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BMC Cancer

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Comparison of detection methods and follow-up study on the tyrosine kinase inhibitors therapy in non-small cell lung cancer patients with ROS1 fusion rearrangement

  • Jieyu Wu1,
  • Yunen Lin1,
  • Xinming He1,
  • Haihong Yang2,
  • Ping He1,
  • Xinge Fu1,
  • Guangqiu Li1 and
  • Xia Gu1Email author
BMC Cancer201616:599

https://doi.org/10.1186/s12885-016-2582-9

Received: 6 December 2015

Accepted: 19 July 2016

Published: 4 August 2016

Abstract

Background

The screening of ROS proto-oncogene 1, receptor tyrosine kinase(ROS1) fusion rearrangement might be potentially beneficial for an effective therapy against non-small cell lung cancer (NSCLC). However, the three main ROS1 rearrangement detection methods have limitations, and no routine protocol for the detection of ROS1 rearrangement in NSCLC is available. In this study, our aims were to compare immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (qRT-PCR) in their ability to detect ROS1 rearrangement in NSCLC, and discuss the clinical characteristics and histopathology of the patients with ROS1 rearrangement. Moreover, the effects of tyrosine kinase inhibitors (TKIs) therapy on the patients with ROS1 rearrangement and advanced stage disease (III b–IV) were investigated.

Methods

Patients with a previously diagnosed NSCLC were recruited in this study from November 2013 to October 2015. IHC was performed using the D4D6 monoclonal antibody (mAb) in an automatic IHC instrument, while FISH and qRT-PCR were carried out to confirm the IHC results. FISH and qRT-PCR positive cases underwent direct sequencing. After detection, patients with advanced ROS1 rearranged NSCLC had received TKI therapy.

Results

Two hundred and thirty-eight patients were included in this study. ROS1 rearrangement was detected in 10 patients. The concordant rate of FISH and qRT-PCR results was 100 %, while in the FISH and IHC results high congruence was present when IHC showed a diffusely (≥60 % tumor cells) 2–3+ cytoplasmic reactivity pattern. Patients harboring ROS1 rearrangement were mostly young (8/10), females (7/10) and non-smokers (7/10) with adenocarcinoma (10/10) and acinar pattern. Most of their tumor were in intermediate grade (6/8). Among these 10 patients, three of them in stage IV with ROS1 rearrangement gained benefits from ROS1 TKI therapy.

Conclusions

IHC, FISH and qRT-PCR can reliably detect ROS1 rearrangement in NSCLC, while IHC can be used as a preliminary screening tool. These results supported the efficacy of ROS1 TKI therapy in treating advanced NSCLC patients with ROS1 rearrangement.

Keywords

ROS1ImmunohistochemistryFluorescent in situ hybridizationQuantitative real-time polymerase chain reactionNon-small cell lung cancerTyrosine kinase inhibitors

Background

Mutations in receptor tyrosine kinases (RTKs) genes have been identified as the main cause of many carcinomas development, since they can lead to proliferation and transformation of cancer cells [1]. In recent years, ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), a gene located on 6q22, which transcripts the protein that belongs to the subfamily of tyrosine kinase insulin receptor, has been recognized as a driver of non-small cell lung cancer (NSCLC) [2] since it can fuse with other genes (e.g. CD74, SLC34A2, FIG, TPM3, SDC4, EZR, LRIG3, CCDC6, and KDELR2 [3, 4]) and consequently activate the downstream growth and survival signaling pathways [37]. In most cases, ROS1 fusion rearrangement is exclusive to other RTK aberrance, such as the anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangement, epidermal growth factor receptor (EGFR) mutations and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations [4]. Moreover, because of the homology between the ROS1 and ALK proteins [8, 9], patients with ROS1 rearrangement are sensitive to ALK tyrosine kinase inhibitors (TKIs). Therefore, despite the incidence of ROS1 rearrangements in NSCLC is low (1–2 %) [4, 10], screening ROS1 rearrangement could be potentially beneficial for NSCLC patients.

In the present work, fluorescent in situ hybridization (FISH), quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) have been used for ROS1 arrangement detection. All of these methods have advantages and limitations. FISH analysis can reveal the genes rearrangement status, but the procedure is inconvenient [11, 12], and it is not suitable for biopsies with insufficient numbers of tumor cells. qRT-PCR analysis can reveal fusion rearrangements by using specific primers and it has a high sensitivity. However, qRT-PCR cannot detect specimens with unknown fusion types [11, 12]. IHC is feasible in large scale screening, and the D4D6 rabbit monoclonal antibody (mAb) has been identified as effective and specific mAb for ROS1 rearrangement protein detection by several studies [3, 8, 11]. In addition, the costs to perform IHC are less compared with qRT-PCR or FISH. However, there is not an accurate cutoff value to define positive ROS1 protein expression using IHC, thus representing a limitation on using this method [1114]. Therefore, the aim of this study was to compare these three analytical methods in their ability to detect ROS1 rearrangement in NSCLC, trying to set up a cutoff value for ROS1 IHC analysis. In addition, we investigated the efficacy of TKI therapy in treating advanced NSCLC patients with ROS1 rearrangement. The characteristics of NSCLC patients harboring ROS1 rearrangement were also discussed.

Methods

Patient selection

Patients admitted to the First Affiliated Hospital of Guangzhou Medical University were screened and recruited for this study from November 2013 to October 2015. Patients were selected upon (1) a previous identification of NSCLC with (2) a confirmed diagnosis by IHC of p63, CK5/6, NapsinA and TTF-1 protein expression [15]. A cohort of 238 NSCLC patients was included. Afterwards, all slides from the chosen cases were independently analyzed by two pathologists (X Gu & JY Wu) blinded to history and prior diagnoses. The histopathological classification was performed according to the 2015 WHO classification of lung tumors [15] and the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) multidisciplinary classification [16]. Appropriate specimens with sufficient tissue (>100 tumor cells) were included.

After recruitment, the clinical information, including age, gender, smoking history, and tumor node metastasis (TNM 7th) staging were collected. In adenocarcinoma cases, the histological grading was performed by analyzing the single most predominant pattern in a case [15]. According to 2015 WHO histological grading of adenocarcinoma, the grading was divided into low, intermediate and high. Another grading score system that combined the most two predominant pattern in a case was also been used, which was worked out by Sica et al. [17]. The results of other genetic markers testing were also collected, such as ALK, EGFR and KRAS. Ventana IHC with D5F3 mAb and FISH with break-apart probe were used in ALK rearrangement detection. Amplification refractory mutation system polymerase chain reaction (ARMS-PCR) was used to detect EGFR and KRAS gene mutation. This study was approved by the Ethic Review Committee of the First Affiliated Hospital of Guangzhou Medical University.

Immunohistochemistry (IHC)

All the specimens were formalin-fixed and paraffin embedded (FFPE). ROS1 IHC was performed on 4 μm slides and completed on a fully automated IHC instrument (BenchMark XT, Roche, Switzerland). D4D6 rabbit mAb (Cell Signaling Technology, Danvers, MA) diluted in 1:200 was used as primary antibody. Detection was using UltraView Universal DAB detection Kit (Roche, Switzerland). IHC was scored using the following score scheme: 0, no staining of tumor cells; 1+, tumor cells with faint cytoplasmic reactivity without any background staining; 2+, tumor cells with moderate cytoplasmic reactivity; and 3+, tumor cells with strong granular cytoplasmic reactivity [11]. When several intensity levels present in a case, it was scored according to the intensity of major tumor cells. The extent of IHC staining was also analyzed by estimating the staining percentage of tumor cells [8]. Moreover, H-score method was used and calculated using the following equation: H-score = ∑[intensity (0, 1, 2, 3) × extent of each staining intensity(%)], with a scoring range from 0 to 300 [14]. Previous lung specimens with ROS1 rearrangements confirmed by FISH and a 3+ staining score, have been used as positive control. IHC was analyzed independently by two pathologists (X Gu & JY Wu), and disagreements were discussed after the analysis. A third pathologist (XG Fu) was invited as the reviewer when an agreement could not be reached. The above results were blinded for the qRT-PCR results.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from FFPE tissue sections (6 μm slides) using the FFPE RNA Kit (Amoy Diagnostics Co., Ltd, Xiamen, China). RNA concentration was measured using a spectrophotometer (Nanodrop 2000c, Thermo-Scientific, Wilmington, US) and reverse transcription was performed to generate complementary DNA (cDNA). The cDNA was used for multiple RT-PCRs that were carried out in an Mx3000p real-time PCR system (Agilent Technologies, California, US) using the ROS1 Gene Fusion Detection Kit (Amoy Diagnostics Co., Ltd, Xiamen, China). The positive and negative reference samples were also used. The PCR procedure was the following: One cycle at 95 °C for 5 min; 15 denaturation cycles at 95 °C for 25 s, annealing at 64 °C for 20 s and elongation at 72 °C for 20 s; 31 cycles at 93 °C for 25 s, 60 °C for 35 s (data collection) and 72 °C for 20 s. The quantification is determined by the fusion fluorescence signals and the assay with a Ct value < 30 cycles was considered as positive. These results were blinded for the IHC and FISH results.

Tissue microarray (TMA) and fluorescent in situ hybridization (FISH)

IHC positive staining areas were evaluated and selected from the slides by a pathologist (JY Wu) to avoid tumor heterogeneity and the tissue microarray (TMA) was performed from the FFPE samples. Two areas of 2 mm diameter were removed from each sample block using a stainless steel stylet (Xinsen, Jieli Biomedicine Co., Ltd, Guangzhou, China). Serial 4 μm TMAs sections were used for FISH detection using 6q22 ROS1 Break Apart FISH Probe RUO Kit (Abbott Molecular Inc, IL, USA). The protocol and interpretation of FISH were the following: TMA slides were submerged in xylene and decreasing gradient of ethanol for deparaffinization and hydration, respectively. Next, they were subjected to a heat-treatment in boiled water (100 °C, 30 min) and digestion using proteinase K (37 °C, 5 min). They were washed in 2 × SSC solution and dehydrated by increasing gradient of ethanol (70 %, 85 % and 100 %) for 3–5 min. After air drying, the probe was added to the target specimens, and coverslips were placed. The slides were placed in the hybridization machine (ThermoBrite, Abbott Molecular Inc, IL, US) and hybridization was performed as follows: denaturation at 75 °C for 8 min and hybridization at 42 °C for 16 h. Next, the slides were washed in 2 × SSC and NP40 solution at 42 °C for 5 min and immersed in 70 % ethanol for 5 min. DAPI 15 μl was applied to counterstain. Analysis was performed in the dark using the fluorescence microscopy (Nikon 80i, Japan). The data analysis was the following: >15 % tumor cells showing split signals (“red” and “green” split signals) or isolated 3′ signals (single “green” signals) belonged to the ROS1 fusion rearrangement. These results were blinded for the qRT-PCR results.

Direct sequencing

The cDNA of FISH and qRT-PCR positive cases were sent to Amoy Diagnostics Co., Ltd for direct sequencing. The results of the sequencing were compared using the Basic Local Alignment Search Tool (BLAST).

Follow-up visits

After ROS1 rearrangement detection using IHC, FISH and qRT-PCR, the patients harboring ROS1 rearrangement in advanced stages (III b–IV) of disease were selected for TKI therapy. In order to track the efficacy of the therapy, information such as patient’s syndromes, vital signs and CT images were collected every two months. The efficacy was evaluated using RECIST guideline 1.1 [18]. The materials of patients were authorized by the recruited patients and (or) their family members.

Statistical analysis

Pearson’s χ 2 and Fisher’s exact test were used to assess the relationship between ROS1 rearrangement, clinical characteristics and clinicopathological patterns. The Kappa value was calculated to assess the concordant rate of FISH and IHC in detecting ROS1 rearrangement. The analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 13.0 (SPSS, Inc., Chicago, IL, US), and P values less than 0.05 were considered statistically significant.

Result

Characteristics of the recruited cases

Two hundred and thirty-eight cases were recruited, of which 215 were surgical resection cases and 23 were needle biopsy cases. The clinical characteristics and histopathology of the patients are shown in Table 1. The median age was 61 years old (range from 27 to 85 years old), and 107 were females and 131 were males. Most of the included cases were in the early stages (114/238, 47.9 %) of the disease, while 48 (48/238, 20.2 %) cases were in the advanced stages (III b–IV). Total 181 resected adenocarcinoma cases were performed histological grading, they mostly obtained score 5 in Sica staging (69/181, 38.1 %) and classified as intermediate grade in the WHO grading (126/181, 69.6 %). Details of the grading were showed in a supplementary table [see Additional file 1: Table S1]. However, there was no statistical difference between ROS1 rearrangement and non-rearrangement cases in clinical characteristics. Two hundred and twenty-eight patients underwent ALK rearrangement detection, 163 and 153 patients underwent EGFR and KRAS mutation detection, respectively. Among these cases, 12 cases (12/228, 5.3 %) were harboring ALK rearrangement, 87 cases (87/163, 53.4 %) and 13 cases (13/153, 8.50 %) were harboring EGFR and KRAS mutation, respectively.
Table 1

Characteristic of included cases

Characteristic

 

Total

Surgery resection

Biopsy

ROS1 rearrangement

ROS1 non–rearrangement

P.

No.

 

238

215

23

10

228

 

Age

      

0.114a

>61

109

101

8

2

107

 

≤61

129

114

15

8

121

 

Gender

      

0.118a

Male

131

118

13

3

128

 

Female

107

97

10

7

100

 

Smoking history

      

0.792a

Smoker

51

48

3

3

48

 

Non–smoker

158

140

18

6

152

 

Previous smoker

29

27

2

1

28

 

Histopathology

      

1.000a

ADC

216 (8)b

195 (6)b

21 (2)b

10

206 (8)b

 

SCC

11

9

2

0

11

 

ASC

2

2

0

2

 

LCLC

9 (1) b

9 (1) b

0

9 (1) b

 

Adenocarcinoma subtypes (predominant pattern)c

      

0.207a

Lepidic

17

17

2

15

 

Acinar

105

105

4

101

 

Papillary

33

33

3

30

 

Micropapillary

16

16

2

14

 

Solid

20

20

0

20

 

Invasive mucinous adenocarcinoma

8

8

1

7

 

Fetal

1

1

 

0

1

 

TMN staged

      

0.175a

I

114

114

0

6

108

 

II

30

29

1

1

29

 

III

55(IIIb:10)

53

2

0

55

 

IV

38

18

20

3

35

 

Sica gradinge

      

0.871a

Score 2

6

6

-

0

6

 

Score 3

48

48

-

3

45

 

Score 4

48

48

-

1

47

 

Score 5

69

69

-

4

65

 

Score 6

10

10

-

0

10

 

WHO gradinge

      

0.597a

Low grade

17

17

-

1

16

 

Intermediate grade

126

126

-

5

121

 

High grade

38

38

-

2

36

 

Another gene status

      

ALK

228 (12)f

206 (11)f

22 (1)f

0

228

 

EGFR

163 (87)f

141 (78)f

22 (9)f

0

163

 

exon 19 deletion

37

33

4

0

37

 

L858R

48g

44g

4

0

48

 

L861Q

1

1

0

0

1

 

exon 20 S768I

2g

1g

1

0

2

 

KRAS

153 (13)f

136 (11)f

17 (2)f

0

153

 

Gly12Asp

3

3

0

0

3

 

Gly12Cys

4

4

0

0

4

 

Gly12Val

4

4

0

0

4

 

Gly12Ala

1

0

1

0

1

 

Gly12Ser

1

0

1

0

1

 

Abbreviations: ADC Adenocarcinoma, SCC Squamous cell carcinoma, ASC Adenosquamous carcinoma, LCLC Large–cell lung carcinoma, ALK anaplastic lymphoma receptor tyrosine kinase, EGFR epidermal growth factor receptor, KRAS Kirsten rat sarcoma viral oncogene homolog

aFisher exact test

bMetastasis cases

cTotal 190 resected adenocarcinoma with 200 predominant patterns were discussed. Some cases were including more than one predominant patterns

dA case was diagnosed as stage 0

eTotal 181 resected adenocarcinoma have been analyzed, excluding variant subtypes

fThe cases with ALK rearrangement, EGFR mutation or KRAS mutation

gThere was a case harboring both exon 21 L858R and exon 20 S768I mutation

Comparison of IHC, FISH and qRT-PCR in ROS1 rearrangement detection

All the recruited patients underwent FISH and IHC detection of ROS1 rearrangement, and qRT-PCR analysis was applied in 159 cases. A total of 10 cases were confirmed as ROS1 rearrangement positive by FISH (10/238, 4.2 %; Table 2). Six of them underwent qRT-PCR detection, which confirmed the presence of ROS1 rearrangement. All qRT-PCR negative cases were also confirmed as ROS1 rearrangement negative by FISH.
Table 2

The characteristics of patients with ROS1 rearrangement

Cases No.

Smokinga

Staging/Gradingb

IHC resultc

H-score

FISH result

Direct sequencing

Histopathology predominant pattern

Staining Pattern

Another gene aberranced

1

N

II B/

Score 5/Grade 2

3+/90 %

260

Fusion positive

SLC34A2-E4; ROS1-E32/SLC34A2-E4; ROS1-E34

Acinar

Cytoplasmic; membrane

ALK\EGFR\KRAS(−)

2

N

I A/Score 3/Grade 2/

2+/65 %

150

Fusion positive

CD74-E6; ROS1-E34

Acinar

Cytoplasmic; focal granular

ALK\EGFR\KRAS(−)

3

P

IV/−/−

2+/70 %

160

Fusion positive

CD74-E6; ROS1-E34

Invasive mucinous adenocarcinoma

Mucinous staining

ALK\EGFR\KRAS(−)

4

N

I A/Score 5/Grade 2

2+/90 %

200

Fusion positive

TPM3-E8; ROS1-E35

Papillary and acinar

Cytoplasmic; focal granular

ALK\EGFR\KRAS(−)

5

N

I A/Socre 5/Grade 3

2+/90 %

200

Fusion positive

SLC34A2-E14del; ROS1-E32/SLC34A2-E14del; ROS1-E34

Papillary and micropapillary

Cytoplasmic; focal granular

ALK\EGFR\KRAS(−)

6

N

IV/ Socre4/Grade 2

3+/85 %

250

Fusion positive

CD74-E6; ROS-E34

Acinar

Cytoplasmic; granular

ALK\EGFR\KRAS(−)

7e

N

IV/−/−

2+/70 %

160

Fusion positive

Invasive adenocarcinoma with acinar pattern

Cytoplasmic; focal granular

ALK\EGFR\KRAS(−)

8

S

I A/Score 5/Grade 3

3+/90 %

250

Fusion positive

Papillary and micropapillary

Cytoplasmic; focal granular

ALK\EGFR\KRAS(−)

9

S

I B/Socre 3/Grade 2

2+/80 %

180

Fusion positive

Acinar and lepidic

Cytoplasmic; focal granular

ALK\EGFR\KRAS(−)

10

N

I B/Score 3/Grade 1

2+/80 %

180

Fusion positive

Lepidic

Cytoplasmic; focal granular

ALK\EGFR\KRAS(−)

a N, non-smoker; S, smoker; P, previous smoker

bTNM staging, SICA grading and WHO grading

cIHC results were containing intensity and extent scores

dALK rearrangement, EGFR and KRAS mutation have been also investigated at the sme time. The cases harboring ROS1 rearrangement were exclusive to ALK rearrangement, EGFR and KRAS mutation

ecases 7 was biopsy sample

Forty-two cases showed cytoplasmic reactivity by IHC. Nevertheless, only ten cases with diffuse 2–3+ tumor cytoplasmic reactivity were confirmed as ROS1 rearrangement when FISH was set as the standard method. The staining was distributed in more than 60 % tumor cells (Table 2; Fig. 1j & n). A setting of 2+ in intensity, 60 % in extent, and an H-score of 150 as the cutoff value represented the optimal IHC settings to reach the highest sensitivity and specificity on ROS1 rearrangement detection (Table 3) [19]. A concordance between FISH and IHC was found when IHC showed moderate to strong cytoplasmic reactivity (2–3+) with diffuse (≥60 %) distribution or H-score≥ 150. (P < 0.01, Kappa value > 0.6; Table 4).
Fig. 1

Comparison of IHC, FISH and qRT-PCR in ROS1 rearrangement and non-rearrangement cases (ad). The H&E staining, IHC, FISH and qRT-PCR results of a non-rearrangement case. a The case presented acinar and papillary patterns in H&E staining (200×); b IHC showed no staining in tumor cells (200×); c, d it was confirmed by FISH and qRT-PCR as non-rearrangement, respectively; eh A case with weak and focal ROS1 IHC staining. e The case presented acinar pattern in H&E staining (200×); f IHC showed weak to moderate focally staining in about 40 % tumor cells (200×); g, h it was also confirmed as non-rearrangement by FISH and qRT-PCR, respectively; il A case with diffusely moderate IHC staining. i The case presented papillary and micropapillary patterns in H&E staining (200×); j IHC showed moderate staining with cytoplasmic and focal granular patterns in about 80 % tumor cells (200×); k and l it was proved as ROS1-rearrangement by FISH and qRT-PCR, respectively; m-p A case with diffusely strong IHC staining. m The case presented papillary and micropapillary pattern in H&E staining (200×); n IHC showed diffusely strong staining with cytoplasmic, membrane and granular patterns in about 90 % tumor cells (200×); o, p it was also proved as ROS1 rearrangement by FISH and qRT-PCR

Table 3

H-score, intensity and extent of IHC

IHC

FISH+

FISH-

Sensitivity

Specificity

H-score

 ≥50

10

18

100 %

92.1 %

 ≥100

10

5

100 %

97.8 %

 ≥150

10

0

100 %

100.0 %

 ≥200

5

0

50.0 %

100.0 %

 ≥250

3

0

30.0 %

100.0 %

Intensity

 1+

10

32

100 %

85.7 %

 2+

10

1

100.0 %

99.6 %

 3+

3

0

30.0 %

100 %

Extent

 ≥10 %

10

29

100.0 %

87.28 %

 ≥20 %

10

25

100.0 %

89.04 %

 ≥30 %

10

20

100.0 %

91.23 %

 ≥40 %

10

15

100.0 %

93.42 %

 ≥50 %

10

14

100.0 %

93.86 %

 ≥60 %

10

9

100.0 %

96.05 %

 ≥70 %

9

9

90.0 %

96.05 %

 ≥80 %

7

4

70.0 %

98.25 %

 ≥90 %

4

1

40.0 %

99.56 %

Table 4

Comparison of FISH and IHC in ROS1 rearrangement detection

 

FISH ROS1 fusion positive

FISH ROS1 fusion negative

P.

Kappa Value

IHC ROS1 2-3+

10

1

< 0.01a

0.950

IHC ROS1-, 1+

0

227

  

IHC extent ≥ 60 %

10

9

< 0.01a

0.672

IHC extent < 60 %

0

219

  

IHC H-score ≥ 150

10

0

< 0.01a

1.000

IHC H-score < 150

0

228

  

aFisher exact test

Characteristics of the positive cases

Ten cases were identified as positive for ROS1 rearrangement. Most of the positive cases were female (Female: Male = 7:3) and non-smokers (7/10) with younger age (<61 year-old, 8/10). All the cases with ROS1 rearrangement were diagnosed as adenocarcinoma (Tables 1 and 2), and acinar pattern was the most predominant observed pattern. Eight cases could be performed histological grading, five of them got score 5 in Sica grading and 6 of them were classified as intermediate grade by WHO grading. Six of the rearrangement cases had been analyzed using direct sequencing (Table 2), revealing that CD74-E6 was the most common mutation type (3/6, 50 %). The images of direct sequencing are shown in an additional figure [see Additional file 2: Figure S1]. Most cases showed cytoplasmic and focal granular reactivity (7/10, 70 %; Table 2) [see Additional file 3: Figure S2, c & d]. No correlation was found between histopathology predominant patterns and IHC staining patterns (P = 0.645, Fisher exact test). All cases with ROS1 rearrangement were not carrying ALK, EGFR and KRAS gene aberrance.

The remaining 32 cases presented weak or focal IHC staining confirmed as ROS1 gene non-rearrangement or non-amplification by FISH. The staining patterns are shown in an additional figure [see Additional file 3: Figure S2]. All of them were diagnosed as adenocarcinoma. A case with an H-score of 90 and 2+ of intensity had been confirmed as ROS1 non-rearrangement by FISH. Its IHC staining was focal and represented approximately the 40 % of the tumor cells (Fig. 1f). Twenty-three of these cases underwent EGFR mutation detection, and 17 of them were confirmed as EGFR mutation (8 with exon 19 deletion and 9 with exon 21 L858R mutation). Thirty of them underwent ALK rearrangement detection, and two cases were confirmed as ALK rearrangement.

Information related to the follow-up studies

Three patients (case 3, 6 and 7) (Table 2) belonging to the ten ROS1 rearrangement cases at the stage IV of their disease, received the therapy of crizotinib, a TKI approved by the Food and Drug Administration (FDA). The information related to these three patients is shown in Table 5. Details of these patients can be found in a supplementary material [see Additional file 4: Figure S3 (a-c)].
Table 5

The details of follow-up studies

Case no.

Surgery

Histopathology

Chemotherapy

ROS1 detection

CT imagine

TKI start time

Response

Reexamination CT imagine

Side effects

Patient 3

Video-assisted thoracic surgery (VATS) of the left upper lobe wedge resection

Invasive mucinous adenocarcinoma with pleural invasion

Pemetrexed, carboplatin

IHC, FISH and qRT-PCR had proved he as ROS1 rearrangement. Fusion type: CD74-E6

The largest lesion in his left thoracic wall was approximate in size to 40.81 × 12.70 mm2

Crizotinib 250 mg bid from December 2014

February 2015, the lesion decreased to 26.66 × 11.69 mm2 in size

October 2015, the largest lesion shrunk to 10.85 × 8.60 mm2 in size

Tiredness and constipation

Patient 6

Lower right lobe radical resection; Biopsy under CT guidance

Invasive adenocarcinoma with acinar predominant pattern

Pematrexed, nadaplatin and bevacizumab

IHC, FISH and qRT-PCR had proved she as ROS1 rearrangement. Fusion type: CD74-E6

The largest lesion was approximated in size to 36.25 × 36.25 mm2 on the pleura

Crizotinib 250 mg bid from April 2014

May 2014, the largest lesion decreased to 11.02 × 8.59 mm2 in size

October 2015, the largest lesion shrunk to 10.48 × 10.33 mm2 in size

Edema in lower limbs, vomiting and tiredness

Patient 7

Biopsy under CT guidance

Invasive adenocarcinoma with acinar pattern

IHC and FISH had proved she as ROS1 rearrangement

The largest lesion was approximate in size to 35.33 × 19.73 mm2

Crizotinib 250 mg bid from July 2014

September 2014, the lesion decreased to 26.97 × 15.12 mm2 in size

November 2015, her largest lesion shrunk to 16.25 × 5.65 mm2 in size

Tiredness

Discussion

In the present study, three methods for ROS1 rearrangement detection have been compared. The results showed that D4D6 mAb IHC can be a reliable and feasible method for preliminary screening of ROS1 rearrangement in NSCLC, since it showed a high sensitivity and specificity. Nevertheless, the IHC cutoff value should be set at 2–3+ cytoplasmic reactivity with diffuse (≥60 % of the tumor cells) distribution or an H-score ≥150, which was similar to the conclusion of Yoshida’s study [14]. When we analyzed the pattern of IHC staining, we realized that the distribution of the cytoplasmic reactivity is one of the most important aspects in ROS1 IHC analysis. Indeed, a 2+ cytoplasmic reactivity intensity could be a false positive when the staining shows a focal distribution.

In order to verify and confirm the weak or focal cytoplasmic reactivity, FISH and qRT-PCR should be used as secondary confirmation. In our study, the concordant rate between qRT-PCR and FISH was 100 %, indicating that qRT-PCR could be a reliable detection method for ROS1 rearrangement. In addition, some studies have indicated that FISH cannot clearly reveal the rearrangements on the same chromosome, such as GOPC (FIG)-ROS1 and EZR-ROS1 [4, 13]. Therefore, qRT-PCR can be used as a second confirmatory test for revealing these rearrangements. In contrast, the cases that resulted negative after qRT-PCR analysis should be confirmed by FISH since the primers of qRT-PCR do not contain unknown fusion partners [12]. Finally, we designed a protocol for the detection of ROS1 rearrangement shown in Fig. 2 that encompassed these considerations.
Fig. 2

The routine protocol for ROS1 arrangements detection in NSCLC. IHC with D4D6 mAb can be preliminary screening tool for ROS1 rearrangement detection in NSCLC, and the IHC cutoff value should be set at 2–3+ cytoplasmic reactivity with diffuse (≥60 % of the tumor cells) distribution or an H-score ≥150; FISH and qRT-PCR should be used as the secondary confirmation in the cases with weak of focal IHC staining

The clinical characteristics and pathological pattern of patients with ROS1 rearrangements were also discussed in this study. Even though there were no significant difference between ROS1 rearrangement and non-rearrangement patients, the cases with ROS1 rearrangement were mostly females at a younger ages and non-smokers with adenocarcinoma, which was similar to the results of some previous studies [2022]. Most cases were classified as intermediate grade by WHO grading, however, five of them obtained score 5 in Sica grading, which indicated these cases containing high grade patterns as well. This result revealed that ROS1 rearrangement might become a prognosis biomarker of NSCLC. However, this finding should be confirmed in future study. Most ROS1 rearrangement cases presented cytoplasmic and focal granular staining pattern in the IHC staining, which was similar to the finding of some previous studies [14, 23]. However, the correlation between histopathology patterns and IHC staining patterns, as well as the correlation between fusion types and IHC reactivity patterns were not found due to the lack of ROS1 rearrangement cases. In addition, all the ROS1 rearrangement cases in our study were not carrying ALK rearrangement, as well as EGFR and KRAS mutations. Even though the overlapping phenomenon has been reported in some rare cases [8, 22], the result in our study indicated that general oncogene mutations not necessarily overlap in the same patient [4, 21].

Thirty-two cases with IHC weak or focal reactivity had been confirmed as ROS1 non-rearrangement. Although we had set the tissue with IHC strong reactivity as the positive control to avoid misunderstanding with the background staining, and chosen two areas with IHC reactivity in each FFPE block to decrease heterogeneity in establishing TMAs, these weak or focal staining might still be related to background staining or tissue heterogeneity. Nevertheless, among these cases, 17 were carrying EGFR mutation and 2 were harboring ALK rearrangement. To explain this phenomenon, Li et al. [24] investigated the expression of ROS1 mRNA in NSCLC, and found that the level of ROS1 mRNA increased either in ALK rearrangements or EGFR mutation specimens. However, the specific mechanism was unknown. We speculated that the weak or focal staining of ROS1 IHC may result from cross-talk mechanism of EGFR, ALK and ROS1 pathways, which is similar to the mechanism of EGFR mutation in NSCLC with MET proto-oncogene protein expression [25, 26]. However, EGFR mutation and ALK rearrangement detection was not possible on the remaining 9 and 2 cases, respectively. Thus, we cannot conclude that there was a correlation between weak or focal staining of ROS1 IHC and other genes aberrance.

After all the analyses, three patients with stage IV and ROS1 rearrangement underwent crizotinib therapy. All of them showed a partial response (PR) after 2 to 8 weeks, which was similar to some previous studies [8, 24]. In addition, even two patients had received chemotherapy before TKI therapy (patient 3 & 6), both of them had the same response to crizotinib as the other patient (patient 7), which indicated that crizotinib is also sensitive to the patients after chemotherapy. These three patients underwent crizotinib therapy for at least 11 months, reaching an average PR after 13.7 months. Since our patients were under a follow-up schedule, we could not estimate the progression-free survival (PFS). A large-scale study showed that the PFS of patients under crizotinib carrying ROS1 rearrangement was longer than the patients carrying ALK rearrangement undergoing the same therapy, suggesting the possible mechanism that imply crizotinib binding more tightly to ROS1 than to ALK [8].

Tiredness was the most common symptom during the therapy, and a patient (patient 6) also suffered from both lower limbs edema without cardiac dysfunction, which is one of the most common side effects observed in a previous study [8]. However, the correlation between TKI response and fusion partners had not been discussed because of the reduced ROS1 rearrangement cases to draw relevant conclusions. Because the FDA has recently approved crizotinib as a TKI to ROS1 rearrangement of NSCLC, we consider that the patients potentially harboring ROS1 rearrangement can be recruited by preliminary screening, therefore, more patients can receive TKI therapy, and the correlation between fusion types and TKI response or histopathology can be analyzed.

There are some limitations in our study. First of all, the qRT-PCR analysis was performed in only 159 cases. In order to analyze the concordance between qRT-PCR and FISH, all the recruited cases should be tested by qRT-PCR. However, some of the biopsied cases had insufficient amount of tissue to perform the qRT-PCR. Moreover, in order to clarify the relationship between fusion partners and TKIs response, and the correlation between fusion types and histopathology, more positive cases are needed. Finally, the relation between faint and weak staining of ROS1 IHC and other gene aberrance was not clear since not all faint and weak staining cases underwent EGFR mutation and ALK rearrangement detection. Future studies will include more patients carrying ROS1 arrangements (i.e. young, female, non-smokers with adenocarcinoma) to obtain more positive cases for the analysis.

Conclusions

In conclusion, IHC can be a reliable and effective method for ROS1 rearrangements preliminary screening in patients with NSCLC thanks to the high sensitivity and specificity of IHC using the D4D6 mAb, while cutoff should be set at diffusely (≥60 % tumor cells) 2–3+ cytoplasmic reactivity and an H-score ≥150. FISH and qRT-PCR can be used as confirmation analysis. Young, female, and non-smoking patients with adenocarcinoma and without other RTKs aberrance should undergo tests for ROS1 rearrangement because patients with these characteristics may carry ROS1 rearrangement. TKI therapy with crizotinib is effective in patients with ROS1 rearrangements at advanced stages.

Abbreviations

ADC, adenocarcinoma; ALK, anaplastic lymphoma receptor tyrosine kinase; ARMS-PCR, amplification refractory mutation system polymerase chain reaction; ASC, adenosquamous carcinoma; EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; H&E staining, hematoxylin and eosin staining; IHC, immunohistochemistry; KRAS, Kirsten rat sarcoma viral oncogene homolog; LCLC, Large-cell lung carcinoma; mAb, monoclonal antibody; MET, MET proto-oncogene, receptor tyrosine kinase; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction; ROS1, ROS proto-oncogene 1, receptor tyrosine kinase; RTKs, receptor tyrosine kinases; SCC, Squamous cell carcinoma; TKIs, tyrosine kinase inhibitors

Declarations

Acknowledgement

Part of the experimental work (direct sequencing) was supported by Amoy Diagnostics Co., Ltd (Xiamen, China). We sincerely thank all the patients for their participation in this study. We special thank the staffs in Department of Pathology, the First Affiliated Hospital of Guangzhou Medical University, including Xiaodong Lin, Wenting Guo, Yiyan Lin and Jianfei Liang for providing consultation on the experiments. We also want to thank the fellows in the First Affiliated Hospital of Guangzhou Medical University, including Jianrong Zhang, Zhiheng Xu, Qihua He and Yingzhi Wang, for providing suggestions in the writing of this manuscript.

Funding

None.

Availability of data and materials

The dataset supporting the conclusions of this article is available in the supplementary file [see Additional file 5: Public data]. The sequencing results of this study were compared using the Basic Local Alignment Search Tool (BLAST). The accession number of ROS1 is NC_000006.12.

Authors’ contributions

JW and XG have contributed equal to this work. The achievement of all authors were the following: study design by XG; cases selection by JW, XG and XF; experiment by JW, YL, XH; data analysis and manuscript drafting by JW; clinical follow-up study by HY; writing consultation by PH, XF and GL, and final approval to submit by XG. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

We had obtained the consents to publish from the participants to report individual patients’ data in any form (including images, videos, voice recordings etc.). Written informed consent for publication of their clinical details and clinical images was obtained from the patients and their family members. A copy of the consent form is available for review by the Editor of this journal.

Ethics approval and consent to participate

This study was approved by the Ethic Review Committee of the First Affiliated Hospital of Guangzhou Medical University. Consents to participate in the study from the participants were obtained.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Pathology, the First Affiliated Hospital of Guangzhou Medical University
(2)
Department of Cardiothoracic Surgery, the First Affiliated Hospital of Guangzhou Medical University

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Copyright

© The Author(s). 2016

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