MOLECULAR CYTOPATHOLOGY Section Editor: Dara Aisner, M.D., and Anders Hjerpe, M.D.
ALK and ROS1 Rearrangements Tested by Fluorescence In Situ Hybridization in Cytological Smears From Advanced NonSmall Cell Lung Cancer Patients Cecilia Bozzetti,1* Rita Nizzoli,1 Marcello Tiseo,1 Anna Squadrilli,1 Costanza Lagrasta,2 Sebastiano Buti,1 Donatello Gasparro,1 Daniele Zanoni,1 Maria Majori,3 Massimo De Filippo,4 Francesca Mazzoni,5 Cristina Maddau,6 Nadia Naldi,1 Gabriella Sammarelli,7 Caterina Frati,8 Carmine Pinto,1 and Andrea Ardizzoni9
Background: The identification of ALK and ROS1 rearrangements
and the availability of an effective target therapy, such as crizotinib, represent a new option in the treatment of advanced non-small cell lung cancer (NSCLC) patients. In light of recent advances in non-invasive diagnostic procedures, we aimed to demonstrate that direct cytological smears are suitable for assessing ALK and ROS1 rearrangements in patients with NSCLC. Methods: Fifty-five patients with a cytological diagnosis of lung adenocarcinoma (ADC) were evaluated for ALK rearrangements by flu1
Medical Oncology Unit, University Hospital of Parma, Parma, Italy Department of Biomedical, Biotechnological and Translational Sciences, University Hospital of Parma, Parma, Italy 3 Pneumology Unit, University Hospital of Parma, Parma, Italy 4 Radiology Unit, University Hospital of Parma, Parma, Italy 5 Department of Medical Oncology, Careggi Hospital of Firenze, Firenze, Italy 6 Cytopathology Unit, Cancer Prevention and Research Institute, Firenze, Italy 7 Department of Clinical and Experimental Medicine, Haematology and Bone Marrow Transplantation Unit, University Hospital of Parma, Parma, Italy 8 Clinical and Experimental Medicine, University Hospital of Parma, Parma, Italy 9 Medical Oncology Unit, S.Orsola-Malpighi Hospital, Bologna, Italy *Correspondence to: Cecilia Bozzetti, Medical Oncology Unit, University Hospital of Parma, Via Gramsci 14, Parma 43126, Italy. E-mail: [email protected] Received 12 March 2015; Revised 6 May 2015; Accepted 22 June 2015 DOI: 10.1002/dc.23318 Published online 7 July 2015 in Wiley Online Library (wileyonlinelibrary.com). 2
C 2015 WILEY PERIODICALS, INC. V
orescence in situ hybridization (FISH) and 12 patients for ROS1 FISH rearrangements. Seventeen of the 55 cytological samples tested for ALK were obtained from the primary tumor and 38 from metastatic lesions. Ten of 12 samples evaluated for ROS1 were obtained from metastatic sites and two from the primary tumor. Results: ALK FISH was successful in 49/55 (89%) cytological ADC samples and ROS1 FISH in all 12 cytological samples. ALK rearrangements were found in 3/13 (23%) primary tumors and 7/36 (19%) metastatic sites. ROS1 rearrangements were found in one of the two primary tumors and in two of the 10 metastases. Two of the three rearranged cases were tested on cytology after knowing that they were rearranged on histology in order to increase representativeness of ROS1 rearranged cases in this study. Conclusion: Whenever cytology represents the only available material for diagnosis and biological characterization of NSCLC, minimally invasive procedures may provide an additional important source of cellular material for FISH assessment of ALK and ROS1 rearrangements.//// Diagn. Cytopathol. 2015;43:941–946. C 2015 Wiley Periodicals, Inc. V Key Words: non-small cell lung cancer; ALK; ROS1; cytology; FISH
Recently, preclinical and clinical data have demonstrated the efficacy of the kinase inhibitor crizotinib to treat a subset of non-small cell lung cancer (NSCLC) patients harbouring ALK or ROS1 gene rearrangements.1–12 Both ALK and ROS1 gene rearrangements, identified in 4–7% and 1-2% of all NSCLC patients respectively1–3,9–11 result in the formation of fusion proteins having constitutive Diagnostic Cytopathology, Vol. 43, No 11
941
Diagnostic Cytopathology DOI 10.1002/dc
BOZZETTI ET AL.
tyrosin-kinase (TK) activity that stimulates downstream signals promoting cell growth and proliferation.1,13 Although biological tumor features may identify subset of candidate patients for TK inhibitors, a significant limitation is that both molecular profiling and assessment of gene rearrangements by fluorescence in situ hybridization (FISH) require adequate histological sampling. Unfortunately, at diagnosis many NSCLCs present at an advanced stage when surgery is not recommended and a considerable fraction of them is diagnosed by cytology only and merely on metastatic sites. In patients where no other tumor material is available, cytology may represent the only source of cellular material for diagnosis and biological characterization. At the moment some studies refer to ALK and none to ROS1 rearrangements detection by FISH on cytology.14–19 This work evaluates ALK and ROS1 rearrangements by FISH on direct cytological smears obtained in routine clinical practice from primary and metastatic lesions of patients with lung adenocarcinoma (ADC).
Materials and Methods ALK rearrangements were tested on direct smears obtained from 55 patients (22 men and 33 women, median age 65 years, range 22–84, 21 never smokers) with a cytological diagnosis of lung ADC who consecutively underwent minimally invasive diagnostic procedure and screened for EGFR mutations previously. Seventeen of the 55 cytological samples were obtained from the primary tumor and 38 from metastatic sites (15 ilarmediastinal lymph nodes, 14 superficial lymph nodes, four pleural effusions, three liver metastases, one rib, and one thyroid metastasis). Eleven of the 17 primary tumors were sampled by transthoracic needle aspiration (TTNA), five by transbronchial needle aspiration (TBNA) and one by ultrasound-guided fine-needle aspiration biopsy (USFNAB). Among the 38 metastatic lesions, 20 were sampled by US-FNAB, 14 by TBNA, and four were pleural fluids. Forty one of the 55 samples tested for ALK FISH were fresh smears and 14 were May-Gr€unwaldGiemsa (May–Grunwald–Giemsa) destained smears. ROS1 rearrangements were evaluated in 10 metastatic lesions (six US-FNAB, three TBNA, one pleural fluid) and in two primary tumors (1 TTNA, 1 TBNA): 10 of the 12 lesions belonged to the series previously tested for ALK status on direct smear while 2/12 had ALK tested on transbronchial biopsy (TBB). These last two samples were selected for ROS1 FISH test on cytology after knowing that ROS1 was rearranged on TBB with the aim to enrich the population of ROS1 rearranged cases in this study. Eight of the 12 smears tested for ROS1 were fresh smears, two were May–Grunwald–Giemsa and two Papanicolaou (PAP) destained smears. After the aspiration procedure, the cellular material was smeared on glass slides and air dried. Rapid on-site 942
Diagnostic Cytopathology, Vol. 43, No 11
evaluation (ROSE) was applied to TBNA, TTNA, and US-FNAB procedures in order to obtain an adequate and representative sample. At this purpose one slide was stained with Diff-Quik stain for extemporary microscopic evaluation. Some slides were successively stained with May–Grunwald–Giemsa for cytological diagnosis and some were stored unstained at 2208C until FISH assessment. In our experience, on average 1–2 May–Grunwald– Giemsa stained slides were adequate for diagnosis for each patient and, in case of malignancy, in 80% of cases it was possible to keep unstained one to two representative slides. When all available slides were stained for diagnosis, FISH was performed on a destained slide. Cellular suspensions obtained from pleural fluids were cytocentrifuged and a mean of four slides with two spots each were obtained: two slides were stained for the cytological diagnosis and two were kept unstained. For FISH assay, unstained slides were fixed in methanol-acetic acid (3:1), air-dried, incubated in wash buffer (23 saline-sodium citrate buffer/0.3% NP-40, pH 7.0-7.5) at 378C for 30 minutes, dehydrated through gradients of 70%, 85%, and 100% ethanol and air dried. Archival cytological slides, stained with May–Grunwald– Giemsa, were treated with xylene to remove the coverslip and, after washing three times for 10 minutes each in Carnoy solution, slides were dehydrated gradually in ethanol, incubated in wash buffer at 378C for 30 minutes, and air dried. PAP stained smears were hydrated through a gradient of 100%, 95%, and 70% ethanol and destained using a distilled H2O/HCl solution. After pretreatment, FISH was performed using a break-apart probe to ALK (Vysis LSI ALK Dual Color, Break-Apart Rearrangement Probe; Abbott Molecular, Abbott Park, IL) and a breakapart probe to ROS1 (6q22) (Kreatech, Amsterdam, The Netherlands). Samples were denaturated at 678C for 5 minutes and hybridized overnight at 378C in a HY-Brite denaturation/hybridization system for FISH (Vysis). After incubation in wash buffer at 738C for 2 minutes, slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Slides were viewed at a magnification of 31000 and images processed utilizing a Nikon Ni-U fluorescence microscope (Nikon Corporation, Tokyo, Japan) equipped with a 100-W mercury lamp. Separate narrow band pass filters for the detection of spectrum orange, spectrum red, spectrum green and DAPI were used. At least 50 valuable tumor cells for each case were scored visually by two independent observers in blind. In case of lack of hybridization, slides were decoverslipped and submitted to an additional pretreatment with protease K (0.5 mg/ml) at 378C for 5 minutes. ALK negative cells were defined as having adjacent or fused orange and green signals. Samples were classified as rearranged when 15% of tumor cells showed split signals, two or more signal diameters apart, or isolated 3’orange (ALK) or red (ROS1) signals.20
Diagnostic Cytopathology DOI 10.1002/dc
ALK AND ROS1 REARRANGEMENTS ON CYTOLOGY
Polysomy was defined as presence of 3 adjacent or fused orange/red and green signals per nucleus. This study was conducted according to a protocol approved by the institutional review board/independent ethics committee, and informed consent was obtained from all patients for the use of tissue samples and the analysis of clinical information.
Results ALK FISH was successful in 89% (49/55) of tested cases: 5/14 (36%) destained smears were not valuable because of lack of hybridization or difficulties in signal visualization and one fresh smear because of an excess of necrosis. In destained smears, a high hematic component often created a green background fluorescence that interfered with signals visualization. In fresh smears, this interference could be avoided with fixation in a solution of methanol-acetic acid. Table I. ALK and ROS1 by FISH on Direct Smears From Primary and Metastatic Lesions of Lung Adenocarcinoma ALK (n 5 55)
ROS1 (n 5 12)
Primary tumor (n 5 17)
Metastatic site (n 5 38)
Primary tumor (n 5 2)
Metastatic site (n 5 10)
13 3 1 9
36 7 5 24
2 1 1 0
10 2 5 3
Adequate Rearranged Diploid Increase copy number (3)
ALK rearrangement was found in three of the 13 (23%) primary tumors and seven of the 36 (19%) metastatic sites (Table I). Among primary tumors, ALK rearrangement was observed in a rate of nuclei ranging from 16% to 21% and among metastatic sites from 16% to 62%. An increased number of fusion signals, due to polysomy of chromosome 2, was a frequent event in almost all rearranged nuclei. Chromosome 2 polysomy was also observed in 9/10 (90%) ALK negative primary tumors and in 24/29 (83%) ALK negative metastases, almost all cases showing a heterogeneous signal pattern distribution. Patients’ and tumor characteristics of ALK positive cases are reported in Table II. An example of a May–Grunwald–Giemsa stained smear and corresponding ALK FISH is shown in Figures 1a and b respectively. Computed tomography (CT) scans that evidence objective response to crizotinib of this case (#1 of ALK series in Table II) are shown in Figures 1c and d. The site of sampling and FISH status of the 12 cytological samples tested for ROS1 are shown in Table I. All 12 samples (eight fresh smears and four destained smears) were valuable by FISH. Split signals, indicating a rearrangement of the ROS1 gene locus, were evident in three of the 12 cases. Patients’ and tumor characteristics of the three ROS1 positive cases are reported in Table II. An example of a May–Grunwald–Giemsa stained cytological sample and corresponding ROS1 FISH assay is shown in Figures 2a and b respectively. CT scans that evidence objective response to crizotinib of this case (#1 of ROS1 series of Table II) are shown in Figures 2c and d.
Table II. Baseline Clinical Characterization of aADC Patients Showing ALK or ROS1 Rearrangements and Summary of Responses to Crizotinib Age
Sex
Smoking status
Hystotype
ALK (n 5 10) 1 22 2 77 3 72 4 68 5 66 6 63 7 84 8 65 9 52 10 62
F F F M F F F F F F
Never smoked Current smoker Former smoker Never smoked Never smoked Current smoker Never smoked Never smoked Current smoker Never smoked
ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC
Superficial lymph Ilar mediastinal Superficial lymph Pleuric effusion Superficial lymph Ilar mediastinal Lung Superficial lymph Lung Lung
ROS1 (n 5 3) 1 75 2 42 3 58
F F F
Never smoked Never smoked Never smoked
ADC ADC ADC
Axillary lymph node Lung Lung
Site sampling nodes nodes nodes nodes
b FISH (% rearrangement)
Crizotinib
60 62 16 22 50 30 16 40 20 21
yes yes e not yet yes e not yet no f ongoing no f ongoing e not yet
68 60 20
yes yes f ongoing
Type of response c d d
PR SD SD
c
PR PR
c
a
ADC: adenocarcinoma. FISH: fluorescence in situ hybridization. c PR: partial response. d SD: stable disease. e Not yet: patients are ongoing of first-line chemotherapy treatment;. f Ongoing: patients are ongoing of crizotinib treatment from
ALK and ROS1 Rearrangements Tested by Fluorescence In Situ Hybridization in Cytological Smears From Advanced NonSmall Cell Lung Cancer Patients Cecilia Bozzetti,1* Rita Nizzoli,1 Marcello Tiseo,1 Anna Squadrilli,1 Costanza Lagrasta,2 Sebastiano Buti,1 Donatello Gasparro,1 Daniele Zanoni,1 Maria Majori,3 Massimo De Filippo,4 Francesca Mazzoni,5 Cristina Maddau,6 Nadia Naldi,1 Gabriella Sammarelli,7 Caterina Frati,8 Carmine Pinto,1 and Andrea Ardizzoni9
Background: The identification of ALK and ROS1 rearrangements
and the availability of an effective target therapy, such as crizotinib, represent a new option in the treatment of advanced non-small cell lung cancer (NSCLC) patients. In light of recent advances in non-invasive diagnostic procedures, we aimed to demonstrate that direct cytological smears are suitable for assessing ALK and ROS1 rearrangements in patients with NSCLC. Methods: Fifty-five patients with a cytological diagnosis of lung adenocarcinoma (ADC) were evaluated for ALK rearrangements by flu1
Medical Oncology Unit, University Hospital of Parma, Parma, Italy Department of Biomedical, Biotechnological and Translational Sciences, University Hospital of Parma, Parma, Italy 3 Pneumology Unit, University Hospital of Parma, Parma, Italy 4 Radiology Unit, University Hospital of Parma, Parma, Italy 5 Department of Medical Oncology, Careggi Hospital of Firenze, Firenze, Italy 6 Cytopathology Unit, Cancer Prevention and Research Institute, Firenze, Italy 7 Department of Clinical and Experimental Medicine, Haematology and Bone Marrow Transplantation Unit, University Hospital of Parma, Parma, Italy 8 Clinical and Experimental Medicine, University Hospital of Parma, Parma, Italy 9 Medical Oncology Unit, S.Orsola-Malpighi Hospital, Bologna, Italy *Correspondence to: Cecilia Bozzetti, Medical Oncology Unit, University Hospital of Parma, Via Gramsci 14, Parma 43126, Italy. E-mail: [email protected] Received 12 March 2015; Revised 6 May 2015; Accepted 22 June 2015 DOI: 10.1002/dc.23318 Published online 7 July 2015 in Wiley Online Library (wileyonlinelibrary.com). 2
C 2015 WILEY PERIODICALS, INC. V
orescence in situ hybridization (FISH) and 12 patients for ROS1 FISH rearrangements. Seventeen of the 55 cytological samples tested for ALK were obtained from the primary tumor and 38 from metastatic lesions. Ten of 12 samples evaluated for ROS1 were obtained from metastatic sites and two from the primary tumor. Results: ALK FISH was successful in 49/55 (89%) cytological ADC samples and ROS1 FISH in all 12 cytological samples. ALK rearrangements were found in 3/13 (23%) primary tumors and 7/36 (19%) metastatic sites. ROS1 rearrangements were found in one of the two primary tumors and in two of the 10 metastases. Two of the three rearranged cases were tested on cytology after knowing that they were rearranged on histology in order to increase representativeness of ROS1 rearranged cases in this study. Conclusion: Whenever cytology represents the only available material for diagnosis and biological characterization of NSCLC, minimally invasive procedures may provide an additional important source of cellular material for FISH assessment of ALK and ROS1 rearrangements.//// Diagn. Cytopathol. 2015;43:941–946. C 2015 Wiley Periodicals, Inc. V Key Words: non-small cell lung cancer; ALK; ROS1; cytology; FISH
Recently, preclinical and clinical data have demonstrated the efficacy of the kinase inhibitor crizotinib to treat a subset of non-small cell lung cancer (NSCLC) patients harbouring ALK or ROS1 gene rearrangements.1–12 Both ALK and ROS1 gene rearrangements, identified in 4–7% and 1-2% of all NSCLC patients respectively1–3,9–11 result in the formation of fusion proteins having constitutive Diagnostic Cytopathology, Vol. 43, No 11
941
Diagnostic Cytopathology DOI 10.1002/dc
BOZZETTI ET AL.
tyrosin-kinase (TK) activity that stimulates downstream signals promoting cell growth and proliferation.1,13 Although biological tumor features may identify subset of candidate patients for TK inhibitors, a significant limitation is that both molecular profiling and assessment of gene rearrangements by fluorescence in situ hybridization (FISH) require adequate histological sampling. Unfortunately, at diagnosis many NSCLCs present at an advanced stage when surgery is not recommended and a considerable fraction of them is diagnosed by cytology only and merely on metastatic sites. In patients where no other tumor material is available, cytology may represent the only source of cellular material for diagnosis and biological characterization. At the moment some studies refer to ALK and none to ROS1 rearrangements detection by FISH on cytology.14–19 This work evaluates ALK and ROS1 rearrangements by FISH on direct cytological smears obtained in routine clinical practice from primary and metastatic lesions of patients with lung adenocarcinoma (ADC).
Materials and Methods ALK rearrangements were tested on direct smears obtained from 55 patients (22 men and 33 women, median age 65 years, range 22–84, 21 never smokers) with a cytological diagnosis of lung ADC who consecutively underwent minimally invasive diagnostic procedure and screened for EGFR mutations previously. Seventeen of the 55 cytological samples were obtained from the primary tumor and 38 from metastatic sites (15 ilarmediastinal lymph nodes, 14 superficial lymph nodes, four pleural effusions, three liver metastases, one rib, and one thyroid metastasis). Eleven of the 17 primary tumors were sampled by transthoracic needle aspiration (TTNA), five by transbronchial needle aspiration (TBNA) and one by ultrasound-guided fine-needle aspiration biopsy (USFNAB). Among the 38 metastatic lesions, 20 were sampled by US-FNAB, 14 by TBNA, and four were pleural fluids. Forty one of the 55 samples tested for ALK FISH were fresh smears and 14 were May-Gr€unwaldGiemsa (May–Grunwald–Giemsa) destained smears. ROS1 rearrangements were evaluated in 10 metastatic lesions (six US-FNAB, three TBNA, one pleural fluid) and in two primary tumors (1 TTNA, 1 TBNA): 10 of the 12 lesions belonged to the series previously tested for ALK status on direct smear while 2/12 had ALK tested on transbronchial biopsy (TBB). These last two samples were selected for ROS1 FISH test on cytology after knowing that ROS1 was rearranged on TBB with the aim to enrich the population of ROS1 rearranged cases in this study. Eight of the 12 smears tested for ROS1 were fresh smears, two were May–Grunwald–Giemsa and two Papanicolaou (PAP) destained smears. After the aspiration procedure, the cellular material was smeared on glass slides and air dried. Rapid on-site 942
Diagnostic Cytopathology, Vol. 43, No 11
evaluation (ROSE) was applied to TBNA, TTNA, and US-FNAB procedures in order to obtain an adequate and representative sample. At this purpose one slide was stained with Diff-Quik stain for extemporary microscopic evaluation. Some slides were successively stained with May–Grunwald–Giemsa for cytological diagnosis and some were stored unstained at 2208C until FISH assessment. In our experience, on average 1–2 May–Grunwald– Giemsa stained slides were adequate for diagnosis for each patient and, in case of malignancy, in 80% of cases it was possible to keep unstained one to two representative slides. When all available slides were stained for diagnosis, FISH was performed on a destained slide. Cellular suspensions obtained from pleural fluids were cytocentrifuged and a mean of four slides with two spots each were obtained: two slides were stained for the cytological diagnosis and two were kept unstained. For FISH assay, unstained slides were fixed in methanol-acetic acid (3:1), air-dried, incubated in wash buffer (23 saline-sodium citrate buffer/0.3% NP-40, pH 7.0-7.5) at 378C for 30 minutes, dehydrated through gradients of 70%, 85%, and 100% ethanol and air dried. Archival cytological slides, stained with May–Grunwald– Giemsa, were treated with xylene to remove the coverslip and, after washing three times for 10 minutes each in Carnoy solution, slides were dehydrated gradually in ethanol, incubated in wash buffer at 378C for 30 minutes, and air dried. PAP stained smears were hydrated through a gradient of 100%, 95%, and 70% ethanol and destained using a distilled H2O/HCl solution. After pretreatment, FISH was performed using a break-apart probe to ALK (Vysis LSI ALK Dual Color, Break-Apart Rearrangement Probe; Abbott Molecular, Abbott Park, IL) and a breakapart probe to ROS1 (6q22) (Kreatech, Amsterdam, The Netherlands). Samples were denaturated at 678C for 5 minutes and hybridized overnight at 378C in a HY-Brite denaturation/hybridization system for FISH (Vysis). After incubation in wash buffer at 738C for 2 minutes, slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Slides were viewed at a magnification of 31000 and images processed utilizing a Nikon Ni-U fluorescence microscope (Nikon Corporation, Tokyo, Japan) equipped with a 100-W mercury lamp. Separate narrow band pass filters for the detection of spectrum orange, spectrum red, spectrum green and DAPI were used. At least 50 valuable tumor cells for each case were scored visually by two independent observers in blind. In case of lack of hybridization, slides were decoverslipped and submitted to an additional pretreatment with protease K (0.5 mg/ml) at 378C for 5 minutes. ALK negative cells were defined as having adjacent or fused orange and green signals. Samples were classified as rearranged when 15% of tumor cells showed split signals, two or more signal diameters apart, or isolated 3’orange (ALK) or red (ROS1) signals.20
Diagnostic Cytopathology DOI 10.1002/dc
ALK AND ROS1 REARRANGEMENTS ON CYTOLOGY
Polysomy was defined as presence of 3 adjacent or fused orange/red and green signals per nucleus. This study was conducted according to a protocol approved by the institutional review board/independent ethics committee, and informed consent was obtained from all patients for the use of tissue samples and the analysis of clinical information.
Results ALK FISH was successful in 89% (49/55) of tested cases: 5/14 (36%) destained smears were not valuable because of lack of hybridization or difficulties in signal visualization and one fresh smear because of an excess of necrosis. In destained smears, a high hematic component often created a green background fluorescence that interfered with signals visualization. In fresh smears, this interference could be avoided with fixation in a solution of methanol-acetic acid. Table I. ALK and ROS1 by FISH on Direct Smears From Primary and Metastatic Lesions of Lung Adenocarcinoma ALK (n 5 55)
ROS1 (n 5 12)
Primary tumor (n 5 17)
Metastatic site (n 5 38)
Primary tumor (n 5 2)
Metastatic site (n 5 10)
13 3 1 9
36 7 5 24
2 1 1 0
10 2 5 3
Adequate Rearranged Diploid Increase copy number (3)
ALK rearrangement was found in three of the 13 (23%) primary tumors and seven of the 36 (19%) metastatic sites (Table I). Among primary tumors, ALK rearrangement was observed in a rate of nuclei ranging from 16% to 21% and among metastatic sites from 16% to 62%. An increased number of fusion signals, due to polysomy of chromosome 2, was a frequent event in almost all rearranged nuclei. Chromosome 2 polysomy was also observed in 9/10 (90%) ALK negative primary tumors and in 24/29 (83%) ALK negative metastases, almost all cases showing a heterogeneous signal pattern distribution. Patients’ and tumor characteristics of ALK positive cases are reported in Table II. An example of a May–Grunwald–Giemsa stained smear and corresponding ALK FISH is shown in Figures 1a and b respectively. Computed tomography (CT) scans that evidence objective response to crizotinib of this case (#1 of ALK series in Table II) are shown in Figures 1c and d. The site of sampling and FISH status of the 12 cytological samples tested for ROS1 are shown in Table I. All 12 samples (eight fresh smears and four destained smears) were valuable by FISH. Split signals, indicating a rearrangement of the ROS1 gene locus, were evident in three of the 12 cases. Patients’ and tumor characteristics of the three ROS1 positive cases are reported in Table II. An example of a May–Grunwald–Giemsa stained cytological sample and corresponding ROS1 FISH assay is shown in Figures 2a and b respectively. CT scans that evidence objective response to crizotinib of this case (#1 of ROS1 series of Table II) are shown in Figures 2c and d.
Table II. Baseline Clinical Characterization of aADC Patients Showing ALK or ROS1 Rearrangements and Summary of Responses to Crizotinib Age
Sex
Smoking status
Hystotype
ALK (n 5 10) 1 22 2 77 3 72 4 68 5 66 6 63 7 84 8 65 9 52 10 62
F F F M F F F F F F
Never smoked Current smoker Former smoker Never smoked Never smoked Current smoker Never smoked Never smoked Current smoker Never smoked
ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC
Superficial lymph Ilar mediastinal Superficial lymph Pleuric effusion Superficial lymph Ilar mediastinal Lung Superficial lymph Lung Lung
ROS1 (n 5 3) 1 75 2 42 3 58
F F F
Never smoked Never smoked Never smoked
ADC ADC ADC
Axillary lymph node Lung Lung
Site sampling nodes nodes nodes nodes
b FISH (% rearrangement)
Crizotinib
60 62 16 22 50 30 16 40 20 21
yes yes e not yet yes e not yet no f ongoing no f ongoing e not yet
68 60 20
yes yes f ongoing
Type of response c d d
PR SD SD
c
PR PR
c
a
ADC: adenocarcinoma. FISH: fluorescence in situ hybridization. c PR: partial response. d SD: stable disease. e Not yet: patients are ongoing of first-line chemotherapy treatment;. f Ongoing: patients are ongoing of crizotinib treatment from
