Anaplastic lymphoma kinase gene rearrangement in non-small-cell lung cancer in a Brazilian population.

INTRODUCTION

Lung cancer is the leading cause of cancer deaths worldwide (1). Non-small-cell lung carcinoma (NSCLC), which represents approximately 80% of all lung cancer cases, is frequently diagnosed at advanced stages of the disease and is associated with short survival time. Although the prognosis of this disease remains poor, significant advances in the genetics and treatment of NSCLC have been made in recent years.

In 2007, Soda et al. (2) identified the transforming EML4-ALK fusion gene in 6.7% of NSCLC patients. This fusion transcript, which presents potent oncogenic activity both in vitro and in vivo (2,3), is formed by the translocation of the echinoderm microtubule-associated protein-like 4 (EML4) gene (2p21) and anaplastic lymphoma kinase (ALK) gene (2p23). Subsequent studies have shown that between two and eight percent of all NSCLC cases may harbor ALK rearrangements, which are almost always mutually exclusive with EGFR and KRAS mutations. Moreover, these patients are frequently younger, non- or light smokers and have adenocarcinomas (2),. Recently, crizotinib, an orally available small-molecule inhibitor of the ALK tyrosine kinase, was approved for the treatment of ALK-rearranged NSCLC patients, with successful results (24-29). The two most widely used methods for the diagnosis of ALK-rearranged NSCLC are fluorescence in situ hybridization (FISH) and immunohistochemistry.

Because there is no information about the prevalence and clinical characteristics of ALK-rearranged NSCLC in Latin America in the literature, the aim of this study was to evaluate NSCLC with ALK rearrangement in Brazilian patients.

MATERIALS AND METHODS

Case Selection

This study included 62 consecutive NSCLC cases received from August 2010 to September 2011 for ALK rearrangement testing using FISH (30) by Consultoria em Patologia, a large Anatomic Pathology and Molecular Pathology reference laboratory located in Botucatu, São Paulo State, Brazil. Tissues from small biopsies and lung resection specimens were used. The ages and genders of the patients were registered.

Fluorescence in situ hybridization (FISH)

FISH was performed on formalin-fixed, paraffin-embedded tumor samples using a probe specific to the ALK locus (Vysis LSI ALK dual color, break apart rearrangement probe; Abbott Molecular, Des Plaines, IL, USA) according to the manufacturer's instructions. The FISH results were analyzed under a fluorescence microscope (Zeiss Axio Imager M1, Carl Zeiss AG, Oberkochen, Germany) with the appropriate filters (Chroma Technology GmbH, Fuerstenfeldbruck, Germany) and Metafer 4 software (MetaSystems, Altlussheim, Germany). FISH-positive cases were defined as those in which more than 15% of the cells (at least 40 neoplastic cells were counted) presented with split orange and green signals or an isolated orange signal, as previously described (31,32).

Immunohistochemistry

For comparison with FISH results, ALK protein expression was determined by immunohistochemistry using the mouse monoclonal antibody 5A4 (code ab17127, ABCAM, Cambridge, MA, USA), which was found to show high concordance with ALK gene status, as determined by molecular tests (13,14,23),. Briefly, 3-µm-thick unstained histologic sections of lung carcinoma tissue were used in all cases. The sections were deparaffinized in xylene and rehydrated in a graded series of alcohol in phosphate-buffered saline. The sections then were submitted to antigen retrieval in a microwave. Subsequently, slides were incubated overnight with the 5A4 antibody at a pre-standardized concentration (1:200), washed with phosphate-buffered saline, and incubated with Novocastra Novolink Polymer Detection System (Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK). Diaminobenzidine was used as the chromogen, and sections were counterstained with hematoxylin and coverslipped. A case was considered positive for ALK if ≥10% of the tumor cells showed intense cytoplasmic immunostaining.

RESULTS

Of the 62 NSCLC cases included in this study, two (3.2%) showed ALK rearrangement by FISH: one case presented an isolated orange signal along with one fusion signal in tumor cells, and the other showed split orange and green signals along with one fusion signal. These two FISH-positive cases were also positive for ALK by immunohistochemistry, with strong cytoplasmic immunostaining in virtually all tumor cells. All of the other sixty FISH-negative cases were also negative for ALK using immunohistochemistry.

Both of the patients with FISH- and ALK immunohistochemistry-positive NSCLC were men (36 years old and 45 years old; mean: 40 years). One of them had no history of tobacco smoking, and the other was a light smoker who had stopped smoking some months prior to the diagnosis. The group of patients (n = 60) presenting ALK-negative tumors by FISH varied in age from 32 to 87 years (mean: 58 years; median: 59 years), and most of these patients were male (33 patients).

According to the new international multidisciplinary classification of lung adenocarcinomas proposed by the International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society (37), tumors from the two patients with ALK-positive FISH were classified as invasive adenocarcinoma, acinar predominant. One tumor had papillary areas, and the other was rich in mucinous areas. The 36-year-old patient with invasive adenocarcinoma, acinar predominant with papillary areas, was also tested for EGFR and KRAS mutations; the tumor was negative for both mutations. Figure 1 presents the morphologic characteristics of the two ALK-positive cases, including FISH and immunohistochemical findings.

Figure 1

ALK-rearranged NSCLCs. A. Invasive adenocarcinoma, acinar predominant with papillary areas, in a 36-year-old male patient (case 1). B. FISH of case 1 showing one isolated orange signal along with one fusion signal (positive for ALK rearrangement). C. ALK expression in case 1: diffuse and intense cytoplasmic immunostaining. D. Invasive adenocarcinoma, acinar predominant with mucinous areas, in a 45-year-old male patient (case 2). E. FISH of case 2 showing split orange and green signals along with one fusion signal (positive for ALK rearrangement). F. ALK expression in case 2: intense cytoplasmic immunostaining in virtually all tumor cells.

DISCUSSION

Clinical characteristics associated with ALK rearrangement in NSCLC include adenocarcinoma histology, younger age at diagnosis, and non- or light smoker status, similar to those described for EGFR mutations. The prevalence of NSCLCs with ALK fusion varies from 2% to 8% in the literature, and most studies have been conducted in Asian countries or in the United States of America. Table 1 presents the frequency of ALK-rearranged NSCLC in several countries. In Japan, the prevalence of lung cancer with ALK rearrangement varied widely, from 2.4% to 6.7% (2,16,19). In Korea, the frequency of ALK-positive NSCLC ranged from 3.7% to 4.2% (11,13,14). In China, it was approximately 5% (4,12,15). In the USA, the prevalence varied from 5.6 to 8.2% (17,23). These findings show that ethnicity likely does not strongly influence the prevalence of ALK-rearranged lung carcinomas, which is in contrast to EGFR- and KRAS-mutated tumors (38-41).

Table 1

Prevalence of ALK-rearranged NSCLC in different countries.

Authors, year of publication	Country	ALK-rearranged tumors
Soda et al., 2007 (2)	Japan	6.7% (n = 75)
Rodig et al., 2009 (17)	USA	5.6% (n = 358)
Wong et al., 2009 (4)	China	4.9% (n = 266)
Sakairi et al., 2010 (19)	Japan	6.4% (n = 109)
Takahashi et al., 2010 (16)	Japan	2.4% (n = 211)
Sun et al., 2010 (15)	China	5.7% (n = 52)
Paik et al., 2011 (11)	Korea	3.8% (n = 735)
Li et al., 2011 (12)	China	5% (n = 202)
Kim et al., 2011 (13)	Korea	3.7% (n = 449)
Paik et al., 2011 (14)	Korea	4.2% (n = 453)
Yang et al., 2012 (23)	USA	8.2% (n = 300)
Lopes et al. (present study)	Brazil	3.2% (n = 62)

To the best of our knowledge, however, no information about the prevalence and clinical characteristics of these tumors in Latin America, including Brazil, is available in the literature.

In the present study, 3.2% (2 out of 62) of NSCLC cases in Brazilian patients were ALK fusion positive by FISH. These two male patients were younger than those with ALK-negative FISH, had a history of no or light smoking, and their tumors presented invasive adenocarcinoma histology similar to that described in the literature. Both tumors were histologically classified as acinar predominant: one tumor had papillary areas, and the other was rich in mucinous areas.

Consistent with the results of other studies (13,14,23), a high concordance between FISH and immunohistochemistry results for the detection of ALK-positive NSCLCs was found in the present study. Although FISH remains the gold standard technique for the diagnosis of ALK-rearranged lung carcinoma, immunohistochemistry may play an important role in the determination of ALK status in NSCLC in the near future. Because data regarding the role of immunohistochemistry in the detection of ALK-positive lung cancer are limited in the literature, additional studies are necessary.

The use of crizotinib has changed the history of NSCLC treatment, with dramatic results in patients with ALK-rearranged tumors. A recent phase I/II clinical trial showed that 47 of 82 (57%) patients with ALK fusion-positive lung tumors confirmed by FISH had objective responses to crizotinib, and an additional 27 (33%) patients displayed stable disease (9). These data highlight that the diagnosis of ALK-rearranged NSCLC is crucial.

As previously stated, the present study is the first in Latin America to present clinicopathologic information regarding ALK-rearranged NSCLC in patients from this geographic area. Further studies are encouraged to expand our knowledge on ALK-positive NSCLC in Latin America and Brazil.