Case Report

A case of microscopic, multiple sclerosing pneumocytoma

Hitomi Kawai,1 Norio Takayashiki,2 Haruo Otani,3 Shingo Sakashita 4 and Masayuki Noguchi4

1Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan, 2Department ofPathology, Mito Kyodo Hospital, Ibaraki, Japan, 3Department of Pathology, Mito Saiseikai Hospital, Ibaraki, Japan,and 4Department of Pathology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

Sclerosing pneumocytoma is a rare tumor of the lung,commonly affecting middle-aged women, and is mostlyisolated. Although this tumor is thought to be derived fromprimitive respiratory epithelial cells, the characteristics ofthe precursor cells are still unknown. A 19-year-old womanpresented with multiple nodules in the right lung. Partialresection of the right middle lobe was performed, andseven sclerosing pneumocytomas, including four thatwere microscopic, were detected. The latter showed asimple papillary pattern, and three of them consisted ofonly round cell-like cells (single population). Interestingly,these round cell-like cells were positive for both p63 andTTF-1, but totally negative for SP-A. On the other hand, thetumor cells of the other four sclerosing pneumocytomasshowing a papillary pattern with a dual population, werediffusely positive for TTF-1 and focally positive for SP-A(only in surface cells), but negative or very focally positivefor p63. It has been reported that p63-positive stem cell-like cells are present in the distal airway and have potentialto differentiate into type II pneumocytes. The immunohisto-chemical features of these multiple microscopic lesionssuggest that the p63-TTF-1 double-positive cells are candi-date precursor cells of sclerosing pneumocytoma.

Key words: distal airway stem cell, microscopic, p63,sclerosing pneumocytoma, SP-A, terminal respiratory unit,TTF-1

Sclerosing pneumocytoma is a rare tumor of pneumocyticorigin with a dual population of surface cells resemblingtype II pneumocytes and round cells, with slightly differenthistogenetic profiles.1–5 Combinations of various histologicalfeatures (papillary, solid, sclerotic and hemorrhagic pat-terns) are also characteristic. Sclerosing pneumocytomausually appears as an isolated solid nodule and never

metastasizes. Sometimes, however, multiple forms of scle-rosing pneumocytoma may be encountered.6–9 Noguchi etal. have reported multiple minute and microscopic scleros-ing pneumocytomas that tended to be composed of onlyepithelial cells arranged in papillary pattern, and suggestedthat the papillary lesions might represent sclerosing pneu-mocytoma at a very early stage.6 Although it has also beensuggested that sclerosing pneumocytoma might be derivedfrom primitive respiratory epithelial cells, the characteristicsof its precursor cells and details of its molecular tumorigen-esis are still unknown.10–18

We have experienced a unique case of multiple scleros-ing pneumocytoma in a 19-year-old woman. Seven scleros-ing pneumocytomas, including four that were microscopic,were present. All of the microscopic tumors exhibited asolely papillary pattern. One of them had a dual populationof surface cells and round cells, but the other three werecomposed solely of round cell-like cells that were positivefor both TTF-1 and p63. We hypothesized that the tumorcells showing double positivity for TTF-1 and p63 were theprecursor cells of sclerosing pneumocytoma.

CLINICAL SUMMARY

An asymptomatic 19-year-old Japanese woman with unre-markable family and medical histories presented for amedical checkup in 2014, and chest X-ray demonstratedmultiple coin lesions in her right lung. A computed tomogra-phy scan of the chest demonstrated dozens of small nodulesup to 18mm in diameter in the right upper and middle lobes.Clinically, granulomatous disease was suspected. To confirmthe diagnosis, partial resection of the right middle lobe wasperformed by video-assisted thoracic surgery.

PATHOLOGICAL FINDINGS

The resected materials were fixed with 10% buffered forma-lin for 2 days and embedded in paraffin. For immunostaining,

Correspondence: Shingo Sakashita, MD, Faculty of Medicine,Department of Pathology, University of Tsukuba, Tennodai, Tsukuba-shi, Ibaraki 305-8575, Japan. Email: sakashingo@hotmail.comReceived 30 March 2017. Accepted for publication 10 January

2018.© 2018 Japanese Society of Pathology and John Wiley & SonsAustralia, Ltd

Pathology International 2018; 68: 196–201 doi:10.1111/pin.12648

These primary antibodies were used: TTF-1 (mouse mono-clonal, 8G7G3/1, diluted; Dako, Glostrup, Denmark), SP-A(mouse monoclonal, PE10, 1:500; Dako) and p63 (mousemonoclonal, Dak-p63, diluted; Dako). The immunohisto-chemical processes were performed with a histostainer(Nichirei Biosciences, Tokyo, Japan). We evaluated thedegree of positivity as follows: negative (�, 0%); very focallypositive (þ, <25% in hot spots); focally positive (þþ, <50%in hot spots); diffusely positive (þþþ, >50% and a uniformpattern). For immunofluorescence, these primary antibodieswere used: TTF-1 (rabbit monoclonal, ab76013, 1:250;Abcam, Cambridge, UK) and p63 (mouse monoclonal,M731729, 1:10; Dako). The secondary antibodies used wereThermo Fisher Scientific Goat anti-rabbit IgG (Alexa Fluor,594, red, 1:50) and Thermo Fisher Scientific Goat anti-mouse IgG (Alexa Fluor, 488, green, 1:50; Thermo FisherScientific, Waltham, MD, USA). Visualization was performedusing a KEYENCE HS ALL-in-one microscope (BZ-900,KEYENCE, Osaka, Japan) with BZ-II observation and analy-sis application. The clones of p63 antibodies (both forimmunostaining and immunofluorescence) are Dak-63,which recognize p63 isoforms both Tap63 and DNp63(p40).Macroscopically, there were three well-circumscribed

tumors (tumor no. 1-A, 1-B, and 1-C, Table 1), the largestmeasuring 16�16� 15mm (Table 1). The surface of thelargest tumor was gray-white and medullary (Fig. 1a).Microscopically, the largest tumor showed three histologicalpatterns: papillary, sclerotic and solid (Fig. 1b). The papil-lary area contained a dual population of surface cellsresembling type II pneumocytes and round cells (Fig. 1c).Immunohistochemistry showed that the surface cells werediffusely positive for TTF-1 and focally positive for SP-A, but

negative for p63. The round cells were positive for TTF-1and focally and slightly positive for p63, but negative for SP-A (Fig. 1d–f).

The other two tumors detected macroscopically were both5� 5mm in size and composed of solid, papillary andhemorrhagic components, and solid and papillary compo-nents, respectively (tumor no. 1-B and 1-C, Table 1). Thetumor cells of No. 1-B were positive for TTF-1 (diffuse) andSP-A (only surface cells), and negative for p63. The tumorcells of No. 1-C were positive for TTF-1 (diffuse), SP-A (onlysurface cells) and p63 (only very focally). We also detectedfour microscopic tumors (0.3–1.5mm in diameter) whichwere not evident macroscopically (tumors No.1-D, 1-E, 1-Fand 1-G, Table 1) and they were distributed at random. All ofthe microscopic tumors showed only a papillary patternhistologically (Fig. 2a,c). Although one microscopictumor No.1-E was composed of surface cells and roundcells (Fig. 2b), the other three (tumors no.1-D, 1-F and 1-G,Table 1) consisted of only round cell-like cells (single popula-tion) (Fig. 2d). Alveolar elastic fibers totally disappeared in thethree macroscopic pneumocytomas, whereas microscopicpneumocytomas maintained some amount of alveolarelastic fibers inside. It might suggest that these lesions werevery early stage of macroscopic pneumocytomas.

The microscopic tumor showing a dual population (no.1-E) was positive for TTF-1 (diffuse), SP-A (only surfacecells), and p63 (only very focally). The others, consisting ofonly round cell-like cells, were diffusely positive for bothTTF-1 and p63 (Fig. 3a–c), but negative for SP-A. Allmacroscopic and microscopic lesions (both dual populationand single population) were negative for CK5/6 (data notshown).

Table 1 Histology and immunohistochemical profile about eight cases of sclerosing pneumocytomas (15 tumors). This case is No. 1(including seven tumors)

Case Sex Age Tumor Size (mm) Histology TTF-1 SP-A p63

This case 1 F 19 1-A 16�16�15 SO>PA>SC (þþþ) (þ) (þ)1-B 5�5 SO>PA>HE (þþþ) (þ) (�)1-C 5�5 SO>PA (þþþ) (þ) (þ)1-D 1.5�1.2 PA� (þþþ) (�) (þþþ)1-E 0.4�0.4 PA (þþþ) (þ) (þ)1-F 0.4�0.4 PA� (þþþ) (�) (þþþ)1-G 0.3�0.3 PA� (þþþ) (�) (þþþ)

Others cases 2 F 73 2 20�15 SO>SC>PA (þþþ) (þþ) (þ)3 F 67 3-A 3�1.3 SO>SC (þþþ) (þ) (þ)

3-B 15�13 SC>SO>PA (þþþ) (þ) (þ)4 F 63 4 13�12 HE>SC>SO (þþþ) (þ) (�)5 F 52 5 11�11 SC>PA>SO (þþþ) (þ) (þ)6 F 60 6 16�13 SC>HE>SO (þþþ) (þ) (þ)7 F 67 7 12�10 SC>HE>SO (þþþ) (þþ) (þ)8 F 50 8 5�5�5 PA>SC (þþþ) (þþ) (þ)

�, papillary pattern consists of only round- cell-like cells (without dual population).(�), 0%; (þ), <25%; (þþ), 25–50%; (þþþ), >50%.SO, solid pattern; PA, papllary pattern; SC, sclerotic pattern; HE, hemorrhagic pattern.

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Figure 1 (a–d) Sclerosing pneumocytoma in a 19-year-old woman. (a) Macroscopically, the surface of the largest sclerosingpneumocytoma is gray-white and medullary. (b) Histologically, the largest sclerosing pneumocytoma shows papillary (�), solid (��) andsclerotic patterns. (c) In the papillary area, a dual cell population is evident (!: surface cell, ): round cell). (d–f) Immunohistochemicalstaining for TTF-1(d), SP-A (e) and p63 (f).

Figure 2 (a–d) Microscopic lesions. (a, b) Tumor No.1-E shows a dual cell population. (c, d) Tumor No.1-D consists of only round cell-likecells and show a single cell population.

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198 H. Kawai et al.

To determine whether the round cells in the microscopiclesions without a dual population were double-positive forboth p63 and TTF-1, we performed an immunofluorescencestudy, and found that the tumor cells were double-positivefor p63 (green) and TTF-1 (red) (Fig. 3d–f). To compare theimmunohistochemical profiles of sclerosing pneumocyto-mas, we reviewed a total of 15 tumors from eight cases(including the present one) diagnosed as sclerosing pneu-mocytoma at Tsukuba University Hospital and Mito KyodoHospital between 1996 and 2016, and examined theirpositivity for TTF-1, SP-A and p63. The results were shownin Table 1. All of the sclerosing pneumocytomas werediffusely positive for TTF-1. Among the macroscopic tu-mors, positivity for SP-A and p63 varied, but p63-positivecells were detected only very focally (0–25%, hot spot). Forreference, we also examined the expression of TTF-1, SP-A, and p63 of normal terminal bronchial tissue. Most of thesurface epithelial cells were positive for p63 and severalp63-positive surface epithelial cells were also positive forTTF-1 (double-positive) (Fig. 3g,h). On the other hand,CK5/6-positive surface epithelial cells were not detected(data not shown).

DISCUSSION

We experienced a 19-year-old female patient with sevensclerosing pneumocytomas, including four microscopic tu-mors. This case had two unique features: the patient wasyounger than usual and had several microscopic tumors.Several cases of multiple sclerosing pneumocytoma of thelung have been reported,6–9 and some of them alsoincluded microscopic lesions.6 Noguchi et al. reportedmultiple sclerosing pneumocytomas containing innumerablelesions varying in size from microscopic to 3.7 cm indiameter.6 They examined 24 tumors microscopically andfound that small lesions, particularly minute ones, tended tobe composed of only epithelial cells arranged in papillarypatterns and round cells in solid areas. They concluded thatboth of these cell types were the main components ofsclerosing peumocytoma. In our case, we also found fourmicroscopic tumors showing just a papillary pattern, andEVG staining showed some amount of elastic fibers remain-ing (tumors no.1-D, 1-E, 1-F and 1-G, Table 1). Thesefindings suggested that the papillary lesions representedthe very early stage of sclerosing pneumocytoma. In this

Figure 3 (a–h) Immunohistochemistry and immunofluorescence of microscopic lesions consisting of only round cell-like cells and terminalrespiratory unit (TRU) cells. (a–c): Immunohistochemical analysis shows that round cell like-cells are positive for both TTF-1 (b) and p63 (c).(d–f): Immunofluorescence reveals cells that are double-positive for p63 and TTF-1(d p63, e TTF-1, f merge). (g) Some amount of p63-TTF-1 double-positive cells are located at the terminal respiratory unit (TRU)(g p63, h TTF-1).

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case, one of the microscopic tumors, no.1-E, showing apapillary pattern was composed of surface cells and roundcells (i.e., dual population), and the other three, no.1-D, 1-Fand 1-G, were composed of round cell-like cells (singlepopulation).

Recently, Kumar et al. reported that p63- and CK5/6-positive cells were present in the distal airway of the lung inmouse models, and suggested that these cells are ofpneumocyte lineage.19 Zuo et al. showed that p63- andCK5/6- positive distal airway stem cells proliferate anddifferentiate into type I and type II pneumocytes in responseto influenza-induced lung damage in mouse models.20

These reports suggested that p63-positive distal airwaystem cells have the potential to differentiate into type IIpneumocytes.

In our reference sample, a proportion of epithelial cellsin the terminal respiratory unit (TRU) were also positivefor both TTF-1 and p63 (Fig. 3g,h), but negative for CK5/6(data not shown). It might indicate that the mouse stemcells were positive for CK5/6, but human stem cells werenot. In other words, in humans, the cells positive for bothTTF-1 and p63 could be regarded as stem cells differenti-ating pneumocytes.

Recently, Zhang J et al. reported that over 75% of TTF-1positive cuboidal cells in sclerosing pneumocytomas werepositive for p63 (16/18 cases)16. And Wu J et al. reportedthat p40 was positive for TTF-1 positive cuboidal cells.17

They suggested sclerosing pneumocytoma was originatedfrom pluripotent original respiratory epithelial cells. In ourcase, the microscopic sclerosing pneumocytomas consistedof p63-TTF-1 double-positive cells, whereas macroscopiclesions had less propotion of p63-TTF-1 double-positivecells. This result corresponded to these previous two reports.

In order to examine the expression profiles of pneomo-cytes stem cell markers and differentiation markers insclerosing pneumocytomas, especially microscopic lesions,

we performed immunostaining for p63, TTF-1 and SP-A(Table 1). In our cases, three microscopic tumors (tumor 1-D, 1-F and 1-G) showed diffuse (over 75%) positivity of p63and TTF-1. All of them showed just papillary pattern withsingle population, composed of round cell-like cells, notcuboidal cells, and totally negative for SP-A. On the otherhand, the tumor cells in three macroscopic tumors and onemicroscopic tumors with dual population were diffuselypositive for TTF-1, focally positive for SP-A and p63. p63positive cells were less numerous than those positive forTTF-1 and two of the four tumors were totally negative forp63. Our findings showed the difference of p63 positivity insclerosing pneumocytomas is related to the size, histologi-cal feature and expression of pneumocyte differentiationmarkers. These results indicated that the round cell-likecells forming the microscopic tumors had the characteristicsof differentiating pneumocytes but might be still immature incomparison to those forming the macroscopic tumors.

Recently, Jung SH et al. examined 44 cases of sclerosingpneumocytoma and found non-silent and reccurent muta-tions of AKT1 (19 of 44, 43.2%), CTNNB1 (b-catenin) (2 of44) and ARID1B (2 of 44).18 All of the AKT1 mutations werelocalized to the pleckstrin homology domain, which werethought to be associated with tumor proliferation. Thebiological relationship between AKT1 mutation and scleros-ing pneumocytoma is still unlnown, but the AKT1 activationmight be associated with the oncogenesis. Although thereare many technical problems to use formalin fixed andmicrodissected materials for genetic analysis, this case maybe suitable for the investigation of the oncogenesis andmultiplicity of multiple sclerosing pneumocytomas.

In the present case, we found that multiple sclerosingpneumocytomas including some microscopic tumors, andwe revealed the biological characteristics of the tumor cellsusing immunohistochemistry. The tumor cells of micro-scopic, single-population tumors showed double positivity

Figure 4 Scheme suggesting the role of p63-TTF-1 double-positive cells. Hypothesis to explain the progression of sclerosingpneumocytoma.

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200 H. Kawai et al.

for p63 and TTF-1, these microscopic tumors seemed to beprecursor lesions of sclerosing pneumocytoma, differentiat-ing and acquiring the characteristics of pneumocytes in thecourse of tumor progression (Fig. 4).

DISCLOSURE STATEMENT

None declared.

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