healthmedicinet

A 38-year-old female patient was admitted to the hospital after suffering upper abdomen
pain and fatigue for about 1 week in 2011. She has no history of smoking and alcohol
or drug abuse. The patient denied immunodeficiency, history of malignancy and toxin
exposure, and family history of genetic disorders. The abdomen computed tomography
(CT) (Fig. 1) has been performed for further examination. The result showed splenomegaly with
multifocal low-density lesions. Other organs including liver, pancreas, renal, and
adrenal were normal. Considering possibility of splenic neoplasm, and for further
clarification, splenectomy was carried out. Histopathology (Fig. 2) evaluation showed well-formed anastomosing vessel channels and proliferated endothelial
cell with mild atypia but inconspicuously malignant. Immunohistochemistry (Fig. 2) revealed positive staining of CD31and CD34 but negative staining of factor VIII,
CD68, p53. The pathology findings supported the diagnosis of hemangioma. Although
no necrosis or hemorrhage was found in macroscopic and histological examination, in
consideration of splenomegaly and anastomosing capillary-sized vessels, the well-differentiated
angiosarcoma cannot be excluded. After splenectomy, the patient was quickly relieved
from the pain and discharged without any postoperative complications. Then, the patient
got lost to follow-up.

Fig. 1. Abdomen CT scan in 2011 (a, b) demonstrated splenomegaly and multiple irregular low-density lesions. Liver, renal,
adrenal, and pancreas are normal. The MRI in 2013 presented multiple cystic lesions
in different sizes diffused in liver. The lesions displayed as inhomogeneous mixed
signal, with high signal intensity on T2-weighted image (T2WI, c) and diffusion-weighted image (DWI, d). Some lesions that indicated eccentric or central low signal intensity were suspected
intertumoral hemorrhage. Nodule lesion with abnormal sign as that in the liver was
also found in remnant accessory spleen (arrow)

Fig. 2. Tumor in spleen shows well-formed anastomosing vessel channels (a, HE 100×), proliferated endothelial cells with mild atypia (b, HE 400×), immunohistochemistry revealed positive staining of CD31 (c, 100×) and CD34 (d, 100×). Tumor cells from bone biopsy showed anastomosing vessel channels with proliferated
endothelial cells without obvious malignant as that in spleen (e, HE 100×; f, HE 400×). Tumor cells from liver biopsy showed a handful of endothelial cells without
obvious malignant (g, HE 100×)

Two years after splenectomy in December 2013, the patient presented with worsening
upper abdomen pain, fatigue, and frequent dizziness for 3 months. On physical examination,
she showed severe anemic appearance. There were no clinically palpable node and no
papilledema. There was no evidence of bruising and easy bleeding. The initial lab
tests were as follows: low hemoglobin (45 g/L, normal range 113–151), an increase
of D-dimer, 78,700 ug/L (normal range 0–700ug/L), and a mild increase of aspartate
aminotransferase (AST), 48 U/L (normal range 8–40U/L). Further examination of upper
abdominal magnetic resonance imaging (MRI) was implemented. The result (Fig. 3) showed multiple nodes in the liver and accessory spleen with rich blood supply and
signals of intratumoral hemorrhage. With these findings in MRI, fine-needle biopsy
was obtained of the hepatic lesions. Due to further clarification of anemia, bone
marrow trephine biopsy as well as smear for cytological examination were performed.
The pathology evaluation (Fig. 2) on hepatic demonstrated a small amount of proliferated endothelial cells. The result
of pathology findings of bone marrow biopsy (Fig. 2) showed anastomosing vascular channels, proliferated endothelial cells with few mitotic
figures and mild nuclear atypia. The similarity of pathological findings in the liver,
spleen, and bone marrow and clinical image presentation supported that the final diagnosis
was well-differentiated angiosarcoma of the spleen with bone and liver metastasis.
The patient was initially treated with analgesia and blood transfusion. Because few
data can be acquired to guide chemotherapy and poor therapeutic effect, she gave up
further chemotherapy after discussing with her family members. She was discharged
in a few days after slight relief from pain and fatigue. Follow-up showed she passed
away 3 months after discharge.

Fig. 3. Summary of chromosomes copy number changes of splenic angiosarcoma detected by aCGH.
The blue color on the right side represents copy number gains and red color on the left side represents copy number losses

DNA was extracted from ten 20-?m thick ribbons of paraffin-embedded tumor of spleen
from FFPE tissue blocks using DNeasy Blood Tissue Kit (Cat#69506, QIAGEN, GmBH, Germany),
following the manufacturer’s instructions. A total of 80 ng sample of DNA was sent
for processing via OconScanTM FFPE Express 2.0 service as manufacturer’s instructions.
The OncoScan assay contains more than 300,000 copy number and single nucleotide polymorphism
(SNP) oligonucleotide probes with a median probe spacing of 4200 kb, with 541 somatic
mutations for known cancer genes. The arrays were scanned by GeneChip® Scanner 3000
(Cat#00-00212, Affymetrix, Santa Clara, CA, USA) and Command Console Software 3.1
(Affymetrix, Santa Clara, CA, USA) with default settings. Raw data that passed quality
control were further analyzed by Affymetrix® OconScan Analysis Suite (Affymetrix,
Santa Clara, CA, USA).

Statistical analysis of the CGH data was performed by FASST2 Segmentation method.
In order to adjust the sensitivity of the segmentation algorithm, we determined the
significant threshold at 1.0E-8, specified 1000 kb being max contiguous probe spacing.
The minimum number of probes per segment required to eliminate small CNVs was five;
gains and losses were defined at ±3?×?SD of all probes, and the threshold was adjusted
at ±0.5 for both.

Copy number change of this angiosarcoma of spleen sample showed diverse DNA copy number
alternations including copy number loss, copy number gain, and homozygous copy loss.
The copy number changes were summarized in Fig. 3 and Table 1. The result (Fig. 4 and Table 1) revealed that copy number gain at 11q23.2, 11q24.3, 12q24.33, 13q34, copy number
loss at 1q24.2-q31.3, 1q41-q42.2, 1 q42.3-q43, 2q36.3-q37.3, 2q37.7, 3q13.33-q26.2,
3q28-q29, 9p11.2, 13q11, 15q11, homozygous copy loss at 8p11.22, 22q11.23.

Table 1. Chromosomes copy number changes of splenic angiosarcoma

Fig. 4. Gene ontology groups enriched by genes with copy number losses and gains. EASE score
higher than 0.1 has been listed in the figure. The gene functional analysis revealed
enrichments for otology terms related to diverse pathways, involved renin-angiotensin
system and ECM-receptor interaction pathways signaling pathways

We found that a total of 1067 genes were affected by copy number changes according
to the result of aCGH. In order to discuss the probably functions of altered genes,
397 out of 1067 genes with known biological functions, we used the Database for Annotation,
Visualization and Integrated Discovery (DAVID) v6.7 (free online bioinformatics resources
at http://david.abcc.ncifcrf.gov/) to clustered Gene Ontology (GO) group. The enriched GO groups (Fig. 4) were ranked according to statistical significance measured by EASE score (EASE score
= ?LgP), a modified Fish’s exact p value. The result illustrates that five relevant pathways have been affected by copy
number changes as EASE score upper than 0.1. It is noteworthy that renin-angiotensin
system and ECM-receptor interaction pathways exhibit statistically EASE score (p??0.05).