Lung Cancer of Unknown Primary Site with EGFR Mutation as Indicated by Frozen Bone Specimen

Boku S¹*, Takase N¹, Onoe T¹, Shibata Y¹, Matsumoto K¹, Satouchi M², Sakuma T³, Sudo T⁴ and Negoro S¹

¹Department of Medical Oncology, Hyogo Cancer Center, Japan

²Department of Thoracic Oncology, Hyogo Cancer Center, Japan

³Department of Diagnostic Pathology, Hyogo Cancer Center, Japan

⁴Section of Translational Research, Hyogo Cancer Center, Japan

*Corresponding author: Shogen Boku, Department of Medical Oncology, Hyogo Cancer Center, 13-70 Kitaojicho, Akashi-City, Hyogo 673-8558, Japan

Received: September 25, 2015; Accepted: November 02, 2015; Published: November 03, 2015

Abstract

We reported a case of lung cancer of unknown primary site harboring EGFR mutation revealed in frozen bone specimens. Tumor cells obtained from bone metastasis are associated with a higher rate of molecular testing failure. We submitted the preserved frozen bone specimens and detected EGFR exon 19 deletions. She has achieved long-term disease-free survival from gefitinib. This result shows the importance of gene analysis of frozen bone specimens in CUP with bone metastases.

Keywords: Lung cancer; EGFR mutation; Bone metastasis; Gefitinib

Case Report

A 75 year-old Japanese women was referred to our hospital complaining of left-sided chest pain. Her performance status (ECOG) was 1. She was a never smoker. On physical examination, there was no palpable mass on her trunk or extremities. Blood examination showed significant elevation of Carcinoembryonic Antigen (CEA) to 209.5 ng/ml. Chest-X rays and Computed Tomography (CT) did not detect the primary lesion in her lung or pleura. Positron Emission Tomography and Computed Tomography (PET-CT) detected abnormal FDG uptake of left 5th and 6th rib and subaortic (Botallo’s) lymph nodes (standardized uptake value-max 5.0, Figure 1A). We performed open biopsy of her left 5th rib in order to ascertain malignancy. We performed decalcification, and some of the samples were H&E (hematoxyline-eosin)-stained. We fixed the rest of the specimens in liquid nitrogen and kept them at -70oC in a deep freezer to be used in any future genetic testing. The HE staining showed the tumor cells to be adenocarcinoma. Immunohistochemical testing showed that the tumor cells to be positive for Thyroid Transcription Factor-1 (TTF-1), Napsin A, and CAM5 2 (Figure 2). Our opinion was that this bony malignancy derived from lung. To examine the gene mutations, we submitted the preserved frozen specimens to the laboratory cooperation (LSI Medience Corporation) and requested processing with protease. EGFR exon 19 deletions were detected by PNA-LNA PCR clamp. We started the patient on gefitinib and bonemodifying agent (zoledolnic acid) in Feb 2013. Skin rash associated with gefitinib was observed on her legs. It was tolerable without dose reduction or discontinuation. Serum levels of CEA normalized four months after she received gefitinib. The PET-CT at the 24th month did not detect the signs of recurrence or progression in terms of either bone metastasis or mediastinal lymph nodes (Figure 1B).

Figure 1a: PET-CT detected abnormal FDG uptake of left rib, and aortic-arch lymph nodes.

    
    
    Figure 1a:  PET-CT detected abnormal FDG uptake of left rib, and aortic-arch
lymph nodes.

Figure 1b: Lower FDG uptake was detected in both lesions 24 months after she received gefitinib. (max SUV 5.0→1.6).

    
    
    Figure 1b:  Lower FDG uptake was detected in both lesions 24 months after
she received gefitinib. (max SUV 5.0→1.6).

Figure 2: Tumor specimens obtained from bony tissue (a. HE, b. TTF-1, c. Napsin, d. CAM5.2).This result of immunostaining presented a phenotype of lung cancer. (All images were taken at 200× original magnification).

    
    
    Figure 2:  Tumor specimens obtained from bony tissue (a. HE, b. TTF-1, c.
Napsin, d. CAM5.2).This result of immunostaining presented a phenotype of
lung cancer. (All images were taken at 200× original magnification).

Discussion/Conclusion

We reported a case of lung cancer of unknown primary site harboring EGFR mutation revealed in frozen bone specimens. The pathological examination suggested lung cancer. The present case characteristics include Asian, female, a history of non-smoking, and histologic evidence of adenocarcinoma. These characteristics are highly associated with Non-Small Cell Lung Cancer (NSCLC) harboring EGFR mutation [1]. Tumor cells obtained from bone metastasis, however, are associated with a higher rate of molecular testing failure [2]. The routine processing of bone specimens incorporates an acid decalcification step. Acid decalcification of bone tissue tends to cause extensive fragmentation of DNA and also interferes with subsequent genetic analysis [3]. We showed that preserved frozen bone specimens were useful for genetic testing and decision making in treatment. We routinely preserved frozen specimens obtained from patients for whom there was a suspicion of Cancer of Unknown Primary (CUP) in our institution. This technique is not common in general hospitals because it is difficult to obtain liquid nitrogen. Recently it was reported that using process of EDTA decalcification or no decalcification, DNA quality was high and sequences could be easily analyzed on bone metastases specimens [4]. PNA-LNA PCR clamp can detect EGFR mutations in the presence of 100-to 1,000-fold background of wild-type EGFR [5]. This system can accurately detect EGFR mutations as much as direct sequencing, Cycleave™, and Scorpion Amplification Refractory Mutation System (ARMS)^®in the analysis of FFPE and cytology samples [6]. In NSCLC harboring EGFR mutations, gefitinib is feasible for elderly patients or patients with poorer PS [7]. Therefore, we selected gefitinib as first-line therapy for this patient. She has achieved long-term diseasefree survival from gefitinib. It was reported that bone metastases and skeletal related events were more frequent in men, heavy smokers and without treatment of EGFR TKI. The presence of bone metastases proved to be an independent prognostic factor related to poor performance status and worse Quality of Life [8]. This result shows the importance of gene analysis of frozen bone specimens in CUP with bone metastases.

References

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Citation: Boku S, Takase N, Onoe T, Shibata Y, Matsumoto K, et al. Lung Cancer of Unknown Primary Site with EGFR Mutation as Indicated by Frozen Bone Specimen. Austin J Pulm Respir Med 2015; 2(3): 1030. ISSN:2381-9022

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