Assessment of cytology based molecular analysis to guide targeted therapy in advanced non-small-cell lung cancer

To investigate the use of molecular testing on cytological specimens in selecting advanced non-small cell lung cancer (NSCLC) patients who are adequate for targeted treatment, a total of 137 NSCLC cases were analyzed by fluorescence in situ hybridization (FISH) for anaplastic lymphoma kinase (ALK) rearrangements, and Epidermal growth factor receptor (EGFR), kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were evaluated by quantitative real-time PCR (qRT-PCR) platform combining amplification refractory mutation system (ARMS) primers and TaqMan probes. Cytological specimens included 91 fine-needle aspirates, 5 fibreoptic bronchoscopic derived samples and 41 pleural effusions. Among 137 NSCLCs analyzed for ALK FISH, 16 (11.7%, of 137) were detected to harbor ALK rearrangement. FISH positive cases were all defined as adenocarcinoma (ADC) histologic subtype and the FNA samples showed the highest ALK positive rate (13.2%, 12/91). Of the 9 ALK FISH positive patients who received crizotinib treatment, 8 (88.9%) patients exhibited tumor regression. In addition, 60 (44.8%, of 134) cases were found to harbor EGFR mutations and 22 patients with EGFR sensitive mutations who received gefitinib or erlotinib treatment showed a median PFS of 16.0 months. Mutations of KRAS occurred in 8 (6.0%, of 134) cases and this was mutually exclusive from EGFR mutation. Our results demonstrated that ALK FISH and EGFR, KRAS mutational analysis on cytological specimens are sensitive methods for screening advanced stage NSCLC patients who are adequate for targeted treatment.


INTRODUCTION
Non-small-cell lung cancer (NSCLC), which accounts for approximately 85% of lung cancers, has been largely identified by oncogenic driver mutations with potential opportunities for targeted therapies [1,2]. Recently, activation of the anaplastic lymphoma kinase (ALK) gene in lung cancer by fusion to echinoderm microtubule-associated protein-like 4 (EML4) or other gene partners (such as BIRC6 [3], TFG [4], KIF5B [5] and KLC1 [6]) has been identified as oncogenic events [7]. Clinical studies have shown that locally advanced or metastatic NSCLC patients harboring ALK gene rearrangement are highly sensitive to Crizotinib, which is a small molecular inhibitor of ALK tyrosine kinase [8,9].
In addition, NSCLC with sensitive epidermal growth factor receptor (EGFR) mutations are well responded to tyrosine kinase inhibitors (TKI) and these patients will have a longer progression-free survival (PFS) than the patients whose tumors do not contain EGFR mutations [1,10].
Approximately 60% of patients with NSCLC are diagnosed at a late stage for the first time [11]. These patients are not suitable for the resection of the primary tumor, and the only pathologic material guiding systemic therapy should be small biopsy or cytological specimens. Recent studies have demonstrated that cytological specimens, including fine-needle aspiration (FNA), fibreoptic bronchoscopic (FOB) and pleural effusion (PLE), are suitable for the molecular testing [12][13][14][15][16]. Although FISH (fluorescent in situ hybridization) is currently the gold standard method to detect ALK gene rearrangement approved by FDA, its application on cytological specimens remains a worth area of investigation. In this study, we investigate the use of ALK FISH and EGFR, KRAS mutational testing on cytological specimens and to evaluate PFS of the patients who received targeted therapies.
The ALK FISH positive cases included 6 men and 10 women and there was no significant difference in gender distribution between FISH positive and negative cases (P = 0.87). The mean age at diagnosis for FISH positive cases was 52.7 ± 11.8 years, which was much younger than that of FISH negative cases (P < 0.05). FISH positive cases were all defined as ADC histologic subtype. In addition, there was one FISH positive case also demonstrated an EGFR L858R mutation ( Table 2).

EGFR and KRAS mutation status
Among 134 NSCLCs tested, 60 (44.8%, of 134) cases carried EGFR mutations, which was 53 (39.5%, of 134) sensitive mutations and 4 (3.0%, of 134) exon 20 mutations with S768I. There was one case demonstrated complex mutation with exon 19 deletion and T790M and the other two cases showed complex mutation with L858R and T790M. Among them, two patients carried acquired T790M mutation after TKI treatment and one patient carried primary coexisting mutations of T790M and L858R. In addition, four patients carried primary exon 20 S768I mutations and did not receive targeted therapies. The mutated EGFR cases of the cytological samples were 36 (60.0%, of 60) FNAs, 1 (1.7%, of 60) FOB and 23 (38.3%, of 60) PLEs (Supplementary Table S2). The PLE samples showed highest EGFR mutation rate (58.9%, 23/39) among three groups, although this did not demonstrate a statistically significant difference (P = 0.41). Mutations of KRAS occurred in 8 (6.0%, of 134) cases and this was mutually exclusive from EGFR mutation. In addition, compared with male patients, female patients were more likely to carry EGFR mutations (55.6% vs 28.3%, P = 0.002) (Supplementary Table S3).

Outcomes of targeted therapies
Of the 9 patients who received crizotinib treatment, there were 5 men and 4 women with mean age of 58.6 years ( Table 3). The median follow-up duration was 9 months (range: 6-15 months). At the end of follow up, 8 (88.9%) patients were still receiving crizotinib treatment and there was one person stopping the treatment because of disease progression.
We performed targeted next-generation sequencing (NGS) in one case who did not response well to crizotinib treatment. Results confirmed the existence of EML4-ALK transloction, and the relative abundance of EML4-ALK fusion was 25.3%. In addition, there were a nonsense mutation (c.991 C > T, p.Q331) in exon 9 of P53 and a deletion of CDKN2A. The mutation in P53 could result in a stop codon and lead to loss of P53 function as a transcription factor. [17] There were 22 patients with EGFR sensitive mutations who received gefitinib or erlotinib treatment. The median PFS was 16.0 months (95% confidence interval, 12.9 -19.1) ( Figure 2). Of them, 8 patients (36.4%) exhibited disease progression after 3 months due to acquired resistance to EGFR-TKI treatment. At the data cutoff point, 14 patients were still taking TKI treatment and tumor regression was observed in these patients.

DISCUSSION
The diagnosis of advanced-stage NSCLC is usually based on a small amount of cytological specimens, consequently, the use of ALK FISH and EGFR, KRAS mutational testing on cytological specimens is gradually becoming a necessity in the routine molecular pathological diagnosis. Although FISH is considered to be the gold standard for detection of ALK rearrangements on histological tissues approved by FDA, its application on cytological specimens remains a worth area of investigation. In this study, for the first time, we demonstrated the possibility of using ALK FISH and EGFR, KRAS mutational testing on cytological specimens in a large cohort of Chinese patients and correlate the analysis results with PFS in patients with targeted therapies.
Targeted therapies are mostly effective in patients with locally advanced or metastatic disease, thus, molecular analysis on cytological samples rather than on resected specimens would be preferred on patients with late stage tumors. The data in previous studies demonstrated that the performance of cytology based  [21]. Cytological specimens of our study demonstrated slightly higher EGFR mutational rates (44.8% vs 36.2%) than that of histological specimens in above study and this could partly be attributed to the patients undertaken cytological mutational analysis often underwent a late stage disease.
Previous studies had rarely evaluated the EGFR-TKI treatment responses on patients with sensitive EGFR mutations tested on cytological specimens, although the outcome of the patients treated with TKI was important to confirm the predictive value of cytological mutational analysis. In our study, fifty-seven patients with EGFR mutations demonstrated an ADC subtype and this population would largely benefit from targeted therapies. Twenty-two patients with ADC subtype and EGFR sensitive mutations received TKI treatment with a median PFS of 16 months and all the patients were observed with tumor regression after 3-months treatment. This was in accordance with previous studies, which demonstrated that the median PFS interval of 16.2 months was observed in 11 EGFR mutated patients who treated with gefitinib [22]. In addition, Maria D. Lozano also observed patients with EGFR mutations had a 12.3-months median PFS [23]. These results indicated that EGFR mutational testing on cytological samples was adequate for patient selection with EGFR-TKI treatment. In addition to guide the targeted therapy for patients with unresected tumors, another possible advantage of cytological mutational analysis was to identify genetic shifts of EGFR acquired mutations during TKI treatment.
It is currently accepted that FISH is the most appropriate method to detect ALK rearrangements and to guide crizotinib treatment. Only a few reports have assessed the adequacy of cytological specimens for ALK FISH analysis, thus, its application on cytological specimens still needs further studies. Weiya Wang, et al analyzed a cohort of 58 patients for ALK rearrangements using pleural effusion cell blocks and found 10.3% positive cases by ALK FISH [16]. Agnese Proietti, et al demonstrated 4.4% cases with ALK FISH rearrangements in both cell blocks and small biopsies [24]. In addition, MJ. Neat, et al compared FISH and IHC methods to  Abbreviations: ALK = anaplastic lymphoma kinase; FISH = fluorescence in situ hybridization; FNA = fine needle aspiration; PLE = pleural effusion; PFS = progression-free survival; PR = partial response; PD = progressive disease. screen for ALK status in endobronchial ultrasound (EBUS)-transnronchial needle aspiration (TBNA) derived cytological specimens and found that FISH was superior to IHC for the detection of ALK rearrangement in these cytological samples [25]. To best of our knowledge, this is the largest report of ALK detection in cytological specimens in Chinese patients and first described the outcome of patients with ALK FISH positive who received crizotinib treatment. In this study, we detected 11.7% ALK rearrangement in 137 cases of various cytological specimens, including FNA, FOB and PLE, which was significantly higher than the rate of 6.8% previously reported in a large Chinese consecutive case series [26]. Our results were in accordance with previous report that 12.7% of ALK rearrangements were observed in malignant pleural effusion cell blocks from patients with advanced NSCLC [16]. The increased percentage of positive cases was likely attributed to the advanced stage of disease among patients with cytological specimens. Of the nine patients who received crizotinib treatment, one patient exhibited disease progression after 5 months and eight patients were still receiving crizotinib treatment at the end of cutoff point. We performed targeted nextgeneration sequencing in one case who did not response well to Crizotinib treatment and found that there was a nonsense mutation in exon 9 of P53 which could lead to loss of P53 function. Previous studies have indicated that P53 alterations might accelerate cancer development and could also lead to shorter survival in patients with P53 mutated NSCLC [27]. Our findings first demonstrated the In summary, we conclude thatALK FISH and EGFR, KRAS mutational analysis on cytological specimens are sensitive methods for screening advanced stage NSCLC patients who are adequate for targeted treatment. Cytological specimens including FNA, FOB and PLE all provide feasible and effective material for the molecular analysis and further studies are required to validate the application in the routine molecular pathological diagnosis.

Patients
One hundred and thirty-seven patients with advanced NSCLC were enrolled in this study from September 2013 to June 2015. All these cytological samples, including FNA, FPB and PLE, were tested for ALK fusion by FISH and 134 were performed with EGFR and KRAS mutational testing because 3 samples were not adequate for DNA extraction. Imaging data were independently reviewed by authors to evaluate their treatment responses according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. PFS was calculated from the date of initiating TKI treatment to a radiologic or clinical observation of disease progression. The study protocol was approved by the Institute Review Board of the Cancer Hospital, Chinese Academy of Medical Sciences (CAMS), Beijing, China. The methods were carried out in accordance with the approved guidelines. Each participant signed an Institutional Review Board approved informed consent in accordance with current guidelines.

Specimen preparation
The cytological specimen preparations were conducted according to a standard specimen processing protocol in our laboratory. The cases included FNA specimens obtained under image guidance with a cytopathologist present for adequacy assessment and pleural fluid specimens obtained by thoracentesis. The percentage of tumor cells more than 5% or over 500 tumor cells were used for DNA extraction and 100 tumor cells for ALK FISH analysis. The algorithm used for molecular testing in our study was depicted in Supplementary Figure S1. Each case had air-dried slides stained with Diff-Quik (DQ stain, Protocol Hema 3; Fisher Scientific, Kalamazoo, MI) and additional slides fixed in 95% alcohol for Papanicolaou staining. Fluid specimens also had a ThinPrep (Hologic, Marlborough, MA) slide prepared.

Fluorescence in situ hybridization
FISH analysis was conducted as previously described [28]. Briefly, FISH analysis was performed using the Vysis LSI ALK Dual color, Break Apart Rearrangement Probe (Abbott/Vysis, Abbott Park, IL, USA). Samples were considered to be FISH positive if more than 15% of the scored tumour cells had split one or both ALK 5' and 3' probe signals or had isolated 3' signals. Slides were evaluated independently by two experts blind to the patient's history and histological findings.

EGFR and KRAS mutational testing
EGFR and KRAS mutational testing was conducted as previously described [29]. Briefly, mutational testing was performed using the HumanEGFR or KRAS Mutation Qualitative Detection Kit (Beijing ACCB Biotech Ltd., China), which applies quantitative real-time PCR (qRT-PCR) platform combining amplification refractory mutation system (ARMS) primers and TaqMan probes. The sensitive mutations were defined as p.G719S/C/A, p.L858R, p.L861Q and insertions in exon 19. The hotspot mutations in KRAS gene were within codon 12 and 13 including p.G12C/V/S/R/D/A and p.G13D. The assay was carried out according to the manufacturer's protocol using the Stratagene Mx3000P real-time PCR system (Agilent technologies Inc, USA). Presence or absence of mutations was assessed from the fluorescence amplification curve.

Targeted next-generation sequencing
For one patient who did not response well to crizotinib treatment, targeted next-generation sequencing was performed. Genomic DNA was profiled by using a capture-based targeted sequencing panel (Burning Rock Biotech, Guangzhou, People's Republic of China). In brief, human genomic regions of 271 kb, including all exons in 56 genes and selected introns in ALK, RET and ROS1 for the detection of translocation events, were captured by using 120-bp probes and were sequenced (Supplementary Table S4). The concentration of the DNA samples was measured with the Qubit dsDNA assay. Fragments of 200 to 400-bp sizes were selected with beads (Agencourt AMPure XP kit; Beckman-Coulter, Brea, CA), followed by hybridization with the capture probes baits, hybrid selection with magnetic beads, and PCR amplification. A bioanalyzer high-sensitivity DNA assay was then used to assess the quality and size range. Available indexed samples were then sequenced on a Nextseq (Illumina, San Diego, CA) with pair-end reads. Sequence data were analyzed by GATK 3.2 (https://www. broadinstitute.org/gatk/) and DNA translocation analysis was performed by using both Tophat2 (http://ccb.jhu.edu/ software/tophat/index.shtml) and Factera 1.4.3 ((http:// factera.stanford.edu) [30].

Statistical analysis
Differences of patient characteristics and clinicopathologic factors in the two-dimensional cross-