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Annexin A2 Expression in the Aerogenous Spread of Pulmonary Invasive Mucinous Adenocarcinoma with Gastric Lineage

Annexin A2 Expression in the Aerogenous Spread of Pulmonary Invasive Mucinous Adenocarcinoma with... Hindawi Case Reports in Oncological Medicine Volume 2020, Article ID 2492636, 9 pages https://doi.org/10.1155/2020/2492636 Case Report Annexin A2 Expression in the Aerogenous Spread of Pulmonary Invasive Mucinous Adenocarcinoma with Gastric Lineage 1 1 1 2 Kazumori Arai , Tomohiro Iwasaki, Chinatsu Tsuchiya, and Akihiro Sonoda Department of Pathology, Shizuoka General Hospital, 4-27-1 Kitaando, Aoi-ku, Shizuoka 420-0881, Japan Department of Clinical Research, Shizuoka General Hospital, Japan Correspondence should be addressed to Kazumori Arai; m-arai@ny.tokai.or.jp Received 17 September 2019; Revised 8 May 2020; Accepted 8 May 2020; Published 19 May 2020 Academic Editor: Constantine Gennatas Copyright © 2020 Kazumori Arai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Spread through air spaces (STAS) is a unique form of lung cancer progression associated with a worse prognosis. However, the mechanisms underlying STAS and the associated proteins remain unclear. Annexin A2 (ANX A2), which is a membrane- binding protein involved in cell adhesion, is known to promote cancer invasion. In this report, we describe the immunohistochemical analysis of ANX A2 expression in an invasive mucinous adenocarcinoma (IMAC) resected from a 63-year-old man in whom the tumor cells had detached from the alveolar wall and exhibited STAS. At the detachment site, we observed cytoplasmic ANX A2 positivity on the basal side and in the exfoliative gap, as well as reduced collagen IV positivity expression. This biomarker pattern suggested an IMAC with gastric lineage. We hypothesize that ANX A2 is secreted from the basal sides of tumor cells and induces tumor cell detachment by degrading the basement membrane. A further comparison of this case with an IMAC with nongastric lineage suggested the following probabilities: (1) ANX A2 likely contributes to STAS in a manner that is dependent on its subcellular localization. (2) Both the subcellular localization of ANX A2 and the detachment site depend on tumor cell characteristics, including the biomarker immunophenotype. 1. Introduction of tumor cell detachment into alveolar spaces and adhesion to the alveolar wall. Spread through air spaces (STAS) is a unique form of lung Invasive mucinous AC (IMAC) frequently progresses via cancer progression from the primary tumor site via the air- STAS/AM [8]. Recently, we reported the expression of 2+ ways [1, 2]. This new category is included in the definition annexin A2 (ANX A2), which is a Ca /membrane-binding of lung adenocarcinoma (AC) invasion, along with stromal protein in a case of IMAC with STAS/AM [9]. To the best (including vascular) and pleural invasion [1]. STAS is also of our knowledge, our report was the first to indicate the considered the origin of aerogenous metastasis (AM) [1, 2]. involvement of this protein in STAS/AM. In the reported Although STAS has elicited great interest in the field of lung case, tumor cell detachment occurred at the tip of a villous- cancer [3–7], its status as either a true invasive form or a shaped tumor component that had projected into the alveo- technical artifact of sample processing remains controversial lar spaces [9]. In contrast to our previous report, other [3]. However, recent reports have provided evidence that researchers described tumor cell detachment from the base- favors STAS as a true invasive pattern and have demon- ment membrane (BM) of the alveolar wall as a common strated a significant association with a worse prognosis origin of STAS/AM from IMAC [10, 11]. [4–7]. Furthermore, STAS is associated with a higher risk of ANX A2 is expressed in various cells and is involved in locoregional recurrence, thus preventing limited resection cell adhesion, cell proliferation, and cell polarity within the [4–6]. The inhibition of STAS is an important research issue, cytoplasm and plasma membranes [12, 13]. This protein is but the underlying pathogenic mechanisms remain unclear. also expressed in various cancers, including lung cancer, We believe that the ability to inhibit STAS will depend on and extracellular membrane-bound ANX A2 acts as a plas- an understanding of the protein(s) involved in the processes minogen activator to promote angiogenesis and cancer cell 2 Case Reports in Oncological Medicine (a) (b) Figure 1: Axial CT images. (a) A chest CT image obtained 7 days after the initial visit shows a large infiltrative shadow, as well as a small infiltrative shadow that is suspected to be a skip lesion (arrow). (b) A chest CT image obtained 2 months after left lobectomy shows an infiltrative shadow with a skip lesion (arrow) in the right lung. (a) (b) (c) (d) Figure 2: Left lung cancer I. (a) Gross view showing an ill-defined tumor with a diffuse pneumonia-like consolidation. Scale bar: 1 cm. (b) The tumor cells exhibit lepidic or papillary growth, with detached tumor cells scattered in the alveolar spaces. Hematoxylin and eosin (H&E) staining, scale bar: 2 mm. (c) Columnar tumor cells with intracytoplasmic vacuoles are shown in a monolayer arrangement. H&E stain, scale bar: 200 μm. (d) Multiple mucin-producing tumor cells are visible. Alcian blue stain, scale bar: 200 μm. invasion in the stroma [14, 15]. The other functions of ANX ANX A2 expression or subcellular localization. In the current A2 in cancer are currently under investigation [15]. We report, we immunohistochemically examined the expression hypothesized that ANX A2 may also be involved in the of ANX A2 in a case of IMAC in which tumor cell detach- STAS/AM of lung AC and that the observed discrepancies ment occurred from the alveolar wall, consistent with previ- in IMAC detachment sites would result from differences in ous reports [10, 11]. Case Reports in Oncological Medicine 3 (a) (b) (c) (d) Figure 3: Left lung cancer II. (a) Small groups of tumor cells that detached from the alveolar wall. Only a slight inflammatory cell infiltration is visible. H&E stain, scale bar: 200 μm. (b) Detached tumor cells with a micropapillary pattern float within the alveolar spaces. H&E stain, scale bar: 200 μm. (c) The invasive component exhibits glandular or papillary growth. H&E stain, scale bar: 1 mm. (d) Many scattered tumor cell aggregates that detached from the primary tumor are visible within the air spaces and on the alveolar wall. The latter aggregates exhibit lepidic or papillary growth that is suggestive of microscopic aerogenous metastases. H&E stain, scale bar: 2 mm. 2. Case Presentation right lung tumor was an AM from the left lung tumor [17]. No metastatic lesion was detected in the left lung. The patient The subject of this case study provided written informed did not undergo surgery for the right lung tumor and instead consent for the publication of the case details. A 63-year- received various chemotherapy regimens for 30 months, old man with no history of smoking presented with fever, including 11 cycles of paclitaxel, carboplatin, and bevacizu- cough, and mucinous sputum. Chest computed tomography mab; 10 cycles of pemetrexed; and 16 cycles of docetaxel. (CT) revealed a large infiltrative shadow that is approxi- However, no distinct therapeutic effect was observed, and mately 10 cm in size in the lower lobe of the left lung. No CT revealed a further enlargement of the shadow. The enlargement of the hilar or mediastinal lymph nodes was patient developed right pleuritis carcinomatosa at 31 months observed. The differential diagnosis was challenging, and after the start of chemotherapy and died of respiratory failure pneumonia or IMAC was suspected [16]. Seven days later, 36 months after surgery. An autopsy was not performed. chest CT demonstrated the expansion of the shadow and In addition to a routine pathological analysis, tissue from a small infiltrative shadow suggestive of a skip lesion the left-sided tumor was subjected to immunohistochemical (Figure 1(a)). The patient was then hospitalized immediately. analyses to detect the expression of various proteins, Full-body CT did not reveal any metastatic lesions in the including ANX A2 and collagen IV. Immunohistochemistry right lung or extrapulmonary organs. Considering that a was performed using Leica Bond-Max (Leica Biosystems, transbronchial lung biopsy revealed a mucus-producing Australia) and a previously described ANX A2 antibody AC, the patient underwent thoracoscopic lobectomy with and staining protocol [9]. A mouse antihuman collagen IV no adjuvant therapy. antibody (clone CIV 22, DAKO, USA) was used as the primary antibody at a dilution ratio of 1 : 25 after a 5 min Two months postoperatively, a follow-up chest CT revealed an infiltrative shadow with an approximate diameter treatment with proteinase K. Only routine immunohisto- of 6 cm and a skip lesion in the lower lobe of the right lung chemistry was performed on the right lung tumor because (Figure 1(b)). A right lung biopsy revealed an AC that shared of the small amount of available tissue. cell morphologic features with the left lung tumor. The com- Macroscopically, the resected tumor was ill-defined and prehensive clinical course led to a clinical suspicion that the gelatinous with a diffuse pneumonia-like consolidation 4 Case Reports in Oncological Medicine (a) (b) (c) (d) Figure 4: Immunostaining of mucin glycoproteins in the left lung tumor. (a) Diffuse cytoplasmic positivity is visible in the lepidic or papillary component, and positive staining is present in the alveolar spaces (arrows). MUC 5AC immunostain, scale bar: 200 μm. (b) Floating tumor cells exhibit the same positive reaction. MUC 5AC immunostain, scale bar: 100 μm. (c) Some tumor cells exhibit cytoplasmic positivity. MUC 6 immunostain, scale bar: 200 μm. (d) In some cells, positive staining is visible on the cell surfaces but not in the cytoplasm. MUC 1 immunostain, scale bar: 100 μm. (Figure 2(a)). Histopathologically, the majority of the tumor scopic AMs (Figure 3(d)) [2]. Tumor cell detachment was also often observed in the larger foci. cells were columnar, contained varying amounts of intracy- toplasmic mucins, and exhibited lepidic or papillary growth In the left lung tumor, the majority of tumor cells were in a single-layer arrangement (Figures 2(b)–2(d)). Small positive for MUC 5AC (Figures 4(a) and 4(b)) and CK7 groups of tumor cells had detached from the alveolar wall (data not shown). Abundant positive MUC 5AC staining and were scattered diffusely (Figure 3(a)). Tumor cell aggre- was also observed in the alveolar spaces (Figures 4(a) and gates floating within the alveolar spaces were round-shaped 4(b)). Some tumor cells exhibited intracytoplasmic positivity but had apical–basal polarity (Figure 3(b)). The apical surface for MUC 6 (antibody clone CLH 5, Abnova, Taiwan; of the tumor cells was located at the outer edge of the Figure 4(c)). Additionally, some tumor cells exhibited a aggregates but naturally lacked central fibrovascular cores positive MUC 1 reaction at the cell surface but not in the (Figure 3(b)), corresponding to the micropapillary pattern cytoplasm (Figure 4(d)), in contrast to our previously of the three morphological patterns of STAS [6]. reported case [9]. The tumor cells were negative for MUC 2, Slight neutrophil and macrophage infiltration was CK20, TTF-1, Napsin A, CDX2, and ALK (data not shown). observed at the detachment site. No desmoplastic reaction The clone and manufacturer of each antibody, except was observed. MUC6, have been described elsewhere [9]. The immunohis- Invasive foci with a maximum diameter of 2.2 cm were tochemical results of the right lung tumor were similar to observed in the tumor. These foci had a glandular or those of the left lung tumor. papillary structure (Figure 3(c)) and greatly reduced levels As shown in Figures 5 and 6 (5(a), 5(c), 6(a), and 6(b) of intracytoplasmic mucins. However, the nonmucinous show ANX A2 staining), granular cytoplasmic ANX A2 positivity was observed on the basal sides of tumor cells component accounted for <10% of the tumor. Lymphatic and vascular permeation and pleural invasion were not exhibiting lepidic or papillary growth (Figure 5(a)), whereas observed. Many scattered tumor cell aggregates that had no positive signal was detected at the cell–cell borders. Some detached from the main tumor were observed within the air tumor cells also exhibited weak ANX A2 positivity near the spaces and on the alveolar wall. The latter aggregates exhib- cell surfaces. A similar expression pattern was observed in the detached tumor cells, and positive expression was also ited lepidic or papillary growth that is suggestive of micro- Case Reports in Oncological Medicine 5 (a) (b) (c) (d) Figure 5: ANX A2 and collagen IV immunostaining of the lepidic or papillary component of the left lung tumor. (a) Tumor cells exhibit cytoplasmic ANX A2 positivity on the basal side. ANX A2 immunostain, scale bar: 400 μm. (b) A linear positive collagen IV reaction corresponding to the basement membrane is seen on the surfaces of both alveolar and vessel walls. Collagen IV immunostain, scale bar: 400 μm. (c) Detached tumor cells also exhibit cytoplasmic ANX A2 positivity on the basal side but not at the cell–cell borders. Positive staining is also observed in the exfoliative gap. ANX A2 immunostain, scale bar: 100 μm. (d) Weak or no collagen IV positivity is visible at the detachment site (arrows). Collagen IV immunostain, scale bar: 100 μm. observed in the exfoliation gap (Figure 5(c)). Noticeably, were seen. Similar findings were also observed at the detach- weaker ANX A2 positivity was observed in the tumor cells ment site (data not shown). floating within the alveolar spaces (Figure 6(a)). In the inva- sive component, stronger cytoplasmic positivity on the basal 3. Discussion side was observed relative to that observed in the lepidic or papillary growth component (Figure 6(b)), and membranous ANX A2 expression in the STAS/AM of the present tumor positivity was observed on the cell–cell borders (Figure 6(b)). differed slightly from that in our previously reported case As shown in Figures 5 and 6 (5(b), 5(d), and 6(c) show [9], particularly with regard to the following four points. (1) collagen IV staining), the lepidic or papillary area exhibited At the tumor cell detachment site, ANX A2 positivity was linear positive collagen IV staining corresponding to the observed in the cytoplasm on the basal side and in the exfo- BM, which was detected in both the surfaces of the alveolar liative gap. (2) No positive staining of the cell–cell borders and vessel walls (Figure 5(b)). However, either weak positiv- was observed in any component, except the invasive compo- ity or no signal was observed at the detachment site nent. (3) No distinct ANX A2 positivity was observed in (Figure 5(d)). In the invasive area, the desmoplastic stroma either the tumor cell aggregates floating within alveolar exhibited positive staining for collagen IV (Figure 6(c)), thus spaces or the minute foci of AM. (4) No ANX A2 positivity suggesting the presence of cancer-associated fibroblasts and was observed in the intracytoplasmic mucus. These discrep- neogenic collagen [18, 19]. No positive reactions were ancies are likely attributable to differences in the cellular observed in the tumor cells irrespective of invasiveness. characteristics and growth patterns between the tumors. In In an analysis of the AM, no positive reaction to ANX A2 the present case, both the mucin immunophenotype and beyond weak staining near the cell surface was observed in negative immunostaining for TTF-1 and CDX 2 suggest an the minute foci (i.e., tumor cells that had newly adhered to IMAC with gastric lineage [20–22]. The IMAC with gastric the alveolar wall; Figure 7(a)). However, the staining patterns lineage was first reported as a mucinous bronchioloalveolar in larger foci became similar to those observed in the lepidic carcinoma, with cell morphology mimicking the mucous or papillary growth component of the primary lesion cells of gastric pyloric mucosa and with its mucin immuno- (Figure 7(b)). Similar findings were observed at the detach- phenotype [21, 22]. Recently, Sonzogni et al. have classified ment site. Regarding collagen IV, the same findings as those the lineage of IMACs based on their immunophenotype, his- of lepidic or papillary growth component of primary lesion tology, and genetic alterations [20]. The IMAC with gastric 6 Case Reports in Oncological Medicine (a) (b) (c) Figure 6: Floating tumor cells (a) and invasive components (b and c) of the left lung tumor. (a) No distinct ANX A2 positivity is visible in the floating tumor cells within alveolar spaces. ANX A2 immunostain, scale bar: 100 μm. (b) Stronger cytoplasmic ANX A2 positivity is observed on the basal side, and positive staining is visible at the cell–cell borders. ANX A2 immunostain, scale bar: 200 μm. (c) The desmoplastic stroma exhibits positive collagen IV staining, particularly around the tumor cells. Collagen IV immunostain, scale bar: 1 mm. (a) (b) Figure 7: ANX A2 immunostaining of microscopic aerogenous metastases in the left lung. (a) No distinct positivity is visible in the minute foci. Scale bar: 200 μm. (b) Basal-side cytoplasmic positivity is detected in the larger metastatic foci. Scale bar: 200 μm. lineage is characterized by expressions of both membranous particularly given the intracytoplasmic expressions of both MUC 1 and MUC 5AC in the AC [9, 23]. MUC 1 and cytoplasmic MUC 6 and is suggested to be derived from the distal terminal bronchiole [20]. In contrast, ANX A2 moves inside the cell according to its roles [13]. our previously reported case was negative for subsequent Cytoplasmic ANX A2 is involved in cytoskeleton remodel- MUC 6 immunostaining (data not shown) and showed non- ing, exocytosis, and endocytosis [13]. In addition, a part of typical immunohistochemical results for any of the classifica- ANX A2 binds to the extracellular surface-membrane or is secreted [13]. This protein is expressed aberrantly in various tion by Sonzogni et al. [9, 20]. If the previous case is categorized, it is considered to exhibit characteristics of the cancers [14, 15] and may also be secreted [14]. A recent pancreatobiliary type in terms of the classification of the report further suggested that secreted ANX A2 contributes intraductal papillary mucinous neoplasms of the pancreas, to the proteolysis of the extracellular matrix [24]. In the Case Reports in Oncological Medicine 7 aggregates stretch to form a monolayer along the alveolar present tumor, we observed reduced collagen IV staining at the detachment site, thus leading us to consider that the wall in a manner independent of ANX A2. ANX A2 secreted from the basal sides of tumor cells might It remains unclear whether the right lung tumor origi- nated as a second primary lesion or was caused by an AM induce cell detachment by degrading the BM [13–15, 24]. Therefore, in IMAC, the detachment site of the tumor cells of the left lung tumor [17, 31, 32]. A similarly rapid clinical and the subcellular localization of ANX A2 might differ course was also reported in a previous case of multifocal depending on the immunophenotype of the detected IMAC [32]. Given that we did not genetically confirm mono- biomarkers [9, 20]. clonality between the tumors and could not detect a recur- rence in the ipsilateral lung, the right tumor might instead Indistinct membranous ANX A2 positivity in contact with the alveolar wall suggests poor capacity of IMAC for have been another lesion of multifocal IMAC. stromal invasion [1, 9, 25]. However, we observed more This report had some additional limitations. Particularly, intense cytoplasmic ANX A2 expression in the invasive com- we analyzed only two cases, including our previously ponent in the present IMAC case, thus suggesting that this reported case. Moreover, our analysis lacked a functional assay and an immunohistochemical investigation of various protein is involved in stromal invasion [9, 12, 13]. Therefore, we presume (1) that whether IMAC tumor cells detach into BM matrices. Further investigations are needed to confirm alveolar spaces or infiltrate toward the stroma depends our suppositions. on the amount of secreted ANX A2, (2) that the stromal In conclusion, our findings from this case suggest the invasion of IMAC requires a stronger cytoplasmic expression following points. (1) ANX A2 plays a dual role in tumor pro- gression and suppression according to its subcellular locali- of ANX A2, and (3) that extracellular membrane-bound ANX A2 enhances the aggressiveness of stromal invasion in zation. (2) The subcellular localization of ANX A2 depends AC [14, 15, 25, 26]. on the characteristics of the tumor cell, including the bio- We note that weaker ANX A2 positivity was observed at marker immunophenotype and tumor cell growth pattern. the cell–cell borders in the detachment site, even in our pre- (3) In the gastric lineage, ANX A2 secretion induces tumor cell detachment by degrading the BM. (4) The expression of viously reported case [9]. The expression of ANX A2 at the cell–cell borders is possibly related to the adhesions between ANX A2 (or lack thereof) at the cell–cell borders influences tumor cells and the suppression of tumor cell detachment the detachment site. [9, 12, 13]. In other words, little or no expression of ANX A2 at the cell–cell borders might facilitate tumor cell Ethical Approval detachment. Thus, we presumed that the lepidic or papil- lary component of the present tumor harbors a high Ethical approval for this study was provided by the ethics capacity for detachment [9, 12, 13]. Nevertheless, the small committee of Shizuoka General Hospital. groups of detached tumor cells showed a micropapillary pattern without the original monolayer arrangement. It Consent has been suggested that tumor cells forming a micropapil- lary pattern acquire resistance to anoikis (cell detachment- Written informed consent to publish the case details was induced apoptosis) and facilitate anchorage-independent obtained from the patient. growth [27]. Therefore, the morphological change of detached tumor cells is considered to be a defense reaction Conflicts of Interest against the loss of interaction with the BM [11, 27]. It has been known that ANX A2 is released extracellularly dur- The authors declare no conflicts of interest regarding the ing various stresses, including hypoxia [13], which is con- publication of this article. sistent with the findings that tumor cell aggregates floating within the alveolar spaces do not show a distinct ANX Acknowledgments A2-immunopositivity. Some reports have suggested that neutrophils and tumor- The authors would like to thank Enago (http://www associated macrophages and fibroblasts promote STAS/AM .enago.jp/) for providing the English language review. This [28, 29]. In the present case, we did not notice a particular report received no specific grant from any funding agency infiltration of any of those cell types into the tumor. Although in the public, commercial, or nonprofit sector. we agree that the tumor microenvironment is an important factor in STAS/AM, the present case suggests that the tumor References cells can induce STAS/AM directly. In this case, we consider that ANX A2 was only weakly [1] W. D. Travis, E. Brambilla, A. P. Burke, A. Marx, and A. G. involved in mediating the adhesions between tumor cells Nicholson, World Health Organization Classification of and in the anchoring of floating tumor cells to the alveolar Tumours of the Lung, Pleura, Thymus and Heart, International wall. 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Annexin A2 Expression in the Aerogenous Spread of Pulmonary Invasive Mucinous Adenocarcinoma with Gastric Lineage

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Copyright © 2020 Kazumori Arai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Case Reports in Oncological Medicine Volume 2020, Article ID 2492636, 9 pages https://doi.org/10.1155/2020/2492636 Case Report Annexin A2 Expression in the Aerogenous Spread of Pulmonary Invasive Mucinous Adenocarcinoma with Gastric Lineage 1 1 1 2 Kazumori Arai , Tomohiro Iwasaki, Chinatsu Tsuchiya, and Akihiro Sonoda Department of Pathology, Shizuoka General Hospital, 4-27-1 Kitaando, Aoi-ku, Shizuoka 420-0881, Japan Department of Clinical Research, Shizuoka General Hospital, Japan Correspondence should be addressed to Kazumori Arai; m-arai@ny.tokai.or.jp Received 17 September 2019; Revised 8 May 2020; Accepted 8 May 2020; Published 19 May 2020 Academic Editor: Constantine Gennatas Copyright © 2020 Kazumori Arai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Spread through air spaces (STAS) is a unique form of lung cancer progression associated with a worse prognosis. However, the mechanisms underlying STAS and the associated proteins remain unclear. Annexin A2 (ANX A2), which is a membrane- binding protein involved in cell adhesion, is known to promote cancer invasion. In this report, we describe the immunohistochemical analysis of ANX A2 expression in an invasive mucinous adenocarcinoma (IMAC) resected from a 63-year-old man in whom the tumor cells had detached from the alveolar wall and exhibited STAS. At the detachment site, we observed cytoplasmic ANX A2 positivity on the basal side and in the exfoliative gap, as well as reduced collagen IV positivity expression. This biomarker pattern suggested an IMAC with gastric lineage. We hypothesize that ANX A2 is secreted from the basal sides of tumor cells and induces tumor cell detachment by degrading the basement membrane. A further comparison of this case with an IMAC with nongastric lineage suggested the following probabilities: (1) ANX A2 likely contributes to STAS in a manner that is dependent on its subcellular localization. (2) Both the subcellular localization of ANX A2 and the detachment site depend on tumor cell characteristics, including the biomarker immunophenotype. 1. Introduction of tumor cell detachment into alveolar spaces and adhesion to the alveolar wall. Spread through air spaces (STAS) is a unique form of lung Invasive mucinous AC (IMAC) frequently progresses via cancer progression from the primary tumor site via the air- STAS/AM [8]. Recently, we reported the expression of 2+ ways [1, 2]. This new category is included in the definition annexin A2 (ANX A2), which is a Ca /membrane-binding of lung adenocarcinoma (AC) invasion, along with stromal protein in a case of IMAC with STAS/AM [9]. To the best (including vascular) and pleural invasion [1]. STAS is also of our knowledge, our report was the first to indicate the considered the origin of aerogenous metastasis (AM) [1, 2]. involvement of this protein in STAS/AM. In the reported Although STAS has elicited great interest in the field of lung case, tumor cell detachment occurred at the tip of a villous- cancer [3–7], its status as either a true invasive form or a shaped tumor component that had projected into the alveo- technical artifact of sample processing remains controversial lar spaces [9]. In contrast to our previous report, other [3]. However, recent reports have provided evidence that researchers described tumor cell detachment from the base- favors STAS as a true invasive pattern and have demon- ment membrane (BM) of the alveolar wall as a common strated a significant association with a worse prognosis origin of STAS/AM from IMAC [10, 11]. [4–7]. Furthermore, STAS is associated with a higher risk of ANX A2 is expressed in various cells and is involved in locoregional recurrence, thus preventing limited resection cell adhesion, cell proliferation, and cell polarity within the [4–6]. The inhibition of STAS is an important research issue, cytoplasm and plasma membranes [12, 13]. This protein is but the underlying pathogenic mechanisms remain unclear. also expressed in various cancers, including lung cancer, We believe that the ability to inhibit STAS will depend on and extracellular membrane-bound ANX A2 acts as a plas- an understanding of the protein(s) involved in the processes minogen activator to promote angiogenesis and cancer cell 2 Case Reports in Oncological Medicine (a) (b) Figure 1: Axial CT images. (a) A chest CT image obtained 7 days after the initial visit shows a large infiltrative shadow, as well as a small infiltrative shadow that is suspected to be a skip lesion (arrow). (b) A chest CT image obtained 2 months after left lobectomy shows an infiltrative shadow with a skip lesion (arrow) in the right lung. (a) (b) (c) (d) Figure 2: Left lung cancer I. (a) Gross view showing an ill-defined tumor with a diffuse pneumonia-like consolidation. Scale bar: 1 cm. (b) The tumor cells exhibit lepidic or papillary growth, with detached tumor cells scattered in the alveolar spaces. Hematoxylin and eosin (H&E) staining, scale bar: 2 mm. (c) Columnar tumor cells with intracytoplasmic vacuoles are shown in a monolayer arrangement. H&E stain, scale bar: 200 μm. (d) Multiple mucin-producing tumor cells are visible. Alcian blue stain, scale bar: 200 μm. invasion in the stroma [14, 15]. The other functions of ANX ANX A2 expression or subcellular localization. In the current A2 in cancer are currently under investigation [15]. We report, we immunohistochemically examined the expression hypothesized that ANX A2 may also be involved in the of ANX A2 in a case of IMAC in which tumor cell detach- STAS/AM of lung AC and that the observed discrepancies ment occurred from the alveolar wall, consistent with previ- in IMAC detachment sites would result from differences in ous reports [10, 11]. Case Reports in Oncological Medicine 3 (a) (b) (c) (d) Figure 3: Left lung cancer II. (a) Small groups of tumor cells that detached from the alveolar wall. Only a slight inflammatory cell infiltration is visible. H&E stain, scale bar: 200 μm. (b) Detached tumor cells with a micropapillary pattern float within the alveolar spaces. H&E stain, scale bar: 200 μm. (c) The invasive component exhibits glandular or papillary growth. H&E stain, scale bar: 1 mm. (d) Many scattered tumor cell aggregates that detached from the primary tumor are visible within the air spaces and on the alveolar wall. The latter aggregates exhibit lepidic or papillary growth that is suggestive of microscopic aerogenous metastases. H&E stain, scale bar: 2 mm. 2. Case Presentation right lung tumor was an AM from the left lung tumor [17]. No metastatic lesion was detected in the left lung. The patient The subject of this case study provided written informed did not undergo surgery for the right lung tumor and instead consent for the publication of the case details. A 63-year- received various chemotherapy regimens for 30 months, old man with no history of smoking presented with fever, including 11 cycles of paclitaxel, carboplatin, and bevacizu- cough, and mucinous sputum. Chest computed tomography mab; 10 cycles of pemetrexed; and 16 cycles of docetaxel. (CT) revealed a large infiltrative shadow that is approxi- However, no distinct therapeutic effect was observed, and mately 10 cm in size in the lower lobe of the left lung. No CT revealed a further enlargement of the shadow. The enlargement of the hilar or mediastinal lymph nodes was patient developed right pleuritis carcinomatosa at 31 months observed. The differential diagnosis was challenging, and after the start of chemotherapy and died of respiratory failure pneumonia or IMAC was suspected [16]. Seven days later, 36 months after surgery. An autopsy was not performed. chest CT demonstrated the expansion of the shadow and In addition to a routine pathological analysis, tissue from a small infiltrative shadow suggestive of a skip lesion the left-sided tumor was subjected to immunohistochemical (Figure 1(a)). The patient was then hospitalized immediately. analyses to detect the expression of various proteins, Full-body CT did not reveal any metastatic lesions in the including ANX A2 and collagen IV. Immunohistochemistry right lung or extrapulmonary organs. Considering that a was performed using Leica Bond-Max (Leica Biosystems, transbronchial lung biopsy revealed a mucus-producing Australia) and a previously described ANX A2 antibody AC, the patient underwent thoracoscopic lobectomy with and staining protocol [9]. A mouse antihuman collagen IV no adjuvant therapy. antibody (clone CIV 22, DAKO, USA) was used as the primary antibody at a dilution ratio of 1 : 25 after a 5 min Two months postoperatively, a follow-up chest CT revealed an infiltrative shadow with an approximate diameter treatment with proteinase K. Only routine immunohisto- of 6 cm and a skip lesion in the lower lobe of the right lung chemistry was performed on the right lung tumor because (Figure 1(b)). A right lung biopsy revealed an AC that shared of the small amount of available tissue. cell morphologic features with the left lung tumor. The com- Macroscopically, the resected tumor was ill-defined and prehensive clinical course led to a clinical suspicion that the gelatinous with a diffuse pneumonia-like consolidation 4 Case Reports in Oncological Medicine (a) (b) (c) (d) Figure 4: Immunostaining of mucin glycoproteins in the left lung tumor. (a) Diffuse cytoplasmic positivity is visible in the lepidic or papillary component, and positive staining is present in the alveolar spaces (arrows). MUC 5AC immunostain, scale bar: 200 μm. (b) Floating tumor cells exhibit the same positive reaction. MUC 5AC immunostain, scale bar: 100 μm. (c) Some tumor cells exhibit cytoplasmic positivity. MUC 6 immunostain, scale bar: 200 μm. (d) In some cells, positive staining is visible on the cell surfaces but not in the cytoplasm. MUC 1 immunostain, scale bar: 100 μm. (Figure 2(a)). Histopathologically, the majority of the tumor scopic AMs (Figure 3(d)) [2]. Tumor cell detachment was also often observed in the larger foci. cells were columnar, contained varying amounts of intracy- toplasmic mucins, and exhibited lepidic or papillary growth In the left lung tumor, the majority of tumor cells were in a single-layer arrangement (Figures 2(b)–2(d)). Small positive for MUC 5AC (Figures 4(a) and 4(b)) and CK7 groups of tumor cells had detached from the alveolar wall (data not shown). Abundant positive MUC 5AC staining and were scattered diffusely (Figure 3(a)). Tumor cell aggre- was also observed in the alveolar spaces (Figures 4(a) and gates floating within the alveolar spaces were round-shaped 4(b)). Some tumor cells exhibited intracytoplasmic positivity but had apical–basal polarity (Figure 3(b)). The apical surface for MUC 6 (antibody clone CLH 5, Abnova, Taiwan; of the tumor cells was located at the outer edge of the Figure 4(c)). Additionally, some tumor cells exhibited a aggregates but naturally lacked central fibrovascular cores positive MUC 1 reaction at the cell surface but not in the (Figure 3(b)), corresponding to the micropapillary pattern cytoplasm (Figure 4(d)), in contrast to our previously of the three morphological patterns of STAS [6]. reported case [9]. The tumor cells were negative for MUC 2, Slight neutrophil and macrophage infiltration was CK20, TTF-1, Napsin A, CDX2, and ALK (data not shown). observed at the detachment site. No desmoplastic reaction The clone and manufacturer of each antibody, except was observed. MUC6, have been described elsewhere [9]. The immunohis- Invasive foci with a maximum diameter of 2.2 cm were tochemical results of the right lung tumor were similar to observed in the tumor. These foci had a glandular or those of the left lung tumor. papillary structure (Figure 3(c)) and greatly reduced levels As shown in Figures 5 and 6 (5(a), 5(c), 6(a), and 6(b) of intracytoplasmic mucins. However, the nonmucinous show ANX A2 staining), granular cytoplasmic ANX A2 positivity was observed on the basal sides of tumor cells component accounted for <10% of the tumor. Lymphatic and vascular permeation and pleural invasion were not exhibiting lepidic or papillary growth (Figure 5(a)), whereas observed. Many scattered tumor cell aggregates that had no positive signal was detected at the cell–cell borders. Some detached from the main tumor were observed within the air tumor cells also exhibited weak ANX A2 positivity near the spaces and on the alveolar wall. The latter aggregates exhib- cell surfaces. A similar expression pattern was observed in the detached tumor cells, and positive expression was also ited lepidic or papillary growth that is suggestive of micro- Case Reports in Oncological Medicine 5 (a) (b) (c) (d) Figure 5: ANX A2 and collagen IV immunostaining of the lepidic or papillary component of the left lung tumor. (a) Tumor cells exhibit cytoplasmic ANX A2 positivity on the basal side. ANX A2 immunostain, scale bar: 400 μm. (b) A linear positive collagen IV reaction corresponding to the basement membrane is seen on the surfaces of both alveolar and vessel walls. Collagen IV immunostain, scale bar: 400 μm. (c) Detached tumor cells also exhibit cytoplasmic ANX A2 positivity on the basal side but not at the cell–cell borders. Positive staining is also observed in the exfoliative gap. ANX A2 immunostain, scale bar: 100 μm. (d) Weak or no collagen IV positivity is visible at the detachment site (arrows). Collagen IV immunostain, scale bar: 100 μm. observed in the exfoliation gap (Figure 5(c)). Noticeably, were seen. Similar findings were also observed at the detach- weaker ANX A2 positivity was observed in the tumor cells ment site (data not shown). floating within the alveolar spaces (Figure 6(a)). In the inva- sive component, stronger cytoplasmic positivity on the basal 3. Discussion side was observed relative to that observed in the lepidic or papillary growth component (Figure 6(b)), and membranous ANX A2 expression in the STAS/AM of the present tumor positivity was observed on the cell–cell borders (Figure 6(b)). differed slightly from that in our previously reported case As shown in Figures 5 and 6 (5(b), 5(d), and 6(c) show [9], particularly with regard to the following four points. (1) collagen IV staining), the lepidic or papillary area exhibited At the tumor cell detachment site, ANX A2 positivity was linear positive collagen IV staining corresponding to the observed in the cytoplasm on the basal side and in the exfo- BM, which was detected in both the surfaces of the alveolar liative gap. (2) No positive staining of the cell–cell borders and vessel walls (Figure 5(b)). However, either weak positiv- was observed in any component, except the invasive compo- ity or no signal was observed at the detachment site nent. (3) No distinct ANX A2 positivity was observed in (Figure 5(d)). In the invasive area, the desmoplastic stroma either the tumor cell aggregates floating within alveolar exhibited positive staining for collagen IV (Figure 6(c)), thus spaces or the minute foci of AM. (4) No ANX A2 positivity suggesting the presence of cancer-associated fibroblasts and was observed in the intracytoplasmic mucus. These discrep- neogenic collagen [18, 19]. No positive reactions were ancies are likely attributable to differences in the cellular observed in the tumor cells irrespective of invasiveness. characteristics and growth patterns between the tumors. In In an analysis of the AM, no positive reaction to ANX A2 the present case, both the mucin immunophenotype and beyond weak staining near the cell surface was observed in negative immunostaining for TTF-1 and CDX 2 suggest an the minute foci (i.e., tumor cells that had newly adhered to IMAC with gastric lineage [20–22]. The IMAC with gastric the alveolar wall; Figure 7(a)). However, the staining patterns lineage was first reported as a mucinous bronchioloalveolar in larger foci became similar to those observed in the lepidic carcinoma, with cell morphology mimicking the mucous or papillary growth component of the primary lesion cells of gastric pyloric mucosa and with its mucin immuno- (Figure 7(b)). Similar findings were observed at the detach- phenotype [21, 22]. Recently, Sonzogni et al. have classified ment site. Regarding collagen IV, the same findings as those the lineage of IMACs based on their immunophenotype, his- of lepidic or papillary growth component of primary lesion tology, and genetic alterations [20]. The IMAC with gastric 6 Case Reports in Oncological Medicine (a) (b) (c) Figure 6: Floating tumor cells (a) and invasive components (b and c) of the left lung tumor. (a) No distinct ANX A2 positivity is visible in the floating tumor cells within alveolar spaces. ANX A2 immunostain, scale bar: 100 μm. (b) Stronger cytoplasmic ANX A2 positivity is observed on the basal side, and positive staining is visible at the cell–cell borders. ANX A2 immunostain, scale bar: 200 μm. (c) The desmoplastic stroma exhibits positive collagen IV staining, particularly around the tumor cells. Collagen IV immunostain, scale bar: 1 mm. (a) (b) Figure 7: ANX A2 immunostaining of microscopic aerogenous metastases in the left lung. (a) No distinct positivity is visible in the minute foci. Scale bar: 200 μm. (b) Basal-side cytoplasmic positivity is detected in the larger metastatic foci. Scale bar: 200 μm. lineage is characterized by expressions of both membranous particularly given the intracytoplasmic expressions of both MUC 1 and MUC 5AC in the AC [9, 23]. MUC 1 and cytoplasmic MUC 6 and is suggested to be derived from the distal terminal bronchiole [20]. In contrast, ANX A2 moves inside the cell according to its roles [13]. our previously reported case was negative for subsequent Cytoplasmic ANX A2 is involved in cytoskeleton remodel- MUC 6 immunostaining (data not shown) and showed non- ing, exocytosis, and endocytosis [13]. In addition, a part of typical immunohistochemical results for any of the classifica- ANX A2 binds to the extracellular surface-membrane or is secreted [13]. This protein is expressed aberrantly in various tion by Sonzogni et al. [9, 20]. If the previous case is categorized, it is considered to exhibit characteristics of the cancers [14, 15] and may also be secreted [14]. A recent pancreatobiliary type in terms of the classification of the report further suggested that secreted ANX A2 contributes intraductal papillary mucinous neoplasms of the pancreas, to the proteolysis of the extracellular matrix [24]. In the Case Reports in Oncological Medicine 7 aggregates stretch to form a monolayer along the alveolar present tumor, we observed reduced collagen IV staining at the detachment site, thus leading us to consider that the wall in a manner independent of ANX A2. ANX A2 secreted from the basal sides of tumor cells might It remains unclear whether the right lung tumor origi- nated as a second primary lesion or was caused by an AM induce cell detachment by degrading the BM [13–15, 24]. Therefore, in IMAC, the detachment site of the tumor cells of the left lung tumor [17, 31, 32]. A similarly rapid clinical and the subcellular localization of ANX A2 might differ course was also reported in a previous case of multifocal depending on the immunophenotype of the detected IMAC [32]. Given that we did not genetically confirm mono- biomarkers [9, 20]. clonality between the tumors and could not detect a recur- rence in the ipsilateral lung, the right tumor might instead Indistinct membranous ANX A2 positivity in contact with the alveolar wall suggests poor capacity of IMAC for have been another lesion of multifocal IMAC. stromal invasion [1, 9, 25]. However, we observed more This report had some additional limitations. Particularly, intense cytoplasmic ANX A2 expression in the invasive com- we analyzed only two cases, including our previously ponent in the present IMAC case, thus suggesting that this reported case. Moreover, our analysis lacked a functional assay and an immunohistochemical investigation of various protein is involved in stromal invasion [9, 12, 13]. Therefore, we presume (1) that whether IMAC tumor cells detach into BM matrices. Further investigations are needed to confirm alveolar spaces or infiltrate toward the stroma depends our suppositions. on the amount of secreted ANX A2, (2) that the stromal In conclusion, our findings from this case suggest the invasion of IMAC requires a stronger cytoplasmic expression following points. (1) ANX A2 plays a dual role in tumor pro- gression and suppression according to its subcellular locali- of ANX A2, and (3) that extracellular membrane-bound ANX A2 enhances the aggressiveness of stromal invasion in zation. (2) The subcellular localization of ANX A2 depends AC [14, 15, 25, 26]. on the characteristics of the tumor cell, including the bio- We note that weaker ANX A2 positivity was observed at marker immunophenotype and tumor cell growth pattern. the cell–cell borders in the detachment site, even in our pre- (3) In the gastric lineage, ANX A2 secretion induces tumor cell detachment by degrading the BM. (4) The expression of viously reported case [9]. The expression of ANX A2 at the cell–cell borders is possibly related to the adhesions between ANX A2 (or lack thereof) at the cell–cell borders influences tumor cells and the suppression of tumor cell detachment the detachment site. [9, 12, 13]. In other words, little or no expression of ANX A2 at the cell–cell borders might facilitate tumor cell Ethical Approval detachment. Thus, we presumed that the lepidic or papil- lary component of the present tumor harbors a high Ethical approval for this study was provided by the ethics capacity for detachment [9, 12, 13]. Nevertheless, the small committee of Shizuoka General Hospital. groups of detached tumor cells showed a micropapillary pattern without the original monolayer arrangement. It Consent has been suggested that tumor cells forming a micropapil- lary pattern acquire resistance to anoikis (cell detachment- Written informed consent to publish the case details was induced apoptosis) and facilitate anchorage-independent obtained from the patient. growth [27]. Therefore, the morphological change of detached tumor cells is considered to be a defense reaction Conflicts of Interest against the loss of interaction with the BM [11, 27]. It has been known that ANX A2 is released extracellularly dur- The authors declare no conflicts of interest regarding the ing various stresses, including hypoxia [13], which is con- publication of this article. sistent with the findings that tumor cell aggregates floating within the alveolar spaces do not show a distinct ANX Acknowledgments A2-immunopositivity. Some reports have suggested that neutrophils and tumor- The authors would like to thank Enago (http://www associated macrophages and fibroblasts promote STAS/AM .enago.jp/) for providing the English language review. This [28, 29]. In the present case, we did not notice a particular report received no specific grant from any funding agency infiltration of any of those cell types into the tumor. Although in the public, commercial, or nonprofit sector. we agree that the tumor microenvironment is an important factor in STAS/AM, the present case suggests that the tumor References cells can induce STAS/AM directly. 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Case Reports in Oncological MedicineHindawi Publishing Corporation

Published: May 19, 2020

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