Genome profile in a extremely rare case of pulmonary sclerosing pneumocytoma presenting with diffusely-scattered nodules in the right lung

ABSTRACT

Background: Pulmonary sclerosing pneumocytoma (PSP) typically presents solitary and peripheral mass, while only rarely cases display unusual multiple lesions. We reported a extremely rare case of PSP with diffusely-scattered nodules in the right lung.

Case presentation: Diffusely round-shaped nodular shadows in the right lung were found by CT scan in a 31-year-old Chinese woman. The patient undergone the right pneumnectomy. Grossly, numerous small nodules, up to 2.5 cm in greatest dimension were identified in the upper, middle and lower lobes of the right lung. Histologically, the tumor presented the typical features of PSP, with a variable proportion of solid, sclerotic and papillary patterns. Immunohistochemical staining further revealed that cuboidal surface epithelial cells were positive for TTF-1, EMA, AE1/3 and vimentin (partially), and round or polygonal cells expressed TTF-1, vimentin, EMA (weakly), synaptophysin (partially), progesterone receptor (partially), and estrogen receptor (scatteredly). The patient has been followed up for 83 months after surgery by annual chest CT and no new lesions are detected in her left lung and other organs. The whole-exome sequencing identified 15 somatic mutations genes (MEGF6, DNAH5, AKT1, GPRIN2, PIK3AP1, FBXO40, HERC1, VPS16, MORN1, ZNF474, CTNNB1, ZNF251, TSC1, ATM, KDR). Pathway analysis showed possible pathways like the components of CTNNB1, AKT1, and TSC1 mutations in the PI3K/AKT signalings and AKT1, KDR and ATM in VEGF signaling pathway and AKT1 activation seemed closely related with these pathways.

Conclusion: According to our and previous data, PSP with diffuse or multiple lesions is very rare, and the patients are most commonly seen in women in Asian countries. The misdiagnosis rate by clinical and intraoperative frozen-section assessment is high because of the multiple nodules in the lung and its confusing histological features. Long time follow up indicates surgical resection should not be considered as the preferred strategy for treating multiple PSP in the intralobar sites. AKT1 activation may contribute to the development of PSP while the pathogenesis of diffuse or multiple PSP still needs to be further analyzed.

KEYWORDS:

• diffuse pulmonary nodules
• genome profile
• sclerosing pneumocytoma

Background

Pulmonary sclerosing pneumocytoma (PSP), also known as sclerosing hemangioma, is a slow-growing tumor, and behaves in a clinically benign fashion. No recurrence or disease-related deaths have been reported. Most tumors are solitary and peripheral, only rare cases display unusual presentations, such as regional lymph node metastases,^1-3 the progressive dissemination of pleura⁴ and multiple lesions.^1,4-17 This report described a unique case of PSP with diffusely-scattered nodules in the right lung, but spared the left lung within a seven-year follow-up in a 31 year-old woman.

Case presentation

A 31-year-old woman was admitted to Drum Tower hospital with abnormal shadows of multiple nodules in the right lung detected by chest X-ray at an annual medical examination 5 months ago. On examination at our center, the patient was afebrile and general physical examination was unremarkable. Laboratory data and tumor markers were all within normal ranges. Chest X-ray showed multiple nodules in the right lung and computed tomography (CT) demonstrated a number of round-shaped nodular shadows in the upper, middle and lower lobes of the right lung, up to 2.5 cm in greatest dimension (Fig. 1A and B). As metastatic lung tumors were highly suspected, wedge resection of the right lung was performed for a frozen section evaluation.

Figure 1. Chest X-ray and CT (CT) demonstrated diffusely round-shaped nodules in the upper, middle and lower lobes of the right lung (A and B). Grossly, multiple demarcated nodules with a maximum diameter up to 2.5 cm within the lung parenchyma (C) and some nodules located just beneath the visceral pleura (C and D).

Frozen section taken from one of the nodules showed that the tumor was composed of low grade neoplastic cells with low mitotic activity, forming solid and papillary architectures. Based on these findings, intraoperative pathology of the lung tumor showed low grade malignant potential and right pneumonectomy was performed. The upper, middle and lower lobes were 12 × 9 × 3cm, 9 × 6 × 3cm and 4.5 × 2.5 × 1.5cm, respectively. There were numerous well-circumscribed nodules with variable sizes, up to 2.5 cm in greatest dimension within the lung parenchyma (Fig. 1C), some of which were located just beneath the visceral pleura (Fig. 1D). In the gross specimen, the cut surfaces of these tumor nodules appeared firm and grayish-white (Fig. 1C) with foci of calcifications.

Microscopically, micronodules were found, and the tumors were well-circumscribed from the surrounding lung parenchyma but not encapsulated, and showed the typical histological features of PSP (Fig. 3), with a variable proportion of sclerotic (Fig. 2A), solid (Fig. 2B and C) and papillary (Fig. 2C and D) patterns, with rarely hemorrhagic cellular patterns. In minute nodules, the tumor mainly presented solid pattern or sclerotic stroma with peripheral papillary architectures. The tumor with the solid pattern was composed of a sheet-like proliferation of round to polygonal cells with pale cytoplasm. The tumor with the papillary pattern was composed of cuboidal surface cells and the round tumor cells presented a solid proliferation beneath the surface cells. In the mostly hyalinized stroma, nests of small polygonal cells with pale cytoplasm and scattered foam-like macrophages were observed. Some tumor nodules were located just beneath the visceral pleura (Fig. 1D).

Figure 2. The tumor showed the typical histological features of PSP, with varied proportions of sclerotic (A), solid (B and C) and papillary (C and D) patterns.

Immunohistochemically, cuboidal surface cells expressed thyroid transcription factor-1 (TTF-1) (Fig. 3A), EMA (Fig. 3B), Cytokeratin AE1/3 (Fig. 3C) and vimentin (partially), round or polygonal cells expressed TTF-1 (Fig. 3A), vimentin, EMA (weakly) (Fig. 3B), progesterone receptor (PR) (partially) (Fig. 3D), synaptophysin (partially), estrogen receptor (ER) (scatteredly), but chromogranin, CD56, CD31 and CD34 were negative in both types of tumor cells (Table 1). Based on the histological and immunohistochemical findings above, final diagnosis of diffuse PSP was made. The patient remained alive without any evidence of recurrence or metastasis for 83 months after the right lung resection, and no new nodules were found in her left lung and other organs.

Figure 3. Two populations of cells in PSP were present: solid growing polygonal round cells with pale cytoplasm and papillary structures covered with cuboidal surface cells. Cuboidal surface cells expressed TTF-1 (A), EMA (B) and AE1/3 (C), and round or polygonal cells expressed TTF-1 (A), EMA (weakly) (B), PR (partially) (D).

Table 1. Immunohistochemical staining pattern of PSP.

Markers	cuboidal surfacecells	round or polygonalcells
CK	+	−
EMA	+	dim +
Vimentin	Partially +	+
TTF-1	+	+
ER	Scattered +	Scattered +
PR	−	Partially +
syn	−	Partially +
CgA	−	−
CD56	−	−
CD34	−	−
CD31	−	−

+: positive, −: negative

To comprehensively elucidate the genetic alternations in this rare case, whole-exome sequencing was performed with the formalin-fixed paraffin-embedded tissue (FFPE, which fixed seven years ago) of PSP and the matched normal sample of peripheral blood. The mean coverage of the sequencing depth were 125X for PSP and 60X for matched normal sample, with an average of 96% and 90％of bases covered by at least 20 reads in each sample, respectively. Somatic mutations were identified using MuTect¹⁸ for point mutations and indels. The ANNOVAR package was used to select somatic mutations located in the exonic sequences and to predict their functional consequences.¹⁹ To obtain reliable and robust mutation calling, the following somatic variants were eliminated: (i) read depth fewer than 10 in either of the samples; (ii) polymorphisms referenced in either 1000 Genomes Project, Exome Aggregation Consortium, or Exome Sequencing Project with a minor allele frequency greater than 1%; and (iii) variants with variant allele fraction(VAF) between 45% and 55% in a copy-neutral region. Based on the above criteria, somatic mutations were identified in 15 genes (MEGF6, DNAH5, AKT1, GPRIN2, PIK3AP1, FBXO40, HERC1,VPS16, MORN1, ZNF474, CTNNB1, ZNF251, TSC1, ATM, KDR), which were overlapped with both the cancer gene census and cancer drivers database (Table 2).

Table 2. Somatic mutations in PSP.

Gene	Chr	Start	End	Ref	Alt	Exonic function	AA change	VAF, ％
MEGF6	chr1	3431990	3431990	G	T	nonsynonymous	Q236K	26.67%
DNAH5	chr5	13719210	13719210	C	A	stopgain	G4094X	25%
AKT1	chr14	105246551	105246551	C	T	nonsynonymous	E17K	22.58%
GPRIN2	chr10	46999524	46999524	C	A	nonsynonymous	P215H	18.52%
PIK3AP1	chr10	98388193	98388193	C	A	nonsynonymous	R478L	17.86%
FBXO40	chr3	121345660	121345660	C	A	nonsynonymous	P678Q	13.79%
HERC1	chr15	63922703	63922703	C	A	stopgain	G4310X	13.33%
VPS16	chr20	2840715	2840715	C	A	nonsynonymous	P53H	11.76%
MORN1	chr1	2318939	2318939	C	A	stopgain	G93X	11.63%
ZNF474	chr5	121487812	121487812	C	A	nonsynonymous	P43T	11.11%
CTNNB1	chr3	41266027	41266027	G	T	nonsynonymous	M8I	10.81%
ZNF251	chr8	145947981	145947981	C	A	nonsynonymous	G355V	10.71%
TSC1	chr9	135771655	135771655	C	A	nonsynonymous	M1103I	10%
ATM	chr11	108122613	108122613	G	T	stopgain	G553X	10%
KDR	chr4	55946269	55946269	C	A	nonsynonymous	G1304C	7.41%

AA, amino acid; Alt, altered allele; Ref, reference allele; VAF, Variant allele frequency

To further gain insights into the role of genomic alterations underlying PSP development, we analyzed the genome data through pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Mutated genes in PSP were significantly enriched in pathways related to cell survival, proliferation, growth, and angiogenesis activities (Table 3). Mutations in multiple components of the PI3K/AKT signaling, including CTNNB1, AKT1, and TSC1 were identified and several gene alterations such as AKT1, KDR, ATM, were also shown to be related to the VEGF signaling pathway (Fig. 4). Of note, AKT1 activation was closely connected to these pathways (Fig. 4).

Table 3. Top 20 important pathways in PSP.

Ingenuity Canonical Pathways	−log(p-value)	Ratio	Molecules
Glioblastoma Multiforme Signaling	5.41	0.0252	TSC1,AKT1,CTNNB1,ATM
Endometrial Cancer Signaling	4.94	0.0469	AKT1,CTNNB1,ATM
HER-2 Signaling in Breast Cancer	4.52	0.0341	TSC1,AKT1,ATM
VEGF Family Ligand-Receptor Interactions	4.52	0.0341	AKT1,KDR,ATM
Prostate Cancer Signaling	4.44	0.0319	AKT1,CTNNB1,ATM
VEGF Signaling	4.32	0.0291	AKT1,KDR,ATM
Mouse Embryonic Stem Cell Pluripotency	4.28	0.0283	AKT1,CTNNB1,ATM
p53 Signaling	4.22	0.027	AKT1,CTNNB1,ATM
Nitric Oxide Signaling in the Cardiovascular System	4.2	0.0265	AKT1,KDR,ATM
DNA damage-induced 14-3-3σ Signaling	4.1	0.105	AKT1,ATM
Role of NANOG in Mammalian Embryonic Stem Cell Pluripotency	4.1	0.0246	AKT1,CTNNB1,ATM
PI3K/AKT Signaling	4.08	0.0242	TSC1,AKT1,CTNNB1
14-3-3-mediated Signaling	4.02	0.0231	TSC1,AKT1,ATM
Gα12/13 Signaling	4.02	0.0231	AKT1,CTNNB1,ATM
Insulin Receptor Signaling	3.91	0.0213	TSC1,AKT1,ATM
Human Embryonic Stem Cell Pluripotency	3.89	0.021	AKT1,CTNNB1,ATM
Ovarian Cancer Signaling	3.88	0.0208	AKT1,CTNNB1,ATM
eNOS Signaling	3.79	0.0194	AKT1,KDR,ATM
Gap Junction Signaling	3.67	0.0176	AKT1,CTNNB1,ATM
Germ Cell-Sertoli Cell Junction Signaling	3.65	0.0173	AKT1,CTNNB1,ATM

Figure 4. AKT1 activation was closely related and seemed to have a vital function inside.

Discussion

PSP is an uncommon neoplasm, and its origin remains controversial but the histological features are distinctive. The majority of patients with PSP were presented with a solitary nodule, whereas multiple lesions have been reported in 4% of all cases in a Japanese cohort²⁰ and 3.6% in a Chinese group.²¹ To our knowledge, only 17 cases of multiple PSP in lung have been reported in the literatures^1,4-17 including the present case, almost of all patients (14/15) were female adults in the age of 16 to 73 years old and a majority of the reported cases (16/17) were from Asia, especially China, Japan and India. In the reported cases, there were two nodules in 3 cases, three in 3 cases, four in 1 case and more than four in 10 cases. The lesions were located at bilateral, right and left lungs in 8, 6 and 3 cases, respectively, and the maximum diameters of the nodules were no more than 5 cm in all the 17 cases (Table 4). Lung metastasis was usually considered as the favorable differential diagnosis against the patients with multiple pulmonary nodules of PSP. A systemic workup was always pursued but the definite clinical diagnosis was failed. PSP was usually an incidental finding by chest X-ray because the patients were often asymptomatic (Table 4), even with multiple pulmonary nodules, as described in our case. The following chest CT usually revealed well-defined and round-shaped multiple pulmonary small nodules. Only in one reported case, the patient complained of an oppressive feeling in her chest because of the enlarged tumor compressing the surrounding tissues.¹³ The physical examination was also asymptomatic, and the serum levels of some tumor markers, such as CEA, SLX, NSE, Pro-GRP, SCC, Cyfra21-1, CA19-9, AFP, CA125 and CA15-3, were normal.^4,6,12 All cases of multiple PSP were reported to be localized within the lung except one exhibiting pleural dissemination.⁴

Table 4. The reported cases of multiple PSP.

Case No.	Gender	Age	Location	Symptoms or signs	Number of nodules (diameter)	Histological features	Progress(follow-up time)	Refs.
1	F	40	Bilateral	Frequent hemoptysis, minimal cough and breathlessness of one year's duration.	Multiple (0.2cm-5cm)	Spindle shaped cells, many vascular spaces, hyalinization of the vessel walls, local aggregates of histiocytes, and infiltration of the stroma by inflammatory cells.	No(2 years after right upper lobectomy)	1980, Joshi et al.^10
2	F	48	Right lower	hemoptysis	Innumerable (microscopic to 3.7 cm)	Hemorrhagic, solid, papillary, and sclerotic features,	No(2 years after surgery)	1986, Noguchi et al.^14
3	F	39	Left lower	an oppressive feeling in her chest	3(NA)	NA	No(4 years after surgery)	1989, Maezato et al.^13
4	F	48	Bilateral: left lower and lingula, right	asymptomatic	4(0.6-5cm.)	Solid and papillary patterns with focal sclerosis and foamy histocytes	NA	1992, Lee et al.^11
5	F	42	Right lower	NA	2(1.2cm and 0.3cm)	Papillary, solid, hemorrhagic and sclerosis; multiple AAH-like area	No (follow-up time not available)	2004, Kim et al.^1
6	M	17	All lobes of the right lung	asymptomatic, sarcoma at leg	Multiple(1-2cm)	NA	NA	2004, Soumil et al.^17
7	F	38	all lobes of the right lung	asymptomatic	Multiple(afew millimeters to 3.1 cm)	A variable proportion of four patterns: solid, hemorrhagic, sclerotic and papillary	No(10 years after biopsy)	2005, Hishida et al.^8
8	F	32	Bilateral	asymptomatic	2(2.7cm and 1.6cm)	Hemorrhagic and sclerotic patterns	No(24 months after partial lung resection)	2005, Hanaoka et al.^6
9	F	16	Bilateral	asymptomatic	Multiple (size up to 5cm)	A mixture of areas with a papillary, solid and sclerosing patterns.	NA^*	2006, Ito et al.^9
10	F	50	Bilateral, all the lobes	asymptomatic	Multiple (NA)	NA#	NA#	2008, Pedro Boleo-Tome et al.^15]
11	F	73	Bilateral, right and left lower lobes.	unremarkable	3(0.3cm and 0.9cm, the third was unknown)	Pappilary and solid patterns	No(9 years after biopsy)	2009, Maeda et al.^12
12	F	37	Bilateral	with synovial sarcoma at hand	Multiple (varying sizes)	cuboidal cells arranged in papillary fronds with focal lymphocytic infiltrates	No(1 year after biopsy)	2010, Purandare et al.^16
13	F	57	Right lower, mediastinal, parietal and visceral pleura	unremarkable.	Multiple(size up to 2.5cm)	A variable proportion of papillary, sclerotic, solid, and hemorrhagic cellular patterns.	Irregular pleural thickness suggested progressive dissemination(3 years after the operation)	2011, Suzuki et al.^4
14	F	54	Middle lobe of the right lung and the basal segment of the left lower lobe.	unremarkable	3(2.5, 0.9and 0.8 cm)	Three of the histological characteristics appeared	No(2 years after biopsy)	2012, He et al.^7
15	NA	NA	Left lower lobe	NA	Multiple(0.8cm-3cm)	NA	No(NA)	2013, Chen et al.^22
16	NA	NA	Left lower and upper lobes	NA	2(NA)	NA	No(NA)	2013, Chen et al.^5

Note: NA, not available.

* Article in Japanese. # Article in Portuguese.

Histologically, being the same as a solitary lesion, multiple PSP was also mainly composed of variable proportions of four patterns and two types of cells. In typical cases, there was a mixture of papillary, solid, sclerotic and hemorrhagic components (Table 4). In these components, there were two populations of cells present, which were solid growing polygonal round cells with pale cytoplasm and cuboidal surface cells covering papillary structures, respectively. In the present case, the tumor predominantly displayed solid, sclerotic and papillary patterns, but rarely had hemorrhagic component. Because the patient exhibited diffuse pulmonary nodules and the tumors had the histological features of solid and papillary patterns, it was very difficult to establish the definitive diagnosis. The differential diagnosis mainly included primary or metastatic well-differentiated adenocarcinomas or carcinoid tumors. The small biopsy or fine needle aspiration can be performed but the diagnosis may not be rendered due to the limitation of sampling and lack of architectures. Intraoperative frozen-section might provide more diagnostic information and guild the operative procedure. Immunohistochemically, both surface-lining cuboidal and pale polygonal cells in the present case were positive for TTF-1, EMA and vimentin. However, only surface-lining cuboidal cells stained positive for AE1/3, whereas only pale polygonal cells were diffusely immunoreactive against vimentin, with ER, PR and synaptophysin partially or scatteredly stained.

Genetically, 15 somatic mutations (MEGF6, DNAH5, AKT1, GPRIN2, PIK3AP1, FBXO40, HERC1, VPS16, MORN1, ZNF474, CTNNB1,ZNF251, TSC1, ATM, KDR) were identified and pathway analysis showed multiple components of CTNNB1, AKT1, and TSC1 mutations in the PI3K/AKT signaling pathway. Notably, AKT1 activation was closely connected to the PI3K/AKT and VEGF pathways and might play an important role (Fig. 4). AKT1 encodes a serine/threonine kinase that regulates many cellular processes, such as proliferation, survival, and growth.²³ AKT1 E17K is localized to the pleckstrin homology domain, which is crucial for membrane localization and downstream activation of AKT1.²⁴ This alteration is the most frequently reported AKT1 mutation and has been found in multiple solid tumor types including breast, skin, urinary tract, and endometrial cancers.²⁵ The frequency of AKT1 mutation in cancer is relatively low while breast cancer having the highest frequency(4-8%).^26,27 In addition, CTNNB1 and beta-catenin, are members of the WNT signaling pathway and component of cadherin-based adherens junctions. Imbalance of beta-catenin has been implicated in cancer progression and metastasis.²⁸ Multiple solid tumor types such as soft tissue, pituitary, liver, endometrium cancers have been reported to carry CTNNB1 mutations. Remarkably, AKT1 and CTNNB1 are recurrent somatic genes that have been previously reported in pulmonary sclerosing hemangiomas (31 of 68, 45.6%, AKT1; 23 of 68, 33.8%, p.E17K; 3 of 68, 4.4%, CTNNB1), implicating that mutations in these genes may act as important tumor promoting event in PSP.²⁹ Interestingly, several gene alterations such as AKT1, KDR and ATM, are also related to the VEGF signaling pathway that activates angiogenesis, suggesting their possible roles in the vascular phenotype of PSP. Moreover, AKT1 has been engaged as a target in ongoing clinical trials,³⁰ suggesting that AKT1 inhibitors may have potential therapeutic value in PSP.

According to the WHO Classification of pulmonary tumors, current consensus to PSP favored a benign or very low grade neoplasm arising from primitive respiratory epithelium. As to the cases presented multiple lesions located at lung, all patients remained alive, well and no evidence of recurrence or metastasis for 1 to 10 years after surgical resection, or the tumor nodules remained unchanged after biopsy. The present case did not display any evidence of occurrence or metastasis either for 83 months after the resection of right lung. However, there was still one reported progressive dissemination case, which showed irregular thickness of the interlobular pleura demonstrated on CT 3 years postoperatively.⁴ Thus, though PSP presented multiple lesions behaved as a benign condition, serious follow-up should be conducted for some cases with pleural dissemination. Additionally, it was very interesting and worthy of further study to explore why this benign or very low grade neoplasm involved unilateral lung lobes in some cases but bilateral lung lobes in other cases.

Conclusion

PSP with multiple lesions is extremely rare, and the patients are most commonly women in Asian countries, such as China and Japan. The misdiagnosis rate by clinical and intraoperative frozen-section assessment is high because the multiple nodules in the lung and its histological features could mimic well-differentiated adenocarcinoma or carcinoids. According to the data of long time follow up, it seems that surgical resection should not be considered as the preferred strategy for treating multiple PSP in the intralobar sites, because of no evidence of recurrence, metastasis, enlargement of tumor nodules or respiratory dysfunction in almost all reported cases, but further follow-up is still necessary based on the limited reported cases. Somatic mutations sequencing and pathway analysis conclude that AKT1 activation may contribute to the development of PSP while the pathogenesis of diffuse or multiple PSP still needs to be further analyzed.

Consent

Written informed consent was obtained from the patient and her family for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Abbreviations
PSP	=	pulmonary sclerosing pneumocytoma
CT	=	computed tomography
TTF-1	=	thyroid transcription factor-1
PR	=	progesterone receptor
ER	=	estrogen receptor