Murakami Y et al. (2012), A Screening Method for the ALK Fusion Gene in N...

ECB-ART-50124

Front Oncol 2012 Mar 16;2:24. doi: 10.3389/fonc.2012.00024.

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A Screening Method for the ALK Fusion Gene in NSCLC.

Murakami Y , Mitsudomi T , Yatabe Y .

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Lung cancer research has recently made significant progress in understanding the molecular pathogenesis of lung cancer and in developing treatments for it. Such achievements are directly utilized in clinical practice. Indeed, the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (ALK) fusion gene was first described in non-small cell lung cancer in 2007, and a molecularly targeted drug against the fusion was approved in 2011. However, lung cancer with the ALK fusion constitutes only a small fraction of lung cancers; therefore, efficient patient selection is crucial for successful treatment using the ALK inhibitor. Currently, RT-PCR, fluorescent in situ hybridization (FISH), and immunohistochemistry are commonly used to detect the ALK fusion. Although FISH is currently the gold standard technique, there are no perfect methods for detecting these genetic alterations. In this article, we discuss the advantages and disadvantages of each method and the possible criteria for selecting patients who are more likely to have the ALK fusion. If we can successfully screen patients, then ALK inhibitor treatment will be the best example of personalized therapy in terms of selecting patients with an uncommon genotype from a larger group with the same tumor phenotype. In other words, the personalized therapy may offer a new challenge for current clinical oncology.

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	Figure 1. Concentration of EML4–ALK translocation using clinicopathologic features (n = 345). Numbers in black squares indicate number of patients with ALK translocation. Incidence of ALK translocation became 25% when patients were confined to those without EGFR, KRAS, HER2, p53 mutations and to female patients younger than 63. However, four patients were not included in this subset (Mitsudomi et al., 2010).
	Figure 2. Morphological characteristics of ALK-positive lung cancer. The cribriform pattern (A) is composed of fused acini or glands. The solid-growth pattern consists of tumor cell nests without structural architecture (B). Solid adenocarcinoma cells with the ALK fusion often have vesicular nuclei with convoluted contours and deep grooves. This subtype may be misdiagnosed as squamous cell carcinoma or mucoepidermoid carcinoma in some cases. Signet ring cell carcinoma (C) is also characteristic of ALK-positive lung cancer. Rodig et al. (2009) found that 71% of the adenocarcinomas that consisted of more than 10% signet ring cells harbored the ALK fusion. One cellular characteristic of ALK-positive lung cancer is mucin production. In extreme cases, the alveolar space is filled with mucin (D), although the lumens of the neoplastic glands seldom open to the alveolar spaces. Although the majority of mucinous tumors are negative for TTF-1, the ALK-positive tumors (i.e., the signet ring cell carcinomas) are an exception.
	Figure 3. Fluorescent in situ hybridization method for ALK fusion detection. The probes, which are labeled with different fluorescences, are designed at telomeric and centromeric sides between break point (A). Representative FISH images of ALK-negative (B) and positive tumors (C) are displayed. When ALK is translocated, the signals are changed from merged yellow to single red and green. The current criteria of the gene rearrangement and positive ALK FISH are as follows. Cells are considered rearrangement positive when: (i) At least one set of orange and green signals are two or more signal diameters apart. (ii) There is a single orange signal without a corresponding green signal in addition to fused and/or broken apart signals. The tumor is considered positive ALK test when: (i) A sample is considered negative if <5 cells out of 50 (<5/50 or <10%) are positive. (ii) A sample is considered positive if >25 cells out of 50 (>25/50 or >50%) are positive. (iii) A sample is considered equivocal, if 5–25 cells (10–50%) are positive. If the sample is equivocal, a second reader should evaluate the slide.
	Figure 4. A case with EML–ALK-positive adenosquamous cell carcinoma, showing negative FISH. The tumor was a well-circumscribed nodule (A), in which both components of adenocarcinoma (B) and squamous cell carcinoma (C) were histologically identified. The chimeric transcript was detected with RT-PCR products (D), in addition to positive ALK-IHC (E). However, FISH results did not fulfill the positive criteria (F).
	Figure 5. Discordant results according to antibody clones and detection system. A biopsy specimen only contains a small number of tumor cells [H&E staining, (A)]. When ALK1 antibody and conventional detection system was used, IHC showed negative results for tumor cells [(B), arrowheads]. However, proper clone (clone 5A4) and highly sensitive detection method (Envision FLEX+) made tumor cells show clearly positive reactions (C). Gene rearrangement and chimeric transcript of EML4–ALK fusion had been shown with FISH and RT-PCR in this specimen.
	Figure 6. A case of ALK-IHC positive small cell lung cancer. A lung nodule was biopsied from the 83-year-old female smoker. H&E section (A) shows typical morphology of small cell lung cancer and CD56/NCAM and synaptophysin were positive. Although it is little reports of SCLC with ALK fusion, the tumor cells were positive for ALK immunohistochemistry using clone 5A4 and Envision FLEX+ (B). No gene rearrangement and chimeric transcript of EML4–ALK have been detected, respectively.
	Figure 7. A pitfall of false negative immunohistochemistry in the mucinous component of adenocarcinoma, including signet ring cell carcinoma. Although the adenocarcinoma (A) was confirmed to harbor ALK fusion with both FISH and RT-PCR, immunohistochemistry using clone D5F3 and Envision FLEX+ showed various intensity of positive reactions (B). An upper part of the tumor cells (D) presented strong positivity, while the lower part showing signet ring cell carcinoma morphology appeared to be negative (C). Large amount of intracellular mucin pushes out cell cytoplasm outward to the rim. This false negative reaction does not indicate heterogeneity of ALK fusion, because split signals was demonstrated in both components.
	Figure 8. Diagnostic algorithm for ALK fusion from the Japanese Lung Cancer Society. The standard procedure for formalin-fixed paraffin embedded specimens involves screening with immunohistochemistry and confirmation with FISH. Even when the ALK immunohistochemistry is negative, FISH is recommended for the patients who are suspected of harboring the ALK fusion based on clinical considerations (age lower than 40 years, for example), and morphology (e.g., a mucinous cribriform pattern, signet ring cells, and TTF-1 positivity in mucinous adenocarcinomas). For pleural effusions and/or cytology samples, a positive RT-PCR can confirm the ALK fusion and directly facilitate crizotinib treatment. However, a negative RT-PCR does not have any clinical significance, and further examinations are required. Because each method has advantages and disadvantages, the use of more than one method to detect ALK fusion is recommended.

References [+] :

Inamura, EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. 2008, Pubmed, Echinobase