- Email: [email protected]
ROS1 Dual Break Apart FISH Probe probe in non-small-cell lung cancer
Accepted Manuscript Title: Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in Non-small-cell lung cancer Authors: Sun Min Lim, Hyun Chang, Yoon Jin Cha, Shile Liang, Yan Chin Tai, Gu Li, Ekaterina Pestova, Frank Policht, Thomas Perez, Ross A. Soo, Won Young Park, Hye Ryun Kim, Hyo Sup Shim, Byoung Chul Cho PII: DOI: Reference:
S0169-5002(17)30395-1 http://dx.doi.org/doi:10.1016/j.lungcan.2017.07.016 LUNG 5413
To appear in:
Lung Cancer
Received date: Revised date: Accepted date:
9-3-2017 23-5-2017 10-7-2017
Please cite this article as: Lim Sun Min, Chang Hyun, Cha Yoon Jin, Liang Shile, Tai Yan Chin, Li Gu, Pestova Ekaterina, Policht Frank, Perez Thomas, Soo Ross A, Park Won Young, Kim Hye Ryun, Shim Hyo Sup, Cho Byoung Chul.Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in Non-small-cell lung cancer.Lung Cancer http://dx.doi.org/10.1016/j.lungcan.2017.07.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in Non-small-cell lung cancer Sun Min Lim1,2*, Hyun Chang3*, Yoon Jin Cha4*, Shile Liang5, Yan Chin Tai6, Gu Li5, Ekaterina Pestova5, Frank Policht5, Thomas Perez5, Ross A. Soo7, 8, Won Young Park9, Hye Ryun Kim1**, Hyo Sup Shim9**, Byoung Chul Cho1**
1
Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea 2 Division of Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, Seongnam-si, Gyeonggi-do, Korea 3 Hematology and Medical Oncology, International St. Mary’s Hospital, Catholic Kwandong University, College of Medicine, Incheon, Korea 4 Department of Pathology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea 5 Abbott Molecular Inc. Des Plaines, IL, USA 6 Abbott Laboratories (Singapore) Pte. Ltd. 7 Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore 8 Cancer Science Institute of Singapore, National University of Singapore, Singapore 9 Department of Pathology, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
*These authors contributed equally as first authors **These authors contributed equally as corresponding authors
Co-corresponding authors: Hye Ryun Kim, MD., PhD. Yonsei Cancer Center, Division of Medical Oncology Yonsei University College of Medicine 50 Yonsei-ro, Seodaemun-Gu, Seoul 03722, Korea. E-mail: [email protected]
Hyo Sup Shim, MD., PhD. Department of Pathology 1
Yonsei University College of Medicine 50-1 Yonsei-ro, Seodaemun-Gu, Seoul 03722, Korea. E-mail: [email protected]
Byoung Chul Cho, MD., PhD. Yonsei Cancer Center, Division of Medical Oncology Yonsei University College of Medicine 50 Yonsei-ro, Seodaemun-Gu, Seoul 03722, Korea. Tel: 82-2-2228-8126 Fax: 82-2-393-3562 E-mail: [email protected]
Highlights It is clinically important to develop effective screening strategy to detect ALK- and ROS1-rearranged patients Our report validated analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. The concordance of Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and revealed 100% concordance with ALK and ROS1 status Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC.
2
Abstract Background: ALK and ROS1 gene rearrangements are distinct molecular subsets of nonsmall-cell lung cancer (NSCLC), and they are strong predictive biomarkers of response to ALK/ROS1 inhibitors, such as crizotinib. Thus, it is clinically important to develop an effective screening strategy to detect patients who will benefit from such treatment. In this study, we aimed to validate analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. Methods: Study population composed of three patient cohorts with histologically confirmed lung adenocarcinoma (patients with ALK rearrangement, patients with ROS1 rearrangement and patients with wild-type ALK and ROS1). Specimens consisted of 12 ALK-positive, 8 ROS1positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement was previously assessed by Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbot Park, IL, USA) and ROS1 rearrangement was previously assessed by ZytoLight® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). All specimens were re-evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit. FISH images were scanned on BioView AllegroPlus system and interpreted via BioView SoloWeb remotely. Results: For a total of 41 patient samples, the concordance of the results by Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and compared to the known ALK and ROS1 rearrangement status of the specimen. Of the 12 ALK-positive cases, hybridization with Vysis ALK/ROS1 Dual Break Apart Probe Kit was successful in 10 cases (success rate 10/12, 83%) and of these 10 cases, all showed ALK rearrangement (100% concordance with the results of 3
Vysis ALK Break Apart FISH Probe Kit). Two of the ALK+ cases were excluded due to weak ROS1 signals that could not be enumerated. Of the 8 ROS1-positive cases, 6 cases were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The success rate was 75% (6/8), and of these 6 cases, all showed ROS1 rearrangement, giving a 100% concordance with ZytoLight® SPEC ROS1 Break Apart Probe. Two of the cases were exclude due to weak ROS1 gold signal or high background. In the cohort of 21 wild-type cases, the success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 85% (18/21) and the concordance with ALK and ROS1 probe kit was 100% (18/18). Conclusion: Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC.
Key words: ALK, ROS1, fluorescent in situ hybridization, non-small-cell lung cancer
4
Introduction Non-small-cell lung cancer (NSCLC) is characterized by distinct molecular sub-classification which is amenable for targeted therapy. Anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement have been described in 3-5% of NSCLC and the identification of ALK rearrangement at the time of diagnosis is essential to select patients for ALK inhibitors [1, 2]. So far, three ALK-targeted tyrosine kinase inhibitors have been approved. ROS1 rearrangement is a therapeutically tractable oncogenic driver that occurs in 1-2% of patients with NSCLC [3]. ROS1-rearranged tumors are highly sensitive to ROS1 inhibition, making such aberrations an important therapeutic target [4]. A phase I trial of crizotinib (NCT00585195) which originally enrolled ALK-positive NSCLC patients, was amended to include ROS1positive NSCLC patients, and treatment with crizotinib elicited an overall response rate (ORR) of 72% and the median progression-free survival with crizotinib was 19.2 months [5]. As a result, crizotinib was approved by the US Food and Drug Administration for ROS1-rearranged NSCLC patients. In addition, other newer ROS1 inhibitors are in development [6, 7]. Different technologies are available to identify ALK and ROS1 gene rearrangements. For ALK rearrangements, fluorescence in situ hybridization (FISH) has been used in clinical trials [1, 8, 9]. On FISH, ALK positivity is defined as at least 15% of tumor cells with the use of breakapart probes. Recently, immunohistochemical (IHC) analysis of ALK has been recognized as a diagnostic test [10]. Different monoclonal antibodies for the detection of ALK protein expression are commercially available, and patient selection can be based on a definitive IHC test result (positive or negative), regardless of the antibody used. Equivocal cases are required to be confirmed by FISH. For
ROS1
rearrangements,
next-generation 5
sequencing or
reverse-transcriptase-
polymerase-chain-reaction assays were used in a recent phase 1 study of crizotinib [5]. Performance of ROS1 IHC and FISH have been also tested which suggested that ROS1 IHC is highly sensitive, but less specific compared with ALK IHC[11]. Five out of 34 ROS1 immunoreactive cases were ROS1-rearranged by FISH (sensitivity 100% and specificity 72.6%). It is clinically important to detect patients who will benefit from ALK- and ROS1-targeting treatments. Until now, FISH has been used as a confirmatory test in the diagnosing ALK and ROS1 rearrangements. Currently, ALK and ROS1 tests are performed separately, requiring more time, effort and more importantly samples. NSCLC patients often present with small biopsy specimens with a limited number of neoplastic cells available for extensive molecular investigation. Vysis ALK/ROS1 Dual Break Apart Probe Kit is developed to address these challenges. However, it is technically challenging to enumerate manually due to limitations of the human eyes and time required for a reviewer to assess signal patterns for two genomic loci on the same slide. The color configuration of the 4-color probe set employs the red/green color combination for the ALK break-apart probe and gold/aqua color combination for ROS1 break-apart probe. The latter color combination poses difficulties in distinguishing translocation from normal from rearranged signal pattern. In order to overcome the visual limitations, increase the accuracy of the results, and to simplify the enumeration process, an automated imaging system was employed. In this study, we aim to validate diagnostic performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit in NSCLC compared to the performances of previous diagnostic approaches. Vysis ALK/ROS1 Dual Break Apart Probe Kit can detect ALK- and ROS1-rearrangement at the same time within the slide. 6
Methods Study population The study populations were composed of histologically confirmed lung adenocarcinoma diagnosed between 2013 and 2015 at Yonsei Cancer Center. All patients were previously tested for ALK and ROS1 rearrangements by FISH and their molecular status was grouped as 1) ALK-rearranged 2) ROS1-rearranged 3) ALK- and ROS1-wildtype. This study was approved by the Institutional Review Board of Severance Hospital and the ethics committee. All patients provided written informed consent for study participation and genetic analysis.
ALK and ROS1 FISH analysis ALK and ROS1 rearrangements were previously identified separately on formalin-fixed, paraffin-embedded (FFPE) tumors using Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbott Park, IL, USA) and ZytoLight® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). For Vysis ALK Break Apart FISH Probe Kit, FFPE specimens were processed manually using Paraffin Pretreatment Kit IV and Post-Hybridization Wash Buffer Kit (Abbott Molecular, Abbott Park, IL, USA) according to manufacturer’s instruction. ALK rearrangement was scored according to the package insert for Vysis ALK Break Apart FISH Probe Kit. A minimum of 50 cells were enumerated. Cells displaying break apart signals (with at least 2-signal diameter apart) and/or single isolated orange signal were deemed positive. A specimen with >50% positive cells was classified as positive for ALK rearrangement; and a specimen with <10% positive cells was classified as negative for ALK rearrangement. Any specimen with 7
positive cells of 10% to 50% is classified as equivocal and an additional 50 cells were enumerated. For these specimens, the specimens with ≥15% positive cells were classified as positive; while those with <15% positive cells were classified as negative for ALK rearrangement. ROS1 rearrangement was previously tested with on-market Class II IVD ZytoLight® SPEC ROS1 Break Apart Probe. The specimen was processed in the same way as those of ALK. According to the package insert of the probe, in interphases of normal cells or cells without a translocation involving the 6q22.1 band, two green/orange fusion signals appear. One 6q22.1 locus affected by a translocation is indicated by one separate green signal and one separate orange signal. Isolated green signals are the result of deletions distal to the ROS1 breakpoint region or due to unbalanced translocations affecting this chromosomal region. ROS1 rearrangement was evaluated as previously described, where cases with 15% of the cells showing break apart signals were classified as positive [3].
ALK/ROS1 RUO FISH In the initial study design, we intend to include 20 ALK-rearranged cases, 20 ROS1rearranged cases and 20 ALK/ROS1 wild-type cases. We retrospectively reviewed the FFPE specimens obtained from surgical resection or excisional biopsy from cases with known ALK and ROS1 rearrangement status. Based on the availability of the tissue specimens for further testing, we finally selected 12 ALK-rearranged, 8 ROS1-rearranged and 21 ALK/ROS1 wildtype FFPE samples for the evaluation of Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit. The selected 41 cases were re-evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit.
8
Vysis ALK/ROS1 Dual Break Apart Probe Kit probe mixture consists of four fluorophorelabeled DNA probes pre-formulated in hybridization buffer with blocking DNA: 1) Vysis LSI 3’ALK SpectrumRed, 2) Vysis LSI 5’-ALK SpectrumGreen, 3) Vysis LSI 3’-ROS1 SpectrumAqua, 4) Vysis LSI 5’-ROS1 SpectrumGold. The hybridization targets of LSI ALK probes were on opposite sides flanking the breakpoint of the ALK gene. The 3’-ALK probe hybridized telomerically of the breakpoint was approximately 300kb and is labeled with SpectrumRed fluorophore. The 5’-ALK probe was hybridized centromerically to the breakpoint is approximately 442kb and was labeled with SpectrumGreen. The hybridization targets of LSI ROS1 were on the opposite sides flanking the breakpoint of ROS1 gene. The 3′-ROS1 probe that hybridizes centromerically of the breakpoint was approximately 557 kb and was labeled with SpectrumAqua fluorophores. The 5′-ROS1 probe that hybridizes telomerically of the breakpoint was approximately 178 kb and was labeled with SpectrumGold fluorophore. A special filter, Vysis Single Bandpass Green(v2) (Abbott Molecular, Abbott Park, IL, USA) was used to avoid fluorescence bleed-through between SpectrumGreen and SpectrumGold. The FFPE specimens were processed manually using Paraffin Pretreatment Kit IV and PostHybridization Wash Buffer Kit (Abbott Molecular, Abbott Park, IL, USA) according to manufacturer’s instruction. The FISH images were uploaded onto BioView Soloweb (BioView Inc., Israel) and interpreted remotely by the pathologists. Since BioView system was not available at Yonsei Cancer Center, the hybridized slides were shipped to Abbott Molecular training facility in Singapore on dry ice. Automatic scanning was performed using Bioview AllegroPlus system (BioView Inc., Israel). The slides were loaded onto a 8-slide automatic stage, and the slides were configured for automatic scanning according to manufacturer’s instruction. The tumor 9
area that had been selected by pathologist was evaluated using 100x to 200x magnification to check for tissue integrity, background and signal quality. If the quality was acceptable, at least 20 FOV (field of view) were selected for the automatic scanning. The slides were then scanned and analyzed according to the predefined ALK/ROS1 scoring algorithm. The color configuration of probe set employs SpectrumRed for the 3’ ALK, SpectrumGreen for the 5’ ALK, SpectrumGold for the 5’ ROS1, and SpectrumAqua for the 3’ ROS1.
The
SpectrumRed/SpectrumGreen ALK probes have excellent contrast and are readily viewed and analyzed, whereas the SpectrumGold/SpectrumAqua ROS1 probes have less contrast, and can pose some difficulty assessing a ROS1 fusion from a single SpectrumAqua ROS1 signal. The SpectrumGold/SpectrumAqua ROS1 fusion may appear “whitish” and have a similar appearance to the single SpectrumAqua ROS1 signal, which may lead to an incorrect rearrangement determination. BioView automated imaging system allowed to overcome this visual limitation. The color of the SpectrumGold ROS1 probe was pseudo-colored as pink and this color combination (pink and aqua) had a higher contrast, allowing and the
ROS1
fusions to be easily distinguished from single SpectrumAqua ROS1 probes. Additionally, the ALK/ROS1 imaging algorithm images all four fluorescent probes in a single scan for the userselected FOVs and subsequently generates two files, allowing for independent analysis of ALK and ROS1, respectively. During cell review and classification, the imaging algorithm automatically calculates the distance between ALK or ROS1 probes, which provides more consistency in rearrangement determination. Two experienced pathologists (H.S.S and Y.J.C), who were blinded to the previous molecular status, reviewed the FISH images and interpreted results. At least 50 cells were evaluated for each case according to the enumeration criteria described above. The assays were 10
considered concordant when the final interpretation is identical (Positive or Negative). In case discrepancy, a RT-PCR method was used to evaluate the specimen.
Results Patient characteristics A total of 41 patients were included in this study. The median age of all patients was 55 (Range, 32-79), and there were 20 (48%) males. By smoking status, there were 24 (58%) never-smokers, 11 former smokers (27%) and 6 (16%) current smokers. By clinical stage, there were 19 (46%) patients with stage IV disease at initial diagnosis. Mutational status was categorized into 5 groups: EGFR, KRAS, ALK, ROS1, and pan-negative (Table 1).
ALK and ROS1 RUO FISH in ALK-positive cohort The concordance of results obtained from Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated in relation to the ALK and ROS1 rearrangement status of the specimen, as previously determined. Even though strict temperature control was adhered to, about 90% of the hybridized slides were affected by shipment on dry ice from Korea to Singapore. Therefore, we sent unhybridized slides Singapore to be repeated. Of the 12 ALK-positive cases, 2 cases showed weak ROS1 signals that could not be enumerated. We were not able to repeat these cases due to insufficient tissue. All the 10 samples were positive by Vysis ALK/ROS1 Dual Break Apart Probe Kit, which is 100% concordant with the previous result. All 10 cases showed break apart green and red signals. An example of ALK-positive case is shown in Figure 1, showing break apart red and green signals. 11
ALK and ROS1 RUO FISH in ROS1-positive cohort Among 8 ROS1-positive cases, 6 cases were successfully evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit, giving a success rate of 75%. All of the 6 cases showed ROS1 rearrangement, which is 100% concordant to the previous result. The remaining two cases showed weak ROS1 gold signal or high background, there was insufficient tissue for repeat. These two were excluded from the analysis. An example of ROS1-positive sample is shown in Figure 2, showing single aqua pattern.
ALK and ROS1 RUO FISH in ALK-/ROS1- wild type cohort Regarding 21 wild type cases, 18 samples (85%) were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The remaining three cases not evaluable due to high background or poor ROS1 signals, and were excluded from the analysis. The high background was caused by interference from the strong auto-fluorescent of the fibrotic tissues surrounding the tumor cells. This was extremely difficult to avoid in biopsy specimens with very limited tumor cells. The 18 successful samples were 100% concordant with the previous results, and were negative for both ALK and ROS1. An example of ALK-wild, ROS1-wild sample is shown in Figure 3.
Discussion In this study, we showed that simultaneous detection of ALK and ROS1 rearrangement is feasible on a single slide using ALK and ROS1 RUO FISH assay. The overall success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 83% (37/41 cases). The results from 12
Vysis ALK/ROS1 Dual Break Apart Probe Kit was in 100% concordant to the previous ALK and ROS1 tests in all the three patients cohorts – ALK-rearranged, ROS1-rearranged and ALK/ROS1 wildtype. The excluded cases were mainly due to insufficient tissue for further testing or repeat. The specimens, mostly small biopsies, had been investigated extensively prior to this study, thus, there was limited remaining tumor cells on the tissue. Even though strict shipping condition was adhered to, the signals, especially aqua signals of ROS1 probe, was significantly affected. The weak ROS1 signals and/or high fluorescent background observed in some of the samples might be attributed to change in pH and temperature during shipment of slides from Korea to Singapore. We repeated the test by performing hybridization directly in Singapore, and we managed to get satisfactory result from most of the cases. Therefore, shipment of hybridized specimens may not be advised for laboratory tests in clinical diagnostics. High fluorescent background was commonly observed in biopsy specimen and in specimens rich with fibrotic and connective tissues. This had a significant impact on automated scanning due to the interference from the auto-fluorescent. In these cases, the system automatically lowered the exposure time due to the high auto-fluorescent, and thus, causing weaker signals being captured. BioView system has an option to perform interactive scanning, where the operator may manually adjust the exposure time to capture stronger signals. This could overcome of the challenges in these difficult cases. Determination of ALK and ROS1 rearrangement status has become mandatory in the treatment of NSCLC patients due to the availability of ALK and ROS1 inhibitors. However, detection strategy of ALK and ROS1 rearrangements has been variable. In June 2015, the U.S. 13
Food and Drug Administration approved the VENTANA ALK (D5F3) CDx Assay as a companion diagnostic to aid the identification of patients eligible for treatment with the ALK inhibitor crizotinib. However, FISH remains the gold standard for detecting ALK rearrangement [12]. Although the break-apart FISH assay requires experience for interpretation, patience, high cost, and technical expertise; this assay is highly sensitive and specific for the detection of ALK gene
rearrangement
regardless
of ALK fusion
partners.
For
ROS1,
immunohistochemistry was evaluated in previous studies [11, 13], and it showed equally high sensitivity and specificity as ALK IHC. However, FISH assay was necessary to confirm equivocal cases in IHC. In this regard, FISH remains the pertinent method in selecting ALKand ROS1-rearranged patients. In conclusion, Vysis ALK/ROS1 Dual Break Apart Probe Kit can detect ALK and ROS1 rearrangement simultaneously in NSCLC. Due to the limited availability of FFPE specimens, we recommend further validation of this probe in prospective analysis by including more ALK- and ROS1-rearranged patient specimens.
Conflict of Interest statement None declared
Acknowledgements The authors would like to thank BioView Inc. for the technical assistance during the study. This study was supported by a faculty research grant of Yonsei University College of Medicine for 2012(6-2012-0134) to B.C.C. 14
References 1.
Shaw AT, Kim DW, Nakagawa K, Seto T, Crino L, Ahn MJ, De Pas T, Besse B, Solomon BJ, Blackhall F, Wu YL, Thomas M, O'Byrne KJ, Moro-Sibilot D, Camidge DR, Mok T, Hirsh V, Riely GJ, Iyer S, Tassell V, Polli A, Wilner KD, Janne PA. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med 2013;368: 23852394.
2.
Lindeman NI, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, Jenkins RB, Kwiatkowski DJ, Saldivar JS, Squire J, Thunnissen E, Ladanyi M, College of American Pathologists International Association for the Study of Lung C, Association for Molecular P. 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. J Mol Diagn 2013;15: 415-453.
3.
Bergethon K, Shaw AT, Ou SH, Katayama R, Lovly CM, McDonald NT, Massion PP, Siwak-Tapp C, Gonzalez A, Fang R, Mark EJ, Batten JM, Chen H, Wilner KD, Kwak EL, Clark JW, Carbone DP, Ji H, Engelman JA, Mino-Kenudson M, Pao W, Iafrate AJ. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 2012;30: 863-870.
4.
Davies KD, Le AT, Theodoro MF, Skokan MC, Aisner DL, Berge EM, Terracciano LM, Cappuzzo F, Incarbone M, Roncalli M, Alloisio M, Santoro A, Camidge DR, VarellaGarcia M, Doebele RC. Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res 2012;18: 4570-4579. 15
5.
Shaw AT, Ou SH, Bang YJ, Camidge DR, Solomon BJ, Salgia R, Riely GJ, Varella-Garcia M, Shapiro GI, Costa DB, Doebele RC, Le LP, Zheng Z, Tan W, Stephenson P, Shreeve SM, Tye LM, Christensen JG, Wilner KD, Clark JW, Iafrate AJ. Crizotinib in ROS1rearranged non-small-cell lung cancer. N Engl J Med 2014;371: 1963-1971.
6.
Ardini E, Menichincheri M, Banfi P, Bosotti R, De Ponti C, Pulci R, Ballinari D, Ciomei M, Texido G, Degrassi A, Avanzi N, Amboldi N, Saccardo MB, Casero D, Orsini P, Bandiera T, Mologni L, Anderson D, Wei G, Harris J, Vernier JM, Li G, Felder E, Donati D, Isacchi A, Pesenti E, Magnaghi P, Galvani A. Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications. Mol Cancer Ther 2016;15: 628-639.
7.
Zou HY, Li Q, Engstrom LD, West M, Appleman V, Wong KA, McTigue M, Deng YL, Liu W, Brooun A, Timofeevski S, McDonnell SR, Jiang P, Falk MD, Lappin PB, Affolter T, Nichols T, Hu W, Lam J, Johnson TW, Smeal T, Charest A, Fantin VR. PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations. Proc Natl Acad Sci U S A 2015;112: 3493-3498.
8.
Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, Ou SH, Dezube BJ, Janne PA, Costa DB, Varella-Garcia M, Kim WH, Lynch TJ, Fidias P, Stubbs H, Engelman JA, Sequist LV, Tan W, Gandhi L, Mino-Kenudson M, Wei GC, Shreeve SM, Ratain MJ, Settleman J, Christensen JG, Haber DA, Wilner K, Salgia R, Shapiro GI, Clark JW, Iafrate AJ. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363: 1693-1703.
9.
Shaw AT, Kim DW, Mehra R, Tan DS, Felip E, Chow LQ, Camidge DR, Vansteenkiste J, Sharma S, De Pas T, Riely GJ, Solomon BJ, Wolf J, Thomas M, Schuler M, Liu G, 16
Santoro A, Lau YY, Goldwasser M, Boral AL, Engelman JA. Ceritinib in ALK-rearranged non-small-cell lung cancer. N Engl J Med 2014;370: 1189-1197. 10.
Marchetti A, Di Lorito A, Pace MV, Iezzi M, Felicioni L, D'Antuono T, Filice G, Guetti L, Mucilli F, Buttitta F. ALK Protein Analysis by IHC Staining after Recent Regulatory Changes: A Comparison of Two Widely Used Approaches, Revision of the Literature, and a New Testing Algorithm. J Thorac Oncol 2016;11: 487-495.
11.
Cha YJ, Lee JS, Kim HR, Lim SM, Cho BC, Lee CY, Shim HS. Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements. PLoS One 2014;9: e103333.
12.
Sholl LM, Weremowicz S, Gray SW, Wong KK, Chirieac LR, Lindeman NI, Hornick JL. Combined use of ALK immunohistochemistry and FISH for optimal detection of ALKrearranged lung adenocarcinomas. J Thorac Oncol 2013;8: 322-328.
13.
Mescam-Mancini L, Lantuejoul S, Moro-Sibilot D, Rouquette I, Souquet PJ, AudigierValette C, Sabourin JC, Decroisette C, Sakhri L, Brambilla E, McLeer-Florin A. On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas. Lung Cancer 2014;83: 168-173.
17
Figure legends Figure 1. Representative Vysis ALK/ROS1 Dual Break Apart Probe Kit FISH image of tumor with ALK rearrangement. Gold arrow highlights positions of FISH signal corresponding to ALK rearrangement, showing red and green break apart signal
Figure 2. Representative Vysis ALK/ROS1 Dual Break Apart Probe Kit FISH image of tumor with ROS1 rearrangement. Gold arrow highlights positions of FISH signal corresponding to ROS1 rearrangement, showing single aqua signal
Figure 3. Representative ALK/ROS1 R Vysis ALK/ROS1 Dual Break Apart Probe Kit FISH image of ALK- and ROS1-wild type tumor
18
19
Table 1. Clinicopathological characteristics of patients Patients (N=41) Median age (range) Sex
Smoking status
Stage
Genotypes
N (%) 55 (32-79) Male
20 (48)
Female
21 (52)
Never
24 (58)
Former
11 (27)
Current
6 (15)
I
6 (15)
II
4 (10)
III
12 (29)
IV
19 (46)
EGFR mutation
6 (15)
KRAS mutation
1 (2)
ALK rearrangement
12 (29)
ROS1 rearrangement
8 (20)
Pan-negative
14 (34)
20
S0169-5002(17)30395-1 http://dx.doi.org/doi:10.1016/j.lungcan.2017.07.016 LUNG 5413
To appear in:
Lung Cancer
Received date: Revised date: Accepted date:
9-3-2017 23-5-2017 10-7-2017
Please cite this article as: Lim Sun Min, Chang Hyun, Cha Yoon Jin, Liang Shile, Tai Yan Chin, Li Gu, Pestova Ekaterina, Policht Frank, Perez Thomas, Soo Ross A, Park Won Young, Kim Hye Ryun, Shim Hyo Sup, Cho Byoung Chul.Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in Non-small-cell lung cancer.Lung Cancer http://dx.doi.org/10.1016/j.lungcan.2017.07.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in Non-small-cell lung cancer Sun Min Lim1,2*, Hyun Chang3*, Yoon Jin Cha4*, Shile Liang5, Yan Chin Tai6, Gu Li5, Ekaterina Pestova5, Frank Policht5, Thomas Perez5, Ross A. Soo7, 8, Won Young Park9, Hye Ryun Kim1**, Hyo Sup Shim9**, Byoung Chul Cho1**
1
Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea 2 Division of Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, Seongnam-si, Gyeonggi-do, Korea 3 Hematology and Medical Oncology, International St. Mary’s Hospital, Catholic Kwandong University, College of Medicine, Incheon, Korea 4 Department of Pathology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea 5 Abbott Molecular Inc. Des Plaines, IL, USA 6 Abbott Laboratories (Singapore) Pte. Ltd. 7 Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore 8 Cancer Science Institute of Singapore, National University of Singapore, Singapore 9 Department of Pathology, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
*These authors contributed equally as first authors **These authors contributed equally as corresponding authors
Co-corresponding authors: Hye Ryun Kim, MD., PhD. Yonsei Cancer Center, Division of Medical Oncology Yonsei University College of Medicine 50 Yonsei-ro, Seodaemun-Gu, Seoul 03722, Korea. E-mail: [email protected]
Hyo Sup Shim, MD., PhD. Department of Pathology 1
Yonsei University College of Medicine 50-1 Yonsei-ro, Seodaemun-Gu, Seoul 03722, Korea. E-mail: [email protected]
Byoung Chul Cho, MD., PhD. Yonsei Cancer Center, Division of Medical Oncology Yonsei University College of Medicine 50 Yonsei-ro, Seodaemun-Gu, Seoul 03722, Korea. Tel: 82-2-2228-8126 Fax: 82-2-393-3562 E-mail: [email protected]
Highlights It is clinically important to develop effective screening strategy to detect ALK- and ROS1-rearranged patients Our report validated analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. The concordance of Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and revealed 100% concordance with ALK and ROS1 status Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC.
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Abstract Background: ALK and ROS1 gene rearrangements are distinct molecular subsets of nonsmall-cell lung cancer (NSCLC), and they are strong predictive biomarkers of response to ALK/ROS1 inhibitors, such as crizotinib. Thus, it is clinically important to develop an effective screening strategy to detect patients who will benefit from such treatment. In this study, we aimed to validate analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. Methods: Study population composed of three patient cohorts with histologically confirmed lung adenocarcinoma (patients with ALK rearrangement, patients with ROS1 rearrangement and patients with wild-type ALK and ROS1). Specimens consisted of 12 ALK-positive, 8 ROS1positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement was previously assessed by Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbot Park, IL, USA) and ROS1 rearrangement was previously assessed by ZytoLight® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). All specimens were re-evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit. FISH images were scanned on BioView AllegroPlus system and interpreted via BioView SoloWeb remotely. Results: For a total of 41 patient samples, the concordance of the results by Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and compared to the known ALK and ROS1 rearrangement status of the specimen. Of the 12 ALK-positive cases, hybridization with Vysis ALK/ROS1 Dual Break Apart Probe Kit was successful in 10 cases (success rate 10/12, 83%) and of these 10 cases, all showed ALK rearrangement (100% concordance with the results of 3
Vysis ALK Break Apart FISH Probe Kit). Two of the ALK+ cases were excluded due to weak ROS1 signals that could not be enumerated. Of the 8 ROS1-positive cases, 6 cases were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The success rate was 75% (6/8), and of these 6 cases, all showed ROS1 rearrangement, giving a 100% concordance with ZytoLight® SPEC ROS1 Break Apart Probe. Two of the cases were exclude due to weak ROS1 gold signal or high background. In the cohort of 21 wild-type cases, the success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 85% (18/21) and the concordance with ALK and ROS1 probe kit was 100% (18/18). Conclusion: Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC.
Key words: ALK, ROS1, fluorescent in situ hybridization, non-small-cell lung cancer
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Introduction Non-small-cell lung cancer (NSCLC) is characterized by distinct molecular sub-classification which is amenable for targeted therapy. Anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement have been described in 3-5% of NSCLC and the identification of ALK rearrangement at the time of diagnosis is essential to select patients for ALK inhibitors [1, 2]. So far, three ALK-targeted tyrosine kinase inhibitors have been approved. ROS1 rearrangement is a therapeutically tractable oncogenic driver that occurs in 1-2% of patients with NSCLC [3]. ROS1-rearranged tumors are highly sensitive to ROS1 inhibition, making such aberrations an important therapeutic target [4]. A phase I trial of crizotinib (NCT00585195) which originally enrolled ALK-positive NSCLC patients, was amended to include ROS1positive NSCLC patients, and treatment with crizotinib elicited an overall response rate (ORR) of 72% and the median progression-free survival with crizotinib was 19.2 months [5]. As a result, crizotinib was approved by the US Food and Drug Administration for ROS1-rearranged NSCLC patients. In addition, other newer ROS1 inhibitors are in development [6, 7]. Different technologies are available to identify ALK and ROS1 gene rearrangements. For ALK rearrangements, fluorescence in situ hybridization (FISH) has been used in clinical trials [1, 8, 9]. On FISH, ALK positivity is defined as at least 15% of tumor cells with the use of breakapart probes. Recently, immunohistochemical (IHC) analysis of ALK has been recognized as a diagnostic test [10]. Different monoclonal antibodies for the detection of ALK protein expression are commercially available, and patient selection can be based on a definitive IHC test result (positive or negative), regardless of the antibody used. Equivocal cases are required to be confirmed by FISH. For
ROS1
rearrangements,
next-generation 5
sequencing or
reverse-transcriptase-
polymerase-chain-reaction assays were used in a recent phase 1 study of crizotinib [5]. Performance of ROS1 IHC and FISH have been also tested which suggested that ROS1 IHC is highly sensitive, but less specific compared with ALK IHC[11]. Five out of 34 ROS1 immunoreactive cases were ROS1-rearranged by FISH (sensitivity 100% and specificity 72.6%). It is clinically important to detect patients who will benefit from ALK- and ROS1-targeting treatments. Until now, FISH has been used as a confirmatory test in the diagnosing ALK and ROS1 rearrangements. Currently, ALK and ROS1 tests are performed separately, requiring more time, effort and more importantly samples. NSCLC patients often present with small biopsy specimens with a limited number of neoplastic cells available for extensive molecular investigation. Vysis ALK/ROS1 Dual Break Apart Probe Kit is developed to address these challenges. However, it is technically challenging to enumerate manually due to limitations of the human eyes and time required for a reviewer to assess signal patterns for two genomic loci on the same slide. The color configuration of the 4-color probe set employs the red/green color combination for the ALK break-apart probe and gold/aqua color combination for ROS1 break-apart probe. The latter color combination poses difficulties in distinguishing translocation from normal from rearranged signal pattern. In order to overcome the visual limitations, increase the accuracy of the results, and to simplify the enumeration process, an automated imaging system was employed. In this study, we aim to validate diagnostic performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit in NSCLC compared to the performances of previous diagnostic approaches. Vysis ALK/ROS1 Dual Break Apart Probe Kit can detect ALK- and ROS1-rearrangement at the same time within the slide. 6
Methods Study population The study populations were composed of histologically confirmed lung adenocarcinoma diagnosed between 2013 and 2015 at Yonsei Cancer Center. All patients were previously tested for ALK and ROS1 rearrangements by FISH and their molecular status was grouped as 1) ALK-rearranged 2) ROS1-rearranged 3) ALK- and ROS1-wildtype. This study was approved by the Institutional Review Board of Severance Hospital and the ethics committee. All patients provided written informed consent for study participation and genetic analysis.
ALK and ROS1 FISH analysis ALK and ROS1 rearrangements were previously identified separately on formalin-fixed, paraffin-embedded (FFPE) tumors using Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbott Park, IL, USA) and ZytoLight® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). For Vysis ALK Break Apart FISH Probe Kit, FFPE specimens were processed manually using Paraffin Pretreatment Kit IV and Post-Hybridization Wash Buffer Kit (Abbott Molecular, Abbott Park, IL, USA) according to manufacturer’s instruction. ALK rearrangement was scored according to the package insert for Vysis ALK Break Apart FISH Probe Kit. A minimum of 50 cells were enumerated. Cells displaying break apart signals (with at least 2-signal diameter apart) and/or single isolated orange signal were deemed positive. A specimen with >50% positive cells was classified as positive for ALK rearrangement; and a specimen with <10% positive cells was classified as negative for ALK rearrangement. Any specimen with 7
positive cells of 10% to 50% is classified as equivocal and an additional 50 cells were enumerated. For these specimens, the specimens with ≥15% positive cells were classified as positive; while those with <15% positive cells were classified as negative for ALK rearrangement. ROS1 rearrangement was previously tested with on-market Class II IVD ZytoLight® SPEC ROS1 Break Apart Probe. The specimen was processed in the same way as those of ALK. According to the package insert of the probe, in interphases of normal cells or cells without a translocation involving the 6q22.1 band, two green/orange fusion signals appear. One 6q22.1 locus affected by a translocation is indicated by one separate green signal and one separate orange signal. Isolated green signals are the result of deletions distal to the ROS1 breakpoint region or due to unbalanced translocations affecting this chromosomal region. ROS1 rearrangement was evaluated as previously described, where cases with 15% of the cells showing break apart signals were classified as positive [3].
ALK/ROS1 RUO FISH In the initial study design, we intend to include 20 ALK-rearranged cases, 20 ROS1rearranged cases and 20 ALK/ROS1 wild-type cases. We retrospectively reviewed the FFPE specimens obtained from surgical resection or excisional biopsy from cases with known ALK and ROS1 rearrangement status. Based on the availability of the tissue specimens for further testing, we finally selected 12 ALK-rearranged, 8 ROS1-rearranged and 21 ALK/ROS1 wildtype FFPE samples for the evaluation of Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit. The selected 41 cases were re-evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit.
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Vysis ALK/ROS1 Dual Break Apart Probe Kit probe mixture consists of four fluorophorelabeled DNA probes pre-formulated in hybridization buffer with blocking DNA: 1) Vysis LSI 3’ALK SpectrumRed, 2) Vysis LSI 5’-ALK SpectrumGreen, 3) Vysis LSI 3’-ROS1 SpectrumAqua, 4) Vysis LSI 5’-ROS1 SpectrumGold. The hybridization targets of LSI ALK probes were on opposite sides flanking the breakpoint of the ALK gene. The 3’-ALK probe hybridized telomerically of the breakpoint was approximately 300kb and is labeled with SpectrumRed fluorophore. The 5’-ALK probe was hybridized centromerically to the breakpoint is approximately 442kb and was labeled with SpectrumGreen. The hybridization targets of LSI ROS1 were on the opposite sides flanking the breakpoint of ROS1 gene. The 3′-ROS1 probe that hybridizes centromerically of the breakpoint was approximately 557 kb and was labeled with SpectrumAqua fluorophores. The 5′-ROS1 probe that hybridizes telomerically of the breakpoint was approximately 178 kb and was labeled with SpectrumGold fluorophore. A special filter, Vysis Single Bandpass Green(v2) (Abbott Molecular, Abbott Park, IL, USA) was used to avoid fluorescence bleed-through between SpectrumGreen and SpectrumGold. The FFPE specimens were processed manually using Paraffin Pretreatment Kit IV and PostHybridization Wash Buffer Kit (Abbott Molecular, Abbott Park, IL, USA) according to manufacturer’s instruction. The FISH images were uploaded onto BioView Soloweb (BioView Inc., Israel) and interpreted remotely by the pathologists. Since BioView system was not available at Yonsei Cancer Center, the hybridized slides were shipped to Abbott Molecular training facility in Singapore on dry ice. Automatic scanning was performed using Bioview AllegroPlus system (BioView Inc., Israel). The slides were loaded onto a 8-slide automatic stage, and the slides were configured for automatic scanning according to manufacturer’s instruction. The tumor 9
area that had been selected by pathologist was evaluated using 100x to 200x magnification to check for tissue integrity, background and signal quality. If the quality was acceptable, at least 20 FOV (field of view) were selected for the automatic scanning. The slides were then scanned and analyzed according to the predefined ALK/ROS1 scoring algorithm. The color configuration of probe set employs SpectrumRed for the 3’ ALK, SpectrumGreen for the 5’ ALK, SpectrumGold for the 5’ ROS1, and SpectrumAqua for the 3’ ROS1.
The
SpectrumRed/SpectrumGreen ALK probes have excellent contrast and are readily viewed and analyzed, whereas the SpectrumGold/SpectrumAqua ROS1 probes have less contrast, and can pose some difficulty assessing a ROS1 fusion from a single SpectrumAqua ROS1 signal. The SpectrumGold/SpectrumAqua ROS1 fusion may appear “whitish” and have a similar appearance to the single SpectrumAqua ROS1 signal, which may lead to an incorrect rearrangement determination. BioView automated imaging system allowed to overcome this visual limitation. The color of the SpectrumGold ROS1 probe was pseudo-colored as pink and this color combination (pink and aqua) had a higher contrast, allowing and the
ROS1
fusions to be easily distinguished from single SpectrumAqua ROS1 probes. Additionally, the ALK/ROS1 imaging algorithm images all four fluorescent probes in a single scan for the userselected FOVs and subsequently generates two files, allowing for independent analysis of ALK and ROS1, respectively. During cell review and classification, the imaging algorithm automatically calculates the distance between ALK or ROS1 probes, which provides more consistency in rearrangement determination. Two experienced pathologists (H.S.S and Y.J.C), who were blinded to the previous molecular status, reviewed the FISH images and interpreted results. At least 50 cells were evaluated for each case according to the enumeration criteria described above. The assays were 10
considered concordant when the final interpretation is identical (Positive or Negative). In case discrepancy, a RT-PCR method was used to evaluate the specimen.
Results Patient characteristics A total of 41 patients were included in this study. The median age of all patients was 55 (Range, 32-79), and there were 20 (48%) males. By smoking status, there were 24 (58%) never-smokers, 11 former smokers (27%) and 6 (16%) current smokers. By clinical stage, there were 19 (46%) patients with stage IV disease at initial diagnosis. Mutational status was categorized into 5 groups: EGFR, KRAS, ALK, ROS1, and pan-negative (Table 1).
ALK and ROS1 RUO FISH in ALK-positive cohort The concordance of results obtained from Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated in relation to the ALK and ROS1 rearrangement status of the specimen, as previously determined. Even though strict temperature control was adhered to, about 90% of the hybridized slides were affected by shipment on dry ice from Korea to Singapore. Therefore, we sent unhybridized slides Singapore to be repeated. Of the 12 ALK-positive cases, 2 cases showed weak ROS1 signals that could not be enumerated. We were not able to repeat these cases due to insufficient tissue. All the 10 samples were positive by Vysis ALK/ROS1 Dual Break Apart Probe Kit, which is 100% concordant with the previous result. All 10 cases showed break apart green and red signals. An example of ALK-positive case is shown in Figure 1, showing break apart red and green signals. 11
ALK and ROS1 RUO FISH in ROS1-positive cohort Among 8 ROS1-positive cases, 6 cases were successfully evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit, giving a success rate of 75%. All of the 6 cases showed ROS1 rearrangement, which is 100% concordant to the previous result. The remaining two cases showed weak ROS1 gold signal or high background, there was insufficient tissue for repeat. These two were excluded from the analysis. An example of ROS1-positive sample is shown in Figure 2, showing single aqua pattern.
ALK and ROS1 RUO FISH in ALK-/ROS1- wild type cohort Regarding 21 wild type cases, 18 samples (85%) were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The remaining three cases not evaluable due to high background or poor ROS1 signals, and were excluded from the analysis. The high background was caused by interference from the strong auto-fluorescent of the fibrotic tissues surrounding the tumor cells. This was extremely difficult to avoid in biopsy specimens with very limited tumor cells. The 18 successful samples were 100% concordant with the previous results, and were negative for both ALK and ROS1. An example of ALK-wild, ROS1-wild sample is shown in Figure 3.
Discussion In this study, we showed that simultaneous detection of ALK and ROS1 rearrangement is feasible on a single slide using ALK and ROS1 RUO FISH assay. The overall success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 83% (37/41 cases). The results from 12
Vysis ALK/ROS1 Dual Break Apart Probe Kit was in 100% concordant to the previous ALK and ROS1 tests in all the three patients cohorts – ALK-rearranged, ROS1-rearranged and ALK/ROS1 wildtype. The excluded cases were mainly due to insufficient tissue for further testing or repeat. The specimens, mostly small biopsies, had been investigated extensively prior to this study, thus, there was limited remaining tumor cells on the tissue. Even though strict shipping condition was adhered to, the signals, especially aqua signals of ROS1 probe, was significantly affected. The weak ROS1 signals and/or high fluorescent background observed in some of the samples might be attributed to change in pH and temperature during shipment of slides from Korea to Singapore. We repeated the test by performing hybridization directly in Singapore, and we managed to get satisfactory result from most of the cases. Therefore, shipment of hybridized specimens may not be advised for laboratory tests in clinical diagnostics. High fluorescent background was commonly observed in biopsy specimen and in specimens rich with fibrotic and connective tissues. This had a significant impact on automated scanning due to the interference from the auto-fluorescent. In these cases, the system automatically lowered the exposure time due to the high auto-fluorescent, and thus, causing weaker signals being captured. BioView system has an option to perform interactive scanning, where the operator may manually adjust the exposure time to capture stronger signals. This could overcome of the challenges in these difficult cases. Determination of ALK and ROS1 rearrangement status has become mandatory in the treatment of NSCLC patients due to the availability of ALK and ROS1 inhibitors. However, detection strategy of ALK and ROS1 rearrangements has been variable. In June 2015, the U.S. 13
Food and Drug Administration approved the VENTANA ALK (D5F3) CDx Assay as a companion diagnostic to aid the identification of patients eligible for treatment with the ALK inhibitor crizotinib. However, FISH remains the gold standard for detecting ALK rearrangement [12]. Although the break-apart FISH assay requires experience for interpretation, patience, high cost, and technical expertise; this assay is highly sensitive and specific for the detection of ALK gene
rearrangement
regardless
of ALK fusion
partners.
For
ROS1,
immunohistochemistry was evaluated in previous studies [11, 13], and it showed equally high sensitivity and specificity as ALK IHC. However, FISH assay was necessary to confirm equivocal cases in IHC. In this regard, FISH remains the pertinent method in selecting ALKand ROS1-rearranged patients. In conclusion, Vysis ALK/ROS1 Dual Break Apart Probe Kit can detect ALK and ROS1 rearrangement simultaneously in NSCLC. Due to the limited availability of FFPE specimens, we recommend further validation of this probe in prospective analysis by including more ALK- and ROS1-rearranged patient specimens.
Conflict of Interest statement None declared
Acknowledgements The authors would like to thank BioView Inc. for the technical assistance during the study. This study was supported by a faculty research grant of Yonsei University College of Medicine for 2012(6-2012-0134) to B.C.C. 14
References 1.
Shaw AT, Kim DW, Nakagawa K, Seto T, Crino L, Ahn MJ, De Pas T, Besse B, Solomon BJ, Blackhall F, Wu YL, Thomas M, O'Byrne KJ, Moro-Sibilot D, Camidge DR, Mok T, Hirsh V, Riely GJ, Iyer S, Tassell V, Polli A, Wilner KD, Janne PA. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med 2013;368: 23852394.
2.
Lindeman NI, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, Jenkins RB, Kwiatkowski DJ, Saldivar JS, Squire J, Thunnissen E, Ladanyi M, College of American Pathologists International Association for the Study of Lung C, Association for Molecular P. 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. J Mol Diagn 2013;15: 415-453.
3.
Bergethon K, Shaw AT, Ou SH, Katayama R, Lovly CM, McDonald NT, Massion PP, Siwak-Tapp C, Gonzalez A, Fang R, Mark EJ, Batten JM, Chen H, Wilner KD, Kwak EL, Clark JW, Carbone DP, Ji H, Engelman JA, Mino-Kenudson M, Pao W, Iafrate AJ. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 2012;30: 863-870.
4.
Davies KD, Le AT, Theodoro MF, Skokan MC, Aisner DL, Berge EM, Terracciano LM, Cappuzzo F, Incarbone M, Roncalli M, Alloisio M, Santoro A, Camidge DR, VarellaGarcia M, Doebele RC. Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res 2012;18: 4570-4579. 15
5.
Shaw AT, Ou SH, Bang YJ, Camidge DR, Solomon BJ, Salgia R, Riely GJ, Varella-Garcia M, Shapiro GI, Costa DB, Doebele RC, Le LP, Zheng Z, Tan W, Stephenson P, Shreeve SM, Tye LM, Christensen JG, Wilner KD, Clark JW, Iafrate AJ. Crizotinib in ROS1rearranged non-small-cell lung cancer. N Engl J Med 2014;371: 1963-1971.
6.
Ardini E, Menichincheri M, Banfi P, Bosotti R, De Ponti C, Pulci R, Ballinari D, Ciomei M, Texido G, Degrassi A, Avanzi N, Amboldi N, Saccardo MB, Casero D, Orsini P, Bandiera T, Mologni L, Anderson D, Wei G, Harris J, Vernier JM, Li G, Felder E, Donati D, Isacchi A, Pesenti E, Magnaghi P, Galvani A. Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications. Mol Cancer Ther 2016;15: 628-639.
7.
Zou HY, Li Q, Engstrom LD, West M, Appleman V, Wong KA, McTigue M, Deng YL, Liu W, Brooun A, Timofeevski S, McDonnell SR, Jiang P, Falk MD, Lappin PB, Affolter T, Nichols T, Hu W, Lam J, Johnson TW, Smeal T, Charest A, Fantin VR. PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations. Proc Natl Acad Sci U S A 2015;112: 3493-3498.
8.
Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, Ou SH, Dezube BJ, Janne PA, Costa DB, Varella-Garcia M, Kim WH, Lynch TJ, Fidias P, Stubbs H, Engelman JA, Sequist LV, Tan W, Gandhi L, Mino-Kenudson M, Wei GC, Shreeve SM, Ratain MJ, Settleman J, Christensen JG, Haber DA, Wilner K, Salgia R, Shapiro GI, Clark JW, Iafrate AJ. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363: 1693-1703.
9.
Shaw AT, Kim DW, Mehra R, Tan DS, Felip E, Chow LQ, Camidge DR, Vansteenkiste J, Sharma S, De Pas T, Riely GJ, Solomon BJ, Wolf J, Thomas M, Schuler M, Liu G, 16
Santoro A, Lau YY, Goldwasser M, Boral AL, Engelman JA. Ceritinib in ALK-rearranged non-small-cell lung cancer. N Engl J Med 2014;370: 1189-1197. 10.
Marchetti A, Di Lorito A, Pace MV, Iezzi M, Felicioni L, D'Antuono T, Filice G, Guetti L, Mucilli F, Buttitta F. ALK Protein Analysis by IHC Staining after Recent Regulatory Changes: A Comparison of Two Widely Used Approaches, Revision of the Literature, and a New Testing Algorithm. J Thorac Oncol 2016;11: 487-495.
11.
Cha YJ, Lee JS, Kim HR, Lim SM, Cho BC, Lee CY, Shim HS. Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements. PLoS One 2014;9: e103333.
12.
Sholl LM, Weremowicz S, Gray SW, Wong KK, Chirieac LR, Lindeman NI, Hornick JL. Combined use of ALK immunohistochemistry and FISH for optimal detection of ALKrearranged lung adenocarcinomas. J Thorac Oncol 2013;8: 322-328.
13.
Mescam-Mancini L, Lantuejoul S, Moro-Sibilot D, Rouquette I, Souquet PJ, AudigierValette C, Sabourin JC, Decroisette C, Sakhri L, Brambilla E, McLeer-Florin A. On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas. Lung Cancer 2014;83: 168-173.
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Figure legends Figure 1. Representative Vysis ALK/ROS1 Dual Break Apart Probe Kit FISH image of tumor with ALK rearrangement. Gold arrow highlights positions of FISH signal corresponding to ALK rearrangement, showing red and green break apart signal
Figure 2. Representative Vysis ALK/ROS1 Dual Break Apart Probe Kit FISH image of tumor with ROS1 rearrangement. Gold arrow highlights positions of FISH signal corresponding to ROS1 rearrangement, showing single aqua signal
Figure 3. Representative ALK/ROS1 R Vysis ALK/ROS1 Dual Break Apart Probe Kit FISH image of ALK- and ROS1-wild type tumor
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Table 1. Clinicopathological characteristics of patients Patients (N=41) Median age (range) Sex
Smoking status
Stage
Genotypes
N (%) 55 (32-79) Male
20 (48)
Female
21 (52)
Never
24 (58)
Former
11 (27)
Current
6 (15)
I
6 (15)
II
4 (10)
III
12 (29)
IV
19 (46)
EGFR mutation
6 (15)
KRAS mutation
1 (2)
ALK rearrangement
12 (29)
ROS1 rearrangement
8 (20)
Pan-negative
14 (34)
20