A Prompt Diagnosis of Ascites and Dramatic Effect of Alectinib for Advanced Lung Adenocarcinoma Harboring EML4-ALK Fusion

Abstract

A 75-year-old never-smoker woman presented with dyspnea and loss of appetite. A mass was identified in the left upper lobe of the lung, and the patient was referred to our hospital. Despite the diagnosis of lung adenocarcinoma via bronchoscopy, anaplastic lymphoma kinase (ALK) immunostaining was negative. Rapid weight gain and abdominal distension caused by ascites prompted fluid testing using the AmoyDx^Ⓡ Pan Lung Cancer PCR Panel. EML4-ALK fusion was confirmed, and alectinib therapy was initiated immediately. The tumor size had decreased significantly, and the patient was discharged on day 34. This case highlights the necessity of multiplex genetic testing even when ALK immunostaining is negative.

Introduction

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene was discovered in 2007 as a driver gene mutation present in approximately 3-5% of lung adenocarcinomas (1). Various ALK inhibitors have been reported to be effective against ALK fusion gene-positive lung cancers. Among these, the second-generation ALK-tyrosine kinase inhibitor (TKI) alectinib has shown a high response rate of 96.2% and is often used as a first-line treatment because of its minimal adverse effects (2,3). Therefore, it is crucial to appropriately test for gene mutations and administer TKI therapy to patients with driver mutations. Studies have indicated that if TKI therapy is not administered to mutation-positive cases, the prognosis is similar to that of mutation-negative cases (4). Furthermore, ALK-positive lung cancer generally progresses rapidly, necessitating a prompt and accurate diagnosis. The commonly used EML4-ALK fusion gene detection methods include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and next-generation sequencing (NGS). Although IHC is the most commonly used method, its sensitivity is not as high as that of RT-PCR or NGS (5).

We herein report a case in which ALK positivity was detected by genetic testing of ascites fluid using the AmoyDx^Ⓡ Pan Lung Cancer PCR Panel (AmoyDx^Ⓡ Panel), leading to the rapid initiation of alectinib treatment, which achieved a dramatic response.

Case Report

A 75-year-old woman presented with worsening dyspnea and loss of appetite over the course of one month. She had no history of smoking but had several comorbidities, including diabetes mellitus, hypertension, and dyslipidemia, along with a history of left breast cancer surgery. The Eastern Cooperative Oncology Group (ECOG) performance status (PS) was 1. Chest computed tomography (CT) revealed a mass shadow in the left upper lobe of the lung with no pleural or peritoneal effusion (Fig. 1). [¹⁸F] fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT demonstrated contralateral mediastinal lymph node metastasis, multiple bone metastases, and peritoneal dissemination, leading to the diagnosis of cT2bN3M1c, cStage IVB lung cancer (Fig. 1).

Figure 1.

Imaging studies at the time of the initial examination. (a) Chest X-ray, (b, c) contrast-enhanced CT, and (d-f) ¹⁸FDG-PET/CT. CT: computed tomography, ¹⁸F-FDG PET: fluorodeoxyglucose-positron emission tomography

One week after the initial consultation, the patient was admitted for bronchoscopy, which resulted in the pathological diagnosis of lung adenocarcinoma (Fig. 2a, B). ALK immunostaining was performed in-house with an anti-ALK antibody (Ventana ALK D5F3) using a fully automated immunostaining system and tested negative by a pathologist (Fig. 2c). Blood investigations on admission revealed marked anemia and hypoalbuminemia (Table 1).

Figure 2.

Pathological findings from a transbronchial tumor biopsy and ascitic fluid cytology samples. (a, b) Hematoxylin and Eosin staining of the transbronchial tumor biopsy specimen. Black circle indicates the tumor cells. (c) ALK-IHC staining of transbronchial tumor biopsy specimens. (d) Papanicolaou stain of the ascitic fluid before the purifying procedure. The red circle indicates the tumor cells. (e) Papanicolaou staining of the ascitic fluid after the purifying procedure.

Table 1.

Laboratory Data on Admission.

[Complete blood count]				[Biochemistry]
WBC	11,960	/μL		TP	5.9	g/dL
Neu	75.4	%		Alb	3.3	g/dL
Lym	17.4	%		AST	13	U/L
Mon	4.8	%		ALT	7	U/L
Eos	2.0	%		ALP	75	U/L
Bas	0.4	%		LDH	420	U/L
RBC	379	×104/μL		γ-GTP	13	U/L
Hb	11.8	g/dL		Na	140	mEq/L
Ht	35.0	%		K	2.9	mEq/L
Plt	22.0	×104/μL		Cl	99	mEq/L
				Ca	8.9	mg/dL
				BUN	12.8	mg/dL
				Cre	0.74	mg/dL
				CRP	3.33	mg/dL
[Coagulation]				CK	36	U/L
PT	12	s		BNP	31.5	pg/mL
PT-INR	1.17			KL-6	315	U/mL
APTT	26.8	s		CEA	1.52	ng/mL
D-dimer	30	μg/mL		CYFRA	3.6	ng/mL
				ProGRP	57.3	pg/mL

Considering the rapid progression of lung cancer, we searched for genetic mutations. Although the submission of a sample from the transbronchial tumor biopsy for the multi-oncogene test was considered, a sufficient nucleic acid quality could not be assured. Meanwhile, the patient exhibited rapidly worsening ascites, and combined with the FDG-PET/CT findings, malignant ascites due to peritoneal dissemination were strongly suspected.

On day 6 of hospitalization, paracentesis was performed, and 1,400 mL of bloody ascitic fluid was drained for the analysis. The findings of the ascitic fluid analysis are presented in Table 2. The sample was bloody, and the following steps were performed to purify the tumor cells (Fig. 2d, e, 3, 4):

Table 2.

The Findings of the Ascitic Fluid.

[Complete blood count]				[Biochemistry]
WBC	2,127	/μL		TP	2.9	g/dL
Mono	31	%		Alb	1.6	g/dL
Poly	69	%		LDH	203	U/L
RBC	59	×104/μL		CEA	0.4	ng/mL
				CYFRA	2.8	ng/mL
				ADA	8.5	U/L
				Hyaluronic acid	51,625	ng/mL

Figure 3.

Methods of concentrating ascites fluid. (a) Place the sample onto the 10-μm strainer (indicated by the red arrow). (b) Pull the syringe to filter the sample. (c) Invert the strainer over a separate 50 mL tube, rinse it with saline solution, and collect the wash solution.

Figure 4.

The quality and concentration of nucleic acid from a transbronchial tumor biopsy and ascitic fluid cytology samples. The results of electropherogram created by the Bioanalyzer (Agilent Technologies, Santa Clara, USA) are shown in a-f (a, c, e; DNA, b, d, f; RNA). (a, b) Fresh frozen sample of a transbronchial biopsy specimen. (c, d) Formalin-fixed paraffin-embedded sample of a transbronchial specimen. (e, f) Purified ascites sample.

1) Preparation of cytological samples from liquid specimens: Liquid specimens such as pleural effusion, ascites, or pericardial fluid can be used to create cytology slides for assessing cellular composition and ratios. The ascites sample was centrifuged at 1,500 rpm for 5 min, and the buffy coat was smeared onto a glass slide, stained with Hemacolor, and examined under a microscope for confirmation.

2) Selection and use of an appropriate pluriStrainer (filter): We selected a pluriStrainer with a pore size suitable for the specimen, set it with a funnel on a 50-mL centrifuge tube, and filtered the specimen. If necessary, a syringe may be used to apply negative pressure for filtration (Fig. 3a, b).

3) The cells were recovered from the filter, and a cell block was prepared. The filter was inverted into a new 50 mL centrifuge tube, and the retained cells were washed off using saline or cell preservation solution (Fig. 3c). Alternatively, using the same setup as described in Step 2, saline or cell preservation solution was added, and a syringe was used to apply positive pressure to suspend and recover cells from the filter. Our hospital has an in-house AmoyDx^Ⓡ panel testing system that allowed us to obtain EML4-ALK fusion gene results three days after testing.

Alectinib was initiated on the day of EML4-ALK fusion gene detection at a dose of 600 mg/day. As the patient's overall condition had deteriorated to ECOG PS 4, along with anemia and hypoxemia, concurrent treatments, including transfusions and oxygen therapy, were administered, leading to considerable improvement in her overall condition (Fig. 5). CT also showed remarkable improvement in the mass, lymph node metastasis, and peritoneal dissemination (Fig. 6), and there were no severe adverse events. On day 26 after the initiation of alectinib treatment, the patient was transferred to the referring physician for continued rehabilitation therapy. Eight months have passed since the start of alectinib treatment, and the patient has been able to continue treatment without any significant adverse events or relapse of lung cancer.

Figure 5.

Clinical course after hospitalization and alectinib treatment.

Figure 6.

Clinical images after alectinib treatment. (a, b) CT and (c) chest X-ray on the alectinib treatment initiation day. (d, e) CT 18 days after alectinib treatment initiation. (f) Chest X-ray three months after the alectinib treatment initiation. CT: computed tomography

Discussion

We herein report a case of lung adenocarcinoma with rapid progression and worsening ascites in which the timely detection of the EML4-ALK fusion gene in an ascitic fluid sample led to a dramatic response to alectinib treatment.

This report presents two novel findings. First, even if transbronchial tumor biopsy samples yield negative results, high-quality nucleic acids extracted from ascitic fluid samples can enable genetic mutation screening using the AmoyDx^Ⓡ panel. Second, in patients with bloody ascites, tumor purification techniques can facilitate the extraction of nucleic acids.

For ALK-positive lung cancer, the detection method for EML4-ALK fusion is crucial, and IHC, FISH, quantitative RT-PCR (qRT-PCR), and NGS are the currently available methods (5). Among these, qRT-PCR and NGS have the highest positive detection rates (5,6). Recently, in addition to EGFR and ALK, various other driver gene mutations, such as ROS1, RET, MET exon14 skipping, and KRAS G12C, have been reported in lung cancer, making multiplex testing more prevalent than single-gene testing (7). IHC can be performed relatively easily in-house if antibodies are available and have a short turnaround time (TAT). However, antibody reactivity may result in weak staining and false negatives, particularly for small samples. In this case, a transbronchial tumor biopsy sample tested negative for ALK immunostaining. This negative result could be due to the small sample size, which may have contained an inadequate number and proportion of tumor cells, affecting the staining accuracy. Given the significant shrinkage of the primary tumor after alectinib treatment, it is likely that the primary tumor was ALK-positive.

Furthermore, even if single-gene testing, such as ALK immunostaining, is negative in the primary tumor, it is crucial to identify malignant cells in ascitic fluid samples and perform a multiplex gene analysis in non-smoking patients with lung adenocarcinoma. ALK is the second-most common driver gene alteration in lung cancer in Japan, and its advantage is that it can be easily tested using immunostaining. However, it is difficult to search for MET exon14 skipping mutations, RET, and other driver gene mutations, for which a single test does not exist at present. Therefore, we believe that retesting with multiple tests is preferable, especially for never-smoker patients with lung adenocarcinoma who have negative single test results.

In the present case, the tumor grew rapidly, and although ALK immunostaining of the primary tumor was negative, the possibility of another driver gene mutation was considered, necessitating urgent screening for multiple genetic alterations. The quality of the nucleic acids extracted from the ascitic fluid sample was superior to that extracted from the primary tumor biopsy, leading to the use of the AmoyDx^Ⓡ panel for testing. Typically, the AmoyDx^Ⓡ panel has a TAT of 4-7 days; however, our hospital's in-house system allowed us to confirm the results within three days (8). The Lung Cancer Compact Panel is another multiplex test available for cytological samples, but its TAT is reportedly 7-12 days (9). Therefore, the AmoyDx^Ⓡ panel was used to obtain the fastest results.

Bloody ascites typically complicate accurate cytology because of the presence of red blood cells. In this case, we successfully used a mesh filter to remove non-tumor cells, allowing for more accurate cytology and nucleic acid extraction. A recent study evaluated the use of cytological samples for the AmoyDx^Ⓡ panel (10). However, that study focused on transbronchial tumor biopsy brush cytology and endobronchial ultrasound-guided transbronchial needle aspiration samples and did not examine ascitic fluid samples, as in our case. Nevertheless, the study suggested that cytological samples may be useful for detecting driver gene mutations when sufficient tissue samples are not available for NGS. Therefore, if high-quality nucleic acids can be obtained from fluids, such as ascites or pleural effusion, the AmoyDx^Ⓡ panel can be used to identify driver gene mutations in a similar manner. This approach has not been previously reported, and we believe that the further accumulation of similar cases is necessary to validate its effectiveness.

A potential limitation of this case is that the ability to quickly perform the AmoyDx^Ⓡ panel test in-house may have contributed to the success of the examination. In situations where it is difficult to conduct the AmoyDx^Ⓡ panel test in-house, an important alternative is to promptly create a cell block and submit it to an external laboratory.

Conclusion

We encountered a case of lung adenocarcinoma in which the EML4-ALK fusion gene was rapidly detected in an ascitic fluid sample, leading to dramatic efficacy of alectinib treatment. In non-smokers with pleural or peritoneal effusion, the detection of driver genes in body cavity fluids, in addition to primary tumor sites, may be beneficial.

This case report was prepared and completed in accordance with the 2013 Declaration of Helsinki guidelines. Written informed consent was obtained from the patient for publication of the case report and accompanying images.

The authors state that they have no Conflict of Interest (COI).