Utilization of Cell-Transfer Technique for Molecular Testing on Hematoxylin-Eosin Stained Sections A Viable Option for Small Biopsies That Lack Tumor Tissues in Paraffin Block Howard H. Wu, MD; Stephen M. Jovonovich, MD; Melissa Randolph, CT(ASCP); Kristin M. Post, MLS(ASCP), MPH; Joyashree D. Sen, MD; Kendra Curless, MLS(ASCP); Liang Cheng, MD Context. In some instances the standard method of doing molecular testing from formalin-fixed, paraffinembedded block is not possible because of limited tissue. Tumor cell enriched cell-transfer technique has been proven useful for performing immunocytochemistry and molecular testing on cytologic smears. Objective. To establish the cell-transfer technique as a viable option for isolating tumor cells from hematoxylineosin (H&E) stained slides. Design. Molecular testing was performed by using the cell-transfer technique on 97 archived H&E-stained slides from a variety of different tumors. Results were compared to the conventional method of molecular testing. Results. Polymerase chain reaction based molecular testing via the cell-transfer technique was successfully performed on 82 of 97 samples (85%). This included 39 of 47 cases for EGFR, 10 of 11 cases for BRAF, and 33 of 39 cases for KRAS mutations. Eighty-one of 82 cell-transfer technique samples (99%) showed agreement with previous standard method results, including 4 mutations and 35 wild-type alleles for EGFR, 4 mutations and 6 wild-type alleles for BRAF, and 11 mutations and 21 wild-type alleles for KRAS. There was only 1 discrepancy: a cell-transfer technique with a false-negative KRAS result (wild type versus G12C). Conclusions. Molecular testing performed on H&Estained sections via cell-transfer technique is useful when tissue from cell blocks and small surgical biopsy samples is exhausted and the only available material for testing is on H&E-stained slides. (Arch Pathol Lab Med. 2016;140:1383 1389; doi: 10.5858/arpa.2015-0454-OA) The understanding of the molecular pathogenesis of cancer has led to the development of chemotherapeutic agents that target specific genetic alterations such as mutations in the epidermal growth factor receptor (EGFR) and BRAF genes. It is estimated that well over a million people are living with colorectal cancer and 50% to 70% of those with advanced-stage disease receive adjuvant chemotherapy. The US Food and Drug Administration has approved the use of specific targeted chemotherapeutic agents such as panitumumab and cetuximab, which are EGFR inhibitors. 1 Retrospective subset analyses of metastatic or advanced colorectal cancer trials showed that patients with a colorectal tumor-bearing mutated KRAS did not benefit from cetuximab, whereas patients with a tumorbearing wild-type KRAS did benefit from cetuximab. 2 Adenocarcinoma represents the most common type of lung cancer, and understanding of its molecular pathogenesis has Accepted for publication April 11, 2016. Published as an Early Online Release May 19, 2016. From the Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Howard H. Wu, MD, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 350 W 11th St, IUHPL-Room 4086, Indianapolis, IN 46202 (email: hhwu@ iupui.edu). led to the development of several novel chemotherapeutic agents that offer potentially effective therapy by targeting specific genetic alterations such as mutations in EGFR and KRAS. EGFR mutations are found in approximately 10% to 15% of non small cell lung cancers with the highest frequency occurring in adenocarcinoma. Lung adenocarcinomas driven by EGFR mutations are sensitive to tyrosine kinase inhibitors such as gefitinib and erlotinib and affected patients will have longer progression-free survival than patients whose tumors do not contain EGFR mutations. 3 8 BRAF mutations, detected in regionally advanced or metastatic melanoma, appear to be one of the initiating steps in the development of primary melanoma. The discovery of activating mutations in BRAF has led to the development of molecular-targeted therapy. 9 The use of these and other targeted chemotherapeutic agents necessitates timely and accurate molecular testing of good-quality tumor cells. In some instances the conventional method of doing molecular testing on formalin-fixed, paraffin-embedded (FFPE) tumor is not possible because of limited tissue. We have shown that the cell-transfer technique (CTT) can be used as one tool to help achieve testing in such circumstances. The CTT has proven to be a reliable and useful method for performing immunocytochemistry and molecular testing by facilitating the use of direct cytologic smears as a source of Arch Pathol Lab Med Vol 140, December 2016 Cell Transfer on H&E-Stained Sections Wu et al 1383
Table 1. tumor cells in cases when the cell blocks lack adequate cellularity. 10 16 This technology can also be applied to hematoxylin-eosin (H&E) stained sections when the cells of interest are exhausted from the paraffin block. In this study we simply set out to establish the CTT as a viable option for isolating tumor cells from H&E-stained slides. This is helpful when tissue from cell blocks and small surgical biopsy samples is exhausted and the only available material for testing is on H&E-stained slides. We did the molecular testing with our standard polymerase chain reaction (PCR) based platforms. To validate this technique, we tested corresponding FFPE tissue in addition to the formalin-fixed, H&E-stained sections. MATERIALS AND METHODS This study was approved by the Institutional Review Board of Indiana University (protocol No. 1401456334). A variety of Comparison of Results of Cell-Transfer and Conventional Method for EGFR Case No. Organ Diagnosis Tissue DNA-FFPE, ng DNA-CTT, ng Conventional CTT 1 Lung Adenoca Biopsy 15.2 5.3 19 deletion 19 deletion 2 Colon Adenoca Biopsy 20 1.3 WT Inadequate 3 Lung Squam ca Core 31.5 11.9 WT Inadequate 4 Lung Adenoca Biopsy 72.2 3.5 WT WT 5 Lung Adenoca Core 1.2 7.4 WT WT 6 Lung Adenoca Biopsy 18.6 2 19 deletion 19 deletion 7 Lung Adenoca Core 28.1 6.8 WT WT 8 Lung Adenoca Biopsy 16.7 5.5 WT WT 9 Colon Adenoca Biopsy 79.9 18.2 WT WT 10 Liver Adenoca Core 12.7 5.6 WT WT 11 Lung Adenoca Biopsy 34.9 7.4 19 deletion 19 deletion 12 Lung Adenoca Biopsy 32.4 6.6 WT Inadequate 13 Lung Adenoca Biopsy 78.5 7.2 WT WT 14 Lung Adenoca Biopsy 97.9 6.7 WT WT 15 Lung Adenoca Biopsy 24.4 9.9 WT WT 16 LN Adenoca Core 77.2 6.2 WT WT 17 Lung Adenoca Core 33.2 0.3 WT WT 18 Lung Adenoca Biopsy 17.2 3.9 WT Inadequate 19 Lung Adenoca Biopsy 9.7 2.7 WT WT 20 Liver Adenoca Core 40.7 2.3 WT WT 21 Lung Adenoca Biopsy 18.7 2.2 WT WT 22 Lung Adenoca Core 39.7 11.1 L858R L858R 23 Liver Adenoca Core 166.3 21.7 WT WT 24 Lung Adenoca Core 10.5 1.7 WT WT 25 Lung Adenoca Biopsy 32.5 2.2 WT WT 26 Lung Adenoca Biopsy 12.6 5 WT WT 27 Lung Adenoca Core 17.4 9.4 WT WT 28 Lung Adenoca Biopsy 21.5 8.9 WT WT 29 Lung Adenoca Biopsy 13.3 6.2 G719X Inadequate 30 Lung Squam ca Biopsy 45.6 2.3 WT Inadequate 31 Lung Adenoca Biopsy 38.5 6.5 WT WT 32 Lung Adenoca Biopsy 71.8 19.1 WT WT 33 Lung Squam ca Biopsy 8.4 2.3 WT Inadequate 34 Bone Adenoca Core 11.1 4.6 WT WT 35 Bone Adenoca Biopsy 100.6 20.4 Inadequate Inadequate 36 Lung Adenoca Biopsy 16.2 7.4 WT WT 37 Lung Adenoca Core 6.3 4.4 WT WT 38 Lung Adenoca Core 8.7 5.8 WT WT 39 Lung Adenoca Core 14 6.8 WT WT 40 Brain Adenoca Biopsy 69.2 14.2 WT WT 41 Lung Adenoca Core 28.6 3.7 WT WT 42 Lung Adenoca Biopsy 128.7 6.3 WT WT 43 Lung Adenoca Biopsy 30.7 1.9 WT WT 44 Bone Carcinoma Biopsy 30.1 2 WT WT 45 Lung Adenoca Core 22 7.3 WT WT 46 Lung Adenoca Core 49.4 5.2 WT WT 47 Pleura Adenoca Biopsy 141.8 22.6 WT WT Average 40.34 7.05 Abbreviations: Adenoca, adenocarcinoma; CTT, cell-transfer technique; FFPE, formalin-fixed, paraffin-embedded; LN, lymph node; Squam ca, squamous carcinoma; WT, wild type. consecutive surgical pathology cases for which molecular testing (EGFR, BRAF, or KRAS) was ordered within a 10-month period were selected for this prospective study. Cases were required to have extra material for this study so as not to compromise diagnostic workups. The cases selected had extra slides cut, were stained (H&E), and set aside. They consisted mainly of core or forcep biopsies of colonic and lung adenocarcinomas with occasional cases of melanoma, squamous cell carcinoma, and papillary thyroid carcinoma (Tables 1 through 3). After cases were signed out, they were blinded to the molecular results from conventional method and sent for molecular testing through CTT. The archived H&E-stained slides were reviewed and areas with higher tumor cell concentration were selected by marking the slide. Most samples contained greater than 50% of tumor nuclei and at least 200 tumor cells. The marked tumor cells were removed from H&E-stained slides through CTT and submitted for EGFR (47 cases), BRAF (11 cases), and KRAS (39 cases) testing. The results were correlated to the previous conventional method for molecular 1384 Arch Pathol Lab Med Vol 140, December 2016 Cell Transfer on H&E-Stained Sections Wu et al
Table 2. Comparison of Results of Cell-Transfer and Conventional Method for BRAF Case No. Organ Diagnosis Tissue DNA-FFPE, ng DNA-CTT, ng Conventional CTT 1 Colon Adenoca Biopsy 23.3 6.2 V600E/Ec V600E/Ec 2 Thyroid Papillary ca CB 28.1 11.4 WT WT 3 Lung Melanoma Core 11.2 6 WT Inadequate 4 Lung Melanoma Core 28.8 11.5 WT WT 5 Breast Melanoma Core 85.6 10.4 WT WT 6 Chest wall Melanoma Core 17.1 1.1 V600K V600K 7 Brain Melanoma Biopsy 29.1 3.8 WT WT 8 Colon Adenoca Biopsy 58.8 4.5 WT WT 9 Lung Adenoca Core 11.3 6.8 WT WT 10 Groin Melanoma Core 15 1.3 V600E/Ec V600E/Ec 11 Liver Adenoca Core 11.2 4.1 V600E/Ec V600E/Ec Average 29.04 6.1 Abbreviations: Adenoca, adenocarcinoma; CB, cell block; CTT, cell-transfer technique; FFPE, formalin-fixed, paraffin-embedded; Papillary ca, papillary carcinoma; WT, wild type. testing. Representative samples of tumor histology and corresponding mutational result are illustrated (Figures 1 through 3). Cell-Transfer Technique The CTT was performed by using clean technique as follows: (1) the coverslip was removed with fresh histologic-grade xylene (Fisher Scientific, Pittsburgh, Pennsylvania); (2) a thin layer of Mount Quick media (Daido Sangyo, Tokyo, Japan) was spread uniformly over the top of the cellular material; (3) the slide was then placed in a 608C heated oven for approximately 2 to 3 hours (or until hardened to the touch); (4) a Sharpie marker was used on the surface of the dried media to divide the slide into multiple areas of interest; (5) the slide was then placed into a clean Coplin jar of deionized water and submerged into a warm water bath at 458 6 38C for 30 minutes to 2 hours, or until the media was soft enough Table 3. Comparison of Results of Cell-Transfer and Conventional Method for KRAS Case No. Organ Diagnosis Tissue DNA-FFPE, ng DNA-CTT, ng Conventional CTT 1 Colon Adenoca Biopsy 23.3 8.1 WT WT 2 Lung Adenoca Biopsy 15.2 5.3 WT Inadequate 3 Lung Squam ca Core 31.5 11.9 WT Inadequate 4 Lung Adenoca Biopsy 72.2 3.5 WT WT 5 Colon Adenoca Biopsy 90.3 8.1 WT WT 6 Colon Adenoca Biopsy 60.8 13.8 WT WT 7 Colon Adenoca Biopsy 42.5 9.5 WT WT 8 Colon Adenoca Biopsy 96.4 2.8 WT WT 9 Lung Adenoca Biopsy 32.4 6.6 G12C G12C 10 Lung Adenoca Biopsy 78.5 7.2 G12C G12C 11 Liver Adenoca Core 108.1 7.3 WT WT 12 Lung Adenoca Biopsy 97.9 6.7 WT WT 13 Lung Adenoca Biopsy 24.4 9.9 G12C WT 14 Colon Adenoca Biopsy 22.9 4.8 WT WT 15 Lung Adenoca Biopsy 17.2 3.9 WT Inadequate 16 Colon Adenoca Biopsy 58.1 17 G12D G12D 17 Lung Adenoca Core 10.5 1.7 G12C G12C 18 Colon Adenoca Biopsy 119.4 6.3 G12R G12R 19 Liver Adenoca Core 24.6 0.8 WT WT 20 Colon Adenoca Biopsy 118.8 4.5 G12A G12A 21 Colon Adenoca Biopsy 172.9 13.1 WT WT 22 Lung Adenoca Biopsy 21.5 8.9 WT WT 23 Lung Squam ca Biopsy 45.6 2.3 WT Inadequate 24 Colon Adenoca Biopsy 43 8.8 G13D G13D 25 Lung Adenoca Core 11.3 6.8 WT WT 26 Lung Adenoca Core 11.8 6.2 WT WT 27 Lung Adenoca Biopsy 71.8 19.1 WT WT 28 Colon Adenoca Biopsy 48.3 5.2 G12S G12S 29 Liver Adenoca Core 34.5 4.5 WT WT 30 Colon Adenoca Biopsy 21.6 9 Inadequate WT 31 Bone Adenoca Biopsy 100.6 20.4 WT Inadequate 32 Colon Adenoca Biopsy 57.8 8.05 WT WT 33 Lung Adenoca Biopsy 79.5 16.4 WT WT 34 Colon Adenoca Resection 111.3 8.1 G13D G13D 35 Liver Adenoca Core 11.2 4.1 WT WT 36 Colon Adenoca Biopsy 26.6 1.7 WT Inadequate 37 Liver Adenoca Core 96.5 12.1 G12C G12C 38 Colon Adenoca Biopsy 141.4 2.6 G12V G12V 39 Liver Adenoca Biopsy 19.2 3.7 WT WT Average 58.25 7.67 Abbreviations: Adenoca, adenocarcinoma; CTT, cell-transfer technique; FFPE, formalin-fixed, paraffin-embedded; Squam ca, squamous carcinoma; WT, wild type. Arch Pathol Lab Med Vol 140, December 2016 Cell Transfer on H&E-Stained Sections Wu et al 1385
laboratory. The areas of interest from 8 to 10 unstained slides are scraped off with razor blades and placed in an Eppendorf 2.0-mL safe-lock centrifuge tube for molecular testing. Figure 1. Adenocarcinoma of lung with EGFR 19 deletion (hematoxylin-eosin, original magnification 3400). to easily peel away from the slide; and (6) the media was cut along the marked areas, and each cut section was peeled off and placed in an Eppendorf 2.0-mL safe-lock centrifuge tube and sent for molecular testing. No extra steps were needed to get the DNA out of Mount Quick medium. If there are unused sections on the slides, the medium can be removed through 4 exchanges of xylene (15 minutes each) and the slides are re-coverslipped for storage. Conventional Technique. The H&E-stained slides are reviewed by a pathologist and the areas with rich tumor cells are circled with a marker. The H&E-stained slides along with 8 to 10 unstained slides are then sent to our molecular pathology DNA Extraction We applied the same DNA extraction method for both standard and CTT samples. It was performed by using the Qiagen QIAamp DNA Formalin-Fixed, Paraffin-Embedded Tissue Kit (Qiagen, Valencia, California). A modification from the manufacturer s recommendations was made. Samples were incubated at room temperature for 5 minutes with 1 ml of xylene and were then centrifuged at 15,000 rpm for 5 minutes. Xylene was removed from the pellet and ethanol wash was then performed as recommended by the manufacturer. DNA concentration was determined by using the NanoDrop Spectrophotometer (Thermo Fisher, Waltham, Massachusetts). EGFR. After DNA concentration was measured, the DNA was adjusted to approximately 10 ng/ml in distilled water. For EGFR mutational analysis, PCR-amplified products were analyzed on the Q24 Pyrosequencer with Qiagen EGFR Pyro kits (Qiagen). The pyrosequencing kit tests for mutations within exon 18 at codon 719, deletions in exon 19, mutations within exon 20 at codons 768 and 790, and mutations within exon 21 at codons 858 through 861. The resulting amplicons were purified, denatured, and sequenced by using mutation adjacent primers. Pyrograms were generated by the software and interpreted for the presence of mutations in the corresponding codons. The analytic sensitivity of this test is 5% (mutant allele detection). KRAS. Samples were run by using the Qiagen therascreen KRAS RGQ PCR on the Rotor-Gene Q MDx following the manufacturer s recommendations. Genomic DNA was used to detect 7 somatic mutations in codons 12 and 13 of the KRAS oncogene by using real-time PCR on the Rotor-Gene Q instrument using both Scorpions and Amplification Refractory Mutation Figure 2. A and B, Pyrogram of EGFR mutation with exon 19 deletion. 1386 Arch Pathol Lab Med Vol 140, December 2016 Cell Transfer on H&E-Stained Sections Wu et al
Figure 3. A, Metastatic melanoma with BRAF V600E mutation. B, Adenocarcinoma of colon with KRAS G12C mutation. C, BRAF V600E mutation corresponding to (A) is detected during cycling for both the sample (purple, patient) and sample control (blue). A difference of 7.00 or less between the crossing threshold cycles is an acceptable cutoff for a positive V600 result. The delta threshold cycle values of these samples (0.34) demonstrate detection of V600E BRAF mutation in relation to the sample control. D, KRAS G12C mutation corresponding to (B) is detected by demonstrating a delta cycle threshold less than the stated cutoff value for the assay (hematoxylin-eosin, original magnification 3400 [A and B]). Abbreviation: Norm. Fluoro., normalized fluorescence. Arch Pathol Lab Med Vol 140, December 2016 Cell Transfer on H&E-Stained Sections Wu et al 1387
System technologies (Qiagen). 17 19 The somatic mutations capable of being detected were Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val, and Gly13Asp. The reaction mixes were duplex, containing reagents labeled with FAM to detect mutant targets and HEX to detect the internal control. The threshold at which the signal is detected above background signaling is called the cycle threshold. Sample delta cycle threshold values are calculated as the difference between the mutation assay cycle threshold and wild-type assay cycle threshold from the same sample. Samples are subsequently classified as mutation positive if they give a delta cycle threshold less than the stated cutoff value for the assay and classified as not detected if above this value. The data were analyzed by using Rotor-Gene Q series software. Appropriate positive and negative controls were run with each sample. The analytic sensitivity of this KRAS test is 1% (mutant allele detection). BRAF. The BRAF RGQ PCR Kit used is a real-time qualitative PCR assay used on the Rotor-Gene Q MDx instrument for the detection of 4 somatic mutations in the human BRAF oncogene. The reagents used in this validation included BRAF RGQ PCR Kit (catalog No. 870801). The histologic cell-transfer material was received in the molecular pathology laboratory in Eppendorf tubes. DNA was extracted by using the QIAamp DNA FFPE Tissue Kit (catalog No. 56404). A modification was made from the protocol to include a 5-minute incubation time after the addition of 1 ml of xylene and then a 5-minute centrifugation was performed. The protocol continued after this modification to match manufacturer recommendations. The analytic sensitivity of this BRAF test is 5% (mutant allele detection). RESULTS The CTT-generated molecular testing results, obtained from archived H&E-stained sections, were compared to those generated by the conventional method (Tables 1 through 3). The average DNA content for BRAF, KRAS, and EGFR was 29.04, 58.25, and 40.34 ng for conventional method versus 6.1, 7.67, and 7.05 ng for CTT samples. Polymerase chain reaction based molecular testing via the CTT was successfully performed on 82 of 97 samples (85%). These included 39 of 47 cases (83%) for EGFR, 10 of 11 cases (91%) for BRAF, and 33 of 39 cases (85%) for KRAS mutations (Tables 1 through 3). The CTT samples showed agreement in 99% (81 of 82) of cases with the conventional method. (The 95% confidence interval extends from 0.9276 to greater than 0.9999, computed by the modified Wald method through an online calculator [http://www.graphpad. com/quickcalcs/confinterval1.cfm]). These included 4 mutations and 35 wild-type alleles for EGFR, 4 mutations and 6 wild-type alleles for BRAF, and 11 mutation and 21 wildtype alleles for KRAS. There was only 1 discrepant case (Table 3, KRAS case No. 13), namely, a false-negative KRAS result. The CTT sample showed wild-type allele, while the corresponding conventional method sample demonstrated Gly12Cys (G12C) mutation. DISCUSSION The CTT is a proven method for obtaining cellular material from fine-needle aspiration direct smears and facilitates additional immunocytochemical staining or molecular testing. 10 13 In this study, we confirmed its utility to capture sufficient tumor cell DNA for molecular testing if the tissue-embedded paraffin block lacks material. Biopsy samples or fine-needle aspiration cell blocks may only contain a minimal amount of tumor tissue and could be exhausted after the initial routine H&E sections and immunohistochemistry studies. It is a common scenario in which a pathologist signs out the final pathologic report and then receives a phone call requesting that molecular studies be performed. This molecular mutational information is essential for the treatment of the patients, especially those with a diagnosis of advanced-stage non small cell lung cancer. If the tumor cells in the paraffin blocks are exhausted, the patients might need to undergo repeated biopsies to get adequate specimens for molecular testing. Using the CTT we are able to use archived sections of previously cut H&E-stained slides to perform the molecular testing. This prevents patients from undergoing unnecessary additional biopsy procedures, which may have associated risk of complication in addition to superfluous costs and time. In our study, we showed a successful rate of 85% in performing molecular testing on the archived H&E-stained sections and there was 99% agreement on the results between CTT and conventional samples. The only discrepant case in our study was a lung biopsy from a patient with a diagnosis of adenocarcinoma with lepidic pattern. Retrospective review of the H&E-stained slide showed that the section was mostly composed of benign alveolar tissues and reactive pneumocytes with fewer than 200 tumor cells present, accounting for less than 10% of the nucleated cells. The small tumor volume was likely contributing to the falsenegative result. Because we used 8 to 10 unstained slides to obtain tumor DNA during the conventional procedure, the original materials definitely contained a higher volume of tumor cells, and therefore we were able to detect KRAS G12C mutation with these samples. In our previous study of cytologic specimens, we also encountered 1 false-negative KRAS mutation case that was also due to low ratio (,10%) of tumor cellularity. 12 The College of American Pathologists molecular testing guidelines suggest, in general, a minimal mutated allele frequency of 25% (50% cancer cell frequency, assuming heterozygosity and disomy) for Sanger sequencing. Polymerase chain reaction based testing requires lower neoplastic cellularity; however, when the volume of tumor is less than 10%, a false-negative result is likely. If results are based on a less-than-adequate amount of cancer cells, any negative finding should warrant a comment for the possibility of a false-negative result. 20 The purpose of CTT is similar to that of laser-capture microdissection. Both methods are able to harvest specific cells and separate them from unwanted cells to give pure enriched cell populations. The cost and complexity of lasercapture instruments are much higher than those of the CTT. Technically, the processing steps for CTT are not complex, and histology or cytology technologists can easily be trained to perform the procedure. Cell-transfer technique can be performed in any laboratory and no special equipment is necessary. The cost of the materials is minimal. After the selected areas have been cut and peeled off, the rest of the tissue section remaining on the slide can be re-coverslipped and kept on file. The advantage of CTT over conventional scraping procedure is obtaining higher concentration and purity of tumor DNA through lifting the selected tumor areas by CTT. It is difficult to scrape off small areas of tumor on slides, especially when the tumor cells are scattered and intimately associated with normal tissues. The scraped material can also fly off the blade owing to electrostatic forces. Although the DNA yields provided by CTT are 5 times lower than those obtained with the conventional method, because of the higher concentration and purity of tumor DNA obtained by CTT, they are adequate for molecular studies in 85% of cases. However, the higher failure rate for CTT may be related to lower DNA yields. The small amounts of DNA 1388 Arch Pathol Lab Med Vol 140, December 2016 Cell Transfer on H&E-Stained Sections Wu et al
obtained by 1 section through CTT may not be sufficient for assays requiring larger input of DNA (eg, next-generation sequencing), but the CTT could be scaled up to more than 1 section to obtain more DNA as long as diagnostic H&E sections are retained. In this study, the strong correlation between molecular assays performed on H&E-stained slide material by using the CTT and the molecular assays performed on conventional, unstained, recut sections from FFPE tissue samples indicates that the CTT is a reliable alternative resource for assessing EGFR, BRAF, and KRAS mutations, especially when tissue from cell blocks and small surgical biopsy samples is exhausted and the only available material for testing is on H&E-stained slides. References 1. Sharma SV, Bell DW, Settleman J, et al. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;7(3):169 181. 2. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 2008; 359(17):1757 1765. 3. Mitsudomi T, Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci. 2007;98(12): 1817 1824. 4. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21):2129 2139. 5. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304(5676):1497 1500. 6. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from never smokers and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004; 101(36):13306 13311. 7. Mitsudomi T, Kosaka T, Endoh H, et al. Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J Clin Oncol. 2005;23(11):2513 2520. 8. Takano T, Fukui T, Ohe Y, et al. EGFR mutations predict survival benefit from gefitinib in patients with advanced lung adenocarcinoma: a historical comparison of patients treated before and after gefitinib approval in Japan. J Clin Oncol. 2008;26(34):5589 5595. 9. Flaherty KT. Dividing and conquering: controlling advanced melanoma by targeting oncogene-defined subsets. Clin Exp Metastasis. 2012;29(7):841 846. 10. Ferguson J, Chamberlain P, Cramer HM, et al. ER, PR, and Her2 immunocytochemistry on cell-transferred cytologic smears of primary and metastatic breast carcinomas: a comparison study with formalin-fixed cell blocks and surgical biopsies. Diagn Cytopathol. 2013;41(7):575 581. 11. Wu HH, Jones KJ, Cramer HM. Immunocytochemistry performed on the cell-transferred direct smears of the fine-needle aspirates: a comparison study with the corresponding formalin-fixed paraffin-embedded tissue. Am J Clin Pathol. 2013;139(6):754 758. 12. Wu HH, Eaton JP, Jones KJ, et al. Utilization of cell-transferred cytologic smears in detection of EGFR and KRAS mutation on adenocarcinoma of lung. Mod Pathol. 2014;27(7):930 935. 13. Marshall AE, Cramer HM, Wu HH. The usefulness of the cell transfer technique for immunocytochemistry of fine-needle aspirates. Cancer Cytopathol. 2014;122(12):898 902. 14. Chen S, Randolph M, Cramer HM, et al. Detection of BRAF mutation in metastatic melanoma utilizing cell-transferred cytological smears. Acta Cytol. 2014;58(5):478 482. 15. Shi Q, Ibrahim A, Herbert K, et al. Detection of BRAF mutations on direct smears of thyroid fine-needle aspirates through cell transfer technique. Am J Clin Pathol. 2015;143(4):500 504. 16. Zhang C, Randolph ML, Jones KJ, et al. Anaplastic lymphoma kinase immunocytochemistry on cell-transferred cytologic smears of lung adenocarcinoma. Acta Cytol. 2015;59(2):213 218. 17. Harbison CT, Horak CE, Ledeine JM, et al. Validation of companion diagnostic for detection of mutations in codons 12 and 13 of the KRAS gene in patients with metastatic colorectal cancer: analysis of the NCIC CTG CO.17 trial. Arch Pathol Lab Med. 2013;137(6):820 827. 18. Whitcombe D, Theaker J, Guy SP, et al. Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol. 1999;17(8):804 807. 19. Thelwell N, Millington S, Solinas A, et al. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 2000;28(19):3752 3761. 20. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Arch Pathol Lab Med. 2013;137(6):828 860. Arch Pathol Lab Med Vol 140, December 2016 Cell Transfer on H&E-Stained Sections Wu et al 1389