rabkin MS, Ronan SG, White WL, Piepkorn M: Atypical Spitz nevi/tumors: lack of consensus for diagnosis, discrimination from melanoma, and prediction of outcome. Hum Pathol 1999, 30:513e520 7. Farmer ER, Gonin R, Hanna MP: Discordance in the histopathologic diagnosis of melanoma and melanocytic nevi between expert pathologists. Hum Pathol 1996, 27:528e531 8. Troxel DB: Trends in pathology malpractice claims. Am J Surg Pathol 2012, 36:e1ee5 9. Carlson JA, Ross JS, Slominski AJ: New techniques in dermatopathology that help to diagnose and prognosticate melanoma. Clin Dermatol 2009, 27:75e102 10. Baudis M: Genomic imbalances in 5918 malignant epithelial tumors: an explorative meta-analysis of chromosomal CGH data. BMC Cancer 2007, 7:226 11. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, Cho KH, Aiba S, Bröcker EB, LeBoit PE, Pinkel D, Bastian BC: Distinct sets of genetic alterations in melanoma. N Engl J Med 2005, 353:2135e2147 12. Gerami P, Jewell SS, Morrison LE, Blondin B, Schulz J, Ruffalo T, Matushek P 4th, Legator M, Jacobson K, Dalton SR, Charzan S, Kolaitis NA, Guitart J, Lertsbarapa T, Boone S, LeBoit PE, Bastian BC: Fluorescence in situ hybridization (FISH) as an ancillary diagnostic tool in the diagnosis of melanoma. Am J Surg Pathol 2009, 33:1146e1156 13. Moore DA, Saldanha G, Ehdode A, Potter L, Dyall L, Bury D, Pringle JH: Accurate detection of copy number changes in DNA extracted from formalin-fixed, paraffin-embedded melanoma tissue using duplex ratio tests. J Mol Diagn 2013, 15:687e694 14. Saldanha G, Potter L, Dyall L, Bury D, Hathiari N, Ehdode A, Hollox E, Pringle JH: Detection of copy number changes in DNA from formalin fixed paraffin embedded tissues using paralogue ratio tests. Anal Chem 2011, 83:3484e3492 15. Rachlin J, Ding C, Cantor C, Kasif S: MuPlex: multi-objective multiplex PCR assay design. Nucleic Acids Res 2005, 33(Web Server issue):W544eW547 16. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403e410
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Duplex Ratio Tests as Diagnostic Biomarkers in Malignant Melanoma Q12 Q1
David A. Moore, Gerald Saldanha, Abdlrzag Ehdode, Mohamed Z. Mughal, Linda Potter, Lovesh Dyall, and James H. Pringle
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From the Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom Accepted for publication May 13, 2015.
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Address correspondence to David A. Moore, Department of Cancer Studies and Molecular Medicine, University of Leicester, Level 3 RK-CSB, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom. E-mail: dam18@le. ac.uk.
Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCRebased assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%e91%) and a specificity of 88% (95% CI, 84%e92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas and 20 metastasizing primary melanomas revealed that DRTs had a marginal benefit as prognostic markers. DRTs have early potential to act as molecular biomarkers of melanoma on FFPE specimens pending validation, and DRTs may have applicability as prognostic markers in melanoma or other tumor types if new DRTs to relevant loci are developed. (J Mol Diagn 2015, -: 1e7; http://dx.doi.org/10.1016/ j.jmoldx.2015.05.001)
Malignant melanoma incidence in the United Kingdom has increased markedly during the past 30 years, with >13,000 new cases diagnosed in 2011 within the United Kingdom alone.1 The prognosis for those with disseminated disease is poor, with a 5-year survival rate of <10%.2 Many thousands of melanocytic lesions, benign and malignant, are excised and undergo histopathologic analysis every year to diagnose or exclude malignancy or dysplasia, for cosmetic reasons, or because lesions are causing physical irritation. Histopathologic analysis is the gold standard for diagnosis of melanocytic tumors. In most melanocytic lesions, a confident diagnosis of benign nevus or malignant melanoma can be made on a standard hematoxylin and eosinestained tissue section alone, and the histologic features of melanocytic lesions are well described and recognized.3 Within a small subset of melanocytic lesions, however, it can be very difficult to differentiate between melanoma and nevus based on hematoxylin and eosin staining, and even specialist dermatopathologists cannot agree over the classification of a subset of cases.4e7 As a result, the misdiagnosis of
melanocytic lesions accounts for a significant proportion of the litigation claims against histopathologists.8 Diagnostically difficult and genuinely histologically ambiguous melanocytic lesions have prompted the search for effective diagnostic biomarkers in melanoma.9 Chromosomal instability is a well-described feature of malignant tumors, with typical patterns of DNA copy number (DCN) change seen across the genome in specific tumor types.10,11 DCN is a useful marker because it is well characterized in malignant tissue and can be assayed in formalin-fixed, paraffin-embedded (FFPE) tissue. Using the information generated from comparative genomic hybridization (CGH) studies, Gerami et al12 found that fluorescent in situ hybridization (FISH) can be used to differentiate between nevi and melanoma. The assay is a combination of four FISH probes (MYB, RREB1, CEP6, and This work was supported by The Pathological Society of Great Britain and Ireland grant SGS 2010/04/01 (Small Grant Scheme 2010). 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.2015.05.001
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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
Moore et al Table 1
Clinical and Pathological Characteristics of the Nevi and Melanoma Included in the Discovery Series Type (no.)
Site (no.)
Series Head and neck Trunk Limb Mean age (range, years) Male/female ratio IDN BCN Breslow depth (mm) Ulcerated (%) Mitotic index Nevi 2 P-M 3 PþM 5
11 5 5
7 12 10
37 (19e67) 65 years (34e88) 69 years (46e88)
6:14 8:12 10:10
15 5
3.9 (2.0e7.4) 5.4 (2.5e9.7)
35 60
5.9 (1e19) 9.2 (1e40)
BCN, benign compound nevus; IDN, intradermal nevus; P-M, primary melanoma without metastasis; PþM, primary melanoma with metastasis.
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CCND1). This is the only melanoma diagnostic biomarker to have been translated into clinical practice and to have become commercially available. Our group has previously described a series of assays using real-time quantitative PCR (qPCR), namely, duplex ratio tests (DRTs) and paralogue ratio tests.13,14 These are used to compare the quantity of DNA at one site in the genome with another site, both in the same qPCR assay. The ratio between a pair of targets should be 1:1 in diploid tissue (assuming equal efficiency of the two reactions), although when the relevant DCN changes are present, such as may occur in cancer, the ratio will differ from 1. A panel of five DRTs was previously able to differentiate between histologically unambiguous cases of nevi and melanoma in a
small set of cases. The analytical validity of DRTs in FFPE Q8 has been found in previous work by our laboratory using both clinical sample and cell line DNA.13 The assays benefit from being relatively simple cheap tests for assessing DCN changes, and multiple assays can be tested with each sample, meaning that a wide range of DNA loci are tested. DRTs may therefore represent a potential alternative to FISH and CGH for the assessment of DCN variation. The assays have disadvantages compared with FISH, however, such as relying on a specific quantity of DNA of adequate quality being extracted from small FFPE lesions into solution and the potential for significant DNA contamination, particularly in small lesions and those lesions with a heavy inflammatory infiltrate.
Table 2 The Duplex Ratio Test Targets for the Series of Seven Existing Duplex Ratio Tests, Including Sequences for the Primers and Probes (Target from Region of Gain Shaded) Assay
Locus
Forward primer
Reverse primer
Probe
5 -TCATGAAGACCTCACAGTAAAAA- 5 -ATCCAGACAACTGTTCAAACTG- VIC-50 -AATCTCGATGGAGTGGGTAGGT-30 ATG-30 TC-30 -MGB 0 0 0 0 PTEN 10q23.3 5 -GCGACTGCGCTCAGTTCTCT-3 5 -TCACAGCGGCTCAACTCTCA-3 FAM-50 -CTCTCGGAAGCTGC-30 MGB RREB1 6p25 50 -TGTCCCAATGACGTCAAGTTC-30 50 -CTACACTCATGACCGCCGAC-30 FAM-50 -GTTGATGGAAGATAGGTCT-30 -MGB 0 0 0 MYB 6q22-23 5 -GCTTGTACAGAAATACGGTCC5 -GCCACCTCTCCCTACATTGTT-3 VIC-50 -TTGCCAAGCACTTAAAGA-30 30 -MGB 0 0 SSR1 6p24.3 5 -CCTTAGATGCCTCATTCCGTT5 -CAGTGTTCAGAGGAAGAGCTFAM-50 -CAGGACTACCAGTTTT0 0 AT-3 GTG-3 AT-30 -MGB PERP 6q24 50 -TACTCAGCGCCATCGCCT-30 50 -AGCATTTCCACCACAGCGAG-30 VIC-50 -TTGCAGTCTAGCGACCAC-30 -MGB 0 0 ASAP1 8q24.1-24.2 5 -CAGGCTAAATCTGGAAAGTTCAA- 5 -GTTTGTCATCCAGATCATCAVIC-50 -AATCTTCGACAGGAGGA0 0 TC-3 TCG-3 GATA-30 -MGB LZTS1 8p22 50 -GTGACCACTCTTCTTTAAGCCAT- 50 -TGGAAAGCCACACCCTCTG-30 FAM-50 -CCTGGGCTGGGTGC-30 AGA-30 MGB 0 CCND1 11q13 5 -TGGTGAACAAGCTCAAGTGGAA-30 50 -CGCCTCTGGCATTTTGGA-30 FAM-50 -CCGCACGATTTCATTGA-30 -MGB LDLRAD3 11p13 50 -ACAACGTCAATAATGGCATCCA-30 50 -GCCTACTTCCGACGCATTCT-30 VIC-50 -TTGCCAAGCACTTAAA30 -MGB 0 0 0 TBX2 17q23 5 -GGCCTAGACCGCGTGATAAA-3 5 -GGTCTACACTGACTTCAGTCGT- VIC-50 -GGTTGAGGGATGCTGGAAACTG-30 30 -MGB HIC1 17p13.3 50 -TGTGCGACGTGATCATCGT-30 50 -CACCACCAGGGACTTGAGG FAM-50 -AAGAGGGCGTTCTGCATAG-30 30 -MGB 0 0 0 AKT3 1q44 5 -CTGGACATCACCAGTCCTAGC5 -ACCCTTGGCTGGTCTGGG-3 VIC-50 -ATAGCAGGGGCACCTT0 TC-3 30 -MGB MIB2 1p36.33 50 -CACCAAGCACCACTCCTTCTG-30 50 -CAGCCGCTTCACTGTGTCAA-30 FAM-50 -CCGGGTCATCGGCGA30 -MGB BRAF
2
7q34
0
0
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Duplex Ratio Tests in Malignant Melanoma Table 3 Means SD DDCt for the Diploid Samples for all Seven Duplex Ratio Tests, with Details of the DDCt Versus Ct Correction
Assay
Means SD DDCt
Significant trend Ct versus Means SD DDCt corrected DDCt
MYB.RREB1 BRAF.PTEN SSR1.PERP ASAP1.LZTS1 TBX2.HIC1 LDLRAD3.CCND1 AKT3.MIB2
0.162 0.220 0.183 0.163 0.040 0.016 0.177
Yes Yes No Yes No No Yes
0.285 0.478 0.377 0.263 0.277 0.443 0.268
0.0197 <0.0001 NA 0.0004 NA NA 0.0002
0.269 0.461 0.242 0.245
NA, not applicable.
Our aim was to test DRTs on an opportunity sample of FFPE tissue comprising nevi, primary melanomas that had not metastasized (P-M), primary melanomas that had metastasized (PþM), and the matched metastases. We made three comparisons, nevi versus primary melanoma, P-M versus PþM, and PþM versus the matched metastases, to determine the value of DRTs for assessing diagnosis, prognosis, and progression, respectively. We found DRTs to be most useful to differentiate between nevi and melanoma. Therefore, we subsequently tested the assays on large representative cohorts of melanomas and nevi to develop a diagnostic algorithm.
Materials and Methods Case Selection FFPE tissue blocks were selected from the University Hospitals of Leicester histopathology archive. The discovery series was intended to verify the findings by Gerami et al12 that DCN assessed on FFPE tissue was a marker of malignancy. This discovery series comprised 20 nevi, 40 primary melanomas, and 20 metastatic melanoma samples. The 20 nevi consisted of unambiguous cases of benign compound nevi and benign intradermal nevi. For inclusion, all nevi required a cross-sectional area of >25 mm2, and there were no junctional nevi included because of the difficulty of microdissecting thin melanocytic lesions. Of the 40 primary melanomas, 20 had no evidence of metastasis with at least 5 years of follow-up, and these were designated P-M. The remaining 20 had histologic evidence of metastasis within 5 years of the primary diagnosis and were designated PþM. The inclusion criteria for both classes of melanoma were sufficient thickness of FFPE tissue block to allow for six sections of tissue to be cut and Breslow thickness >2 mm. The PþMs also required matched FFPE metastatic melanoma tissue to be available for DNA extraction. Exclusion criteria for both classes of melanoma were equivocal diagnosis of primary melanoma, tumor representing <5% sectional area, and a sectional area of <25 mm2. The mean
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311 Breslow depth was 3.9 mm for the P-Ms and 5.4 mm for the 312 PþMs. The mean period from primary to metastasis for the 313 PþMs was 16 months. Clinicopathologic details of these 314 nevi and melanoma are summarized in Table 1. Diploid ½T1 315 control samples were selected to develop a reference range of 316 values for the assays. This consisted of 53 benign reactive 317 tonsils, 26 skin samples from wide local excisions for mel318 anoma (none of which contained histologic evidence of 319 tumor), and 23 histologically tumor-free lymph nodes from 320 321 lymph node dissections for melanoma. 322 Retrospective cohorts of sequentially submitted mela323 noma and nevi were also assembled. The same inclusion 324 and exclusion criteria used in the discovery series were also 325 used for the cohort of nevi. The melanoma inclusion and 326 exclusion criteria were as for the discovery series with the 327 exception that all melanomas >1 mm in thickness were 328 included in an attempt to make the cohort as representative 329 as possible. 330 From January 1, 2000, onward, all melanomas and nevi 331 sequentially diagnosed in the Cellular Pathology Depart332 333 ment of the University Hospitals of Leicester NHS Trust 334 that met these criteria were included in the cohort; hema335 toxylin and eosin histologic findings were reviewed and the 336 paraffin-embedded tissue block inspected to establish 337 whether the criteria were met. The cohorts of melanoma and 338 nevi extended up to the point where >100 cases were 339 included in each. The melanoma cohort consisted of 106 340 tumors from 44 men and 62 women. Breslow depth of the 341 lesions ranged from 1.1 to 21.0 mm (mean, 4.2 mm; median, 342 2.6 mm). The age range was 30 to 95 years (mean, 71 years; 343 median, 75 years). The nevus cohort consisted of 102 344 345 benign nevi (intradermal or compound type) from 51 men 346 and 51 women. The age range was 18 to 84 years (mean, 35 347 years; median, 32 years). The tissue samples underwent 348 tissue microdissection and DNA extraction as previously 349 described.13 Table 4 Interclass Correlation, Bias, Limit of Agreement, CV, and t-Test P Value for Each of the Seven Duplex Ratio Tests
Assay
Interclass correlation coefficient Bias
MYB RREB1 BRAF PTEN SSR1 PERP TBX2 HIC1 ASAP1 LZTS1 LDLRAD3 CCND1 AKT3 MIB2
0.95 0.95 0.95 0.93 0.95 0.96 0.93 0.94 0.95 0.95 0.96 0.96 0.98 0.97
Limit of agreement
0.00091 0.21 to 0.21
t-Test CV (%) P value 8.4
0.98
0.0010
0.27 to 0.27 10.2
0.98
0.0022
0.22 to 0.22
8.2
0.95
0.0043
0.31 to 0.30 12.9
0.94
0.0045
0.17 to 0.18
7.3
0.88
0.0072
0.28 to 0.30 11.0
0.89
0.0053
0.18 to 0.19
0.87
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350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
Moore et al 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 Figure 1 Plot of corrected DDCt (cDDCt) values for the seven duplex ratio tests in 397 the comparison of nevi, primary melanomas that had not metastasized (P-M), and 398 primary melanomas that had metastasized (PþM) in the discovery set. 399 400 401 402 403 404 95 C for 10 minutes followed by 40 cycles of 95 C for DRT Selection 405 15 seconds and 60 C for 60 seconds. Fluorescence was 406 Target genes for the DRTs were identified as previously detected by the automated system, and Ct values were 407 described.13 The selected genes were paired such that a gene 408 determined when the fluorescence reached a DRN value 409 from a region of DCN gain and a gene from a region of of 0.05. 410 DCN loss comprised a single DRT. Ten FFPE tonsil DNA samples were selected as plate 411 Duplex PCR primers and Taqman MGB probes were controls and were present on each of the 96 well plates run 412 designed for these regions using the online applications with each of the assays. 413 Muplex15 and NetPrimer and the PCR design software 414 Q9 Primer Express (Applied Biosystems, Carlsbad, CA). Statistical Analysis 415 Specificity of the assays was checked using the National 416 Center for Biotechnology Information BLAST applicaFor each sample, the DCt value was created by calculating 417 16 Q10 tion . Details of the seven DRTs developed and tested are the difference between the Ct values for the two probes (the 418 419 ½T2 given in Table 2. Ct for target in the region of DNA gain minus the Ct from 420 the target in the region of DNA loss). A DDCt value was 421 then created by calculating the difference between the inqPCR 422 dividual DCt values and the mean DCt of the 10 plate 423 controls. qPCR was performed using the ABI Step One Fast Real 424 SPSS statistical software version 18.0. software (SPSS Time PCR System. Each reaction comprised 10 ng of 425 Inc., Chicago, IL) was used for binary logistic regression extracted DNA from the FFPE sample (in 3 mL made to 426 analysis and to calculate the interclass correlation coefficoncentration of 3.33 ng/mL), 5 mL of Genotyping 427 cient. GraphPad Prism software version 5.02 (GraphPad Mastermix (Applied Biosystems), and 0.3 mL of for428 Software, San Diego, CA) was used to perform paired t-test ward and reverse primers for both targets, each at a 429 comparisons and the Bland-Altman agreement analysis, concentration of 20 pmol/L, 0.2 mL each of the Taqman 430 431 which determined the bias and 95% limits of agreement. MGB probes at a concentration of 50 to 200 nmol/L. 432 Local research ethics committee approval was obtained The reactions were all performed in triplicate. qPCR 433 (IRAS reference 2952). was then performed, with the reactions incubated at 434
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435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496
497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
Duplex Ratio Tests in Malignant Melanoma Table 5 Correlation Coefficients and Corresponding P Values for Comparison of Primary Melanomas That Had Metastasized and Corresponding Metastases Assay
Correlation coefficient
Correlation coefficient P value
MYB.RREB1 BRAF.PTEN SSR1.PERP ASAP1.LZTS1 TBX2.HIC1 LDLRAD3.CCND1 AKT3.MIB2
0.663 0.520 0.753 0.532 0.675 0.137 0.654
0.002 0.019 0.0001 0.019 0.001 0.565 0.002
Results Determination of Reference Range in Nonneoplastic Diploid Tissues To develop a range of values for the DRTs in nonneoplastic diploid tissue, the assays were tested against a series of 108 FFPE samples of benign skin, lymph node, and tonsil. Although a DRT would be expected to give a DDCt of zero for any benign tissue sample, the reference range was determined to identify whether differences in assay efficiency occurred, which would be expected to generate skewed Ct values. The DDCt values for the diploid samples, using each of the ½T3 seven DRTs, are summarized in Table 3. When the DDCt values for the diploid samples were compared with the corresponding mean Ct values, for four of the seven assays a significant trend was detected (Table 3). This finding indicates that the DDCt result for these assays is dependent on the mean Ct, which may indicate that the DDCt is influenced by DNA quality because a high Ct for the same starting quantity of DNA reflects poor amplification. This finding suggests that as DNA quality changes, there is an unequal change in the reaction efficiency between the two PCR reactions comprising each assay. Because mean Ct values may vary among different types of lesions (with smaller lesions, such as nevi, with lower DNA yields typically having higher mean Ct values), this represents a potential source of bias. In an attempt to eliminate any potential bias, for the four DRTs for which a significant trend was identified, a correction factor was calculated based on the equation of the line when the two values were plotted. This correction factor was applied to all DDCt values for the diploid, nevi, and melanoma samples, producing a corrected DDCt (cDDCt) for these four assays, and these cDDCt values were used to calculate the DRT score (Table 3). In two of the three DRTs without a significant trend, the mean DDCt was close to zero (<0.05). In those with a significant trend, the cDDCt returned the mean value close to zero (Table 3).
Analytical Validity To determine the reliability of the assays, statistical tests were performed on repeat assays on the same samples.
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559 This is a crucial step in ensuring the analytical validity 560 of DRTs. 561 The Ct and DRT values for 10 plate control samples, 562 which were tested every time an assay was performed, were 563 used to determine assay reliability. The Ct values for the 564 plate control samples on the first two occasions the DRTs 565 were performed were used to determine an intraclass cor566 relation coefficient for each DRT to provide a measure of 567 agreement between assays on the same sample run on 568 569 separate occasions. The DRT values for these samples were 570 also used to determine bias, limits of agreement, coefficient 571 of variation, and paired t-test result. The bias is the mean 572 difference in DRT values between the two occasions. The 573 limits of agreement provide a further measure of analytical 574 error expressed as 2 SDs from the mean difference be575 tween repeat assays. A paired t-test is performed to establish 576 that there is no significant difference in DRT values on the 577 separate occasions, as expected with systematic rather than 578 random error. There are good agreement statistics for all 579 seven DRTs, with low levels of bias and nonsignificant P 580 values on paired t-test comparison (Table 4). ½T4 581 582 Both the MYB.RREB1 and SSR1.PERP assays targeted one 583 locus on the gained p arm and one locus on lost q arm of 584 chromosome 6. The 80 nevi and melanoma DRT results were 585 correlated for these two assays, returning an intraclass cor586 relation coefficient of 0.77. This gives a good indication that 587 both assays are detecting the same changes in chromosome 6 588 and supports the validity of the DRTs. 589 In the 288 melanoma and nevi tested, the initial failure 590 rate for the assays ranged from 3.5% (AKT.MIB2) to 11.1% 591 (ASAP1.LZTS1) on the basis of poor agreement among 592 593 replicate values, with an overall failure rate of 6.0%.
Nevi Versus Melanoma Comparison in the Discovery Series This first comparison made with the discovery series was 20 nevi versus 40 melanomas, half of which had metastasized. This was tested to establish the diagnostic potential of the assays. The assays revealed a significant difference between nevi and primary melanoma samples with all the assays, apart from TBX2.HIC1. Plots of the values generated for all seven Table 6 Means SD Values from Seven Duplex Ratio Tests from Cohorts of 145 Melanoma and 123 Nevi Means SD corrected DDCt Assay
Nevi
MYB.RREB1 BRAF.PTEN SSR1.PERP ASAP1.LZTS1 TBX2.HIC1 LDLRAD3.CCND1 AKT3.MIB2
0.11 0.21 0.09 0.14 0.01 0.15 0.10
Melanoma
0.28 0.24 0.39 0.3 0.28 0.28 0.27
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0.52 0.53 0.67 0.05 0.02 0.40 0.27
0.46 0.57 0.59 0.55 0.48 0.80 0.41
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Moore et al 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 Figure 2 Receiver operating characteristic (ROC) curve of the speci640 ficity and sensitivity of the full panel of duplex ratio tests to differentiate 641 between melanoma and nevi from the 268 cases using the DDCt scores. 642 Area under the curve Z 0.933. 643 644 645 ½F1 assays are shown in Figure 1. Binary logistic regression 646 analysis using the seven DRT values generated b values as 647 follows for each of the assays. With these b values, the 648 results of the seven assays were able to correctly differen649 tiate between nevi and primary melanoma with 97.5% 650 sensitivity (95% CI, 94%e100%) and 95% specificity (95% 651 CI, 89%e100%). A receiver operating characteristic curve 652 using the predicted probability values derived from DDCt 653 654 values from all seven DRTs in the comparison between nevi 655 and melanomas yielded an area under the curve value of 656 0.995. 657 658 659 Prognostic Value of DRTs in Melanoma in the Discovery 660 Series and Correlation between Primary Tumor and 661 Corresponding Metastasis 662 663 The comparison of those melanomas that did and which did not 664 metastasize in the discovery series allowed the prognostic value 665 of the DRTs to be tested. There was no statistically significant 666 667 difference between the DRT results for the P-Ms and PþMs 668 with any of the seven DRTs on unpaired t-test analysis. Binary 669 logistic regression analysis using the results of all seven DRTs 670 on the 40 primary melanomas was performed to determine the 671 sensitivity and specificity for separation of PþMs and P-Ms and 672 the percentage correctly predicted. The same analysis was 673 performed using Breslow depth, mitotic index, and age. Both 674 clinical information alone and DRT score alone were able to 675 correctly predict the outcome in 28 (70%) of 40 cases. This was 676 marginally improved to 31 and 10 when DRT scores were used 677 in addition to clinical data. No single DRT score was an inde678 679 pendent predictor of prognosis on multivariate analysis when all 680 clinical variables were included. The results indicate that there 681 may be some, limited, discriminating difference in genetic 682
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instability between PþMs and P-Ms at the loci targeted. The discrimination between metastasizing and nonmetastasizing lesions with DRTs is not as successful as the discrimination between nevi and melanoma on this series. This might be expected because the DRT targets were selected from genomic regions of somatic copy number alteration common to all primary melanomas in general. There was significant correlation between the DRT values for the PþM cases and the corresponding metastases with all the DRTs apart from LDLRAD3.CCND1 (Table 5). ½T5 Paired t-test analysis using the same data revealed no significant differences between the set of PþMs and their matched metastases, with BRAF.PTEN coming closest to significance (P Z 0.07).
Diagnostic Value of DRTs in Large Cohorts of Melanomas and Nevi In the discovery series, the diagnostic potential of the DRT panel was the most striking result. To test this further, the panel was applied to large cohorts of >100 nevi and >100 melanomas. The results from these were combined with those of the discovery series to provide 122 nevi and 146 melanomas for comparison. Details of the cDDCt values generated are listed in Table 6. ½T6 With these b values from regression analysis, the results of the seven assays was able to correctly predict lesions as malignant with a sensitivity of 87% (95% CI, 83%e91%) and a specificity of 88% (95% CI, 84%e92%), with 88% of cases correctly predicted as melanoma or nevus. Separation between nevi and melanoma using predicted probability values developed from all seven assays is represented in a receiver operating characteristic curve (Figure 2), with an ½F2 area under the curve value of 0.933.
Discussion The agreement statistics for the DRTs reveal these assays to be reliable on FFPE tissue. The accuracy of the assays is supported by copy number ratios clustering around 1:1 for the diploid samples (although some DRTs required a correction factor) and the good agreement between the two DRT assays targeting 6p.6q. This study reveals that this panel of DRTs can largely distinguish unambiguous melanomas from nevi when using DNA extracted from FFPE tissues. When applied to the four main categories of melanocytic lesions, the comparison between nevi and primary melanomas revealed the most striking difference, with separation in the discovery series for identifying malignancy using logistic regression analysis with 97.5% sensitivity and 95% specificity using all seven DRTs. When applied to representative cohort specimens, the sensitivity and specificity for detecting malignancy were 87% and 88%, respectively. Comparison of P-Ms and PþMs was less discriminatory, although when combined
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683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744
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Duplex Ratio Tests in Malignant Melanoma with clinical stage, the seven DRT scores correctly separated P-Ms and PþMs in 77.5% of cases. This work supports the concept developed by Bastian et al12 that DCN can be used to differentiate nevi from melanomas when FFPE tissue is used as the starting material. Unlike a FISH assay, DRTs are not limited by the number of targets that can be tested simultaneously, provided there is a suitable yield of DNA from the sample, although the reliance on a suitable DNA yield and the possibility of DNA contamination, particularly in thin or inflamed lesions are both potential limitations of this method. An overall failure rate of 6.0% for the assays is a further limitation. The discovery series was an opportunity sample of cases from a diagnostic archive. For the discovery set only, melanomas >2 mm were used, and for the cohort, only those >1-mm thick were used. This is a potential source of selection bias. For four of the seven assays, there was a significant association between Ct and DDCt values for the diploid samples, implying that DNA quality unequally affected the efficiency of the two duplex reactions. A correction factor was applied to the results of the nevus and melanoma samples to eliminate a potential source of bias. The differentiation between nevi and melanoma using a panel of DRTs is clear grounds for testing this method against difficult and genuinely ambiguous lesions that have follow-up data. This approach would determine their diagnostic potential in clinically problematic cases. This DRT panel also needs to be tested on ambiguous and unambiguous melanocytic lesions in direct comparison with the FISH assay and CGH before any claims can be made as to how it may perform as an alternative assay in clinical practice. Although these DRTs reveal some separation between P-Ms and PþMs, it seems that the benefit of a DRT panel as a prognostic marker is marginal at best. Although it is possible that an alternative panel of DRTs would be better suited to development as prognostic markers, there appears to be greater promise for these assays as diagnostic biomarkers in melanoma. Comparison of PþM and the corresponding metastatic tumor suggests that regions of DCN gain and loss are generally conserved as the primary tumor progresses to metastasis and that there is not a many-fold change in DCN at these loci during this progression, making the assays developed on primary tumor candidates for markers of progression.
Conclusions These DRTs are accurate and reliable and have the capability of differentiating between melanomas and nevi with good sensitivity and specificity using DNA derived from FFPE tissue. The next stage of development for these assays will be to increase the sample size to determine a selection algorithm with cutoff values and then apply the prespecified
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algorithm to problematic melanocytic lesions with clinical follow-up to further test their diagnostic potential. DRTs may have a role as markers of disease progression if they can be applied to circulating cell-free tumor DNA in blood, although further information regarding DCN changes that differentiate the classes of melanomas may be required before these markers can be developed.
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