Astroblastoma: beside being a tumor entity, an occasional phenotype of astrocytic gliomas?

Figures & data

Figure 1 T1-weighted contrast enhancement magnetic resonance imaging scan of Case 1.

Table 1 List of primary antibodies used for immunohistochemistry

Antibody	Source	Dilution	Code	Manufacturer
Ki-67/MIB-1a	Mouse	1:100	M7240	Dako Denmark A/S, Glostrup, Denmark
GFAP	Mouse	1:200	M0761	Dako Denmark A/S, Glostrup, Denmark
IDH1^RI32Ha	Mouse	1:20	DIA H09	Dianova International, Lausanne, Switzerland
Vimentina	Mouse	Prediluted	790–2917	Ventana Medical Systems Inc., Tucson, AZ, USA
CD34a	Mouse	Prediluted	790–2927	Ventana Medical Systems Inc., Tucson, AZ, USA
CD68a	Mouse	Prediluted	790–2931	Ventana Medical Systems Inc., Tucson, AZ, USA

Note:

a Heat-induced epitope retrieval required.

Abbreviations: GFAP, glial fibrillary acidic protein; IDH1, isocitrate dehydrogenase 1.

Table 2 List of primary antibodies used for immunofluorescence

Antibody	Source	Dilution	Code	Manufacturer
GFAP	Rabbit	1:200	Z0334	Dako Denmark A/S, Glostrup, Denmark
Nestina	Rabbit	1:200	AB5922	EMD Millipore, Billerica, MA, USA
CD133/1 (AC133)	Mouse	1:20	130-090-422	Miltenyi Biotec, Bergisch Gladbach, Germany
SOX2a	Mouse	1:100	MAB2018	R&E Systems Inc., Billerica, MA, USA
Musashi-1	Rabbit	1:200	AB5977	EMD Millipore, Billerica, MA, USA
RESTa	Rabbit	1:150	IHC-00141	Bethyl Laboratories Inc., Montgomery, TX, USA
GalCa	Mouse	1:100	MAB342	Chemicon, EMD Millipore, Billerica, MA, USA
βIII-tubulina	Mouse	1:250	MAB1637	Chemicon, EMD Millipore, Billerica, MA, USA

Note:

a Heat-induced epitope retrieval required.

Abbreviations: GalC, galactosylceramidase; GFAP, glial fibrillary acidic protein; REST, RE1-silencing transcription factor; SOX2, sex determining region Y-box 2.

Figure 2 Immunohistochemistry.

Notes: Case 1: (A) perivascular cell arrangement; H&E, 10×; (B) necrosis and polymorphic cells; H&E, 10×; (C) GFAP-positive perivascular cells; DAB, 10×; (D) Ki-67/MIB-1 LI; DAB, 20×. Case 2 (first intervention): (E) aspect of oligodendroglioma; H&E, 10×; (F) GFAP-negative palisades; DAB, 10×; (G) rhythms and palisades; H&E, 10×; (H) Ki-67/MIB-1 LI; DAB, 20×. Case 2 (second intervention): (I) perivascular arrangement of astrocytic cells; H&E, 20×; (J) id, H&E; 20×; (K) GFAP-positive perivascular cells; DAB, 20×; (L) Ki-67/MIB-1 LI; DAB, 10×. Case 3 (M) perivascular arrangement of large cells; H&E, 20×; (N) polymorphic cells and vessel hyalinization; H&E, 10×; (O) GFAP-positive perivascular cells; DAB, 20×; (P) Ki-67/MIB-1 LI; DAB, 20×.
Abbreviations: DAB, 3,3’-Diaminobenzidine; H&E, hematoxylin and eosin; LI, labeling index; id, perivascular arrangement of astrocytic cells.

Figure 3 Sanger direct sequencing.

Notes: Case 1: (A) electropherograms for the stop mutation c.822G>A (p.Trp274*) in PTEN exon 8 in lymphocytes, primary tumor, neurospheres, and adherent cells; (B) electropherograms for the missense mutation c.824G>A (p.Cys275Tyr) in TP53 exon 8 in lymphocytes, primary tumor, neurospheres, and adherent cells. Case 2 (first intervention): (C) electropherograms for the splice variant c.209 + 1G>T in PTEN intron 3 in lymphocytes, primary tumor, and adherent cells.

Table 3 Genetic and epigenetic alterations in tumors and matched cell lines

Case	LOH	EGFR amplification	IDH1 (Arg132)/IDH2 (Arg172)	TP53 mutations	MGMT methylation	PTEN mutations	1p/19q co-deletion	BRAF (Val600)	EMP3 methylation
Case 1, PT	10q	NA	Wild type	p.CYs275Tyra	Meth	p.Trp274*,b	nd	Wild type	Unmeth
Case 1, NS	10q	NA	Wild type	p.CYs275Tyra	Meth	p.Trp274*,b	nd	Wild type	Unmeth
Case 1, AC	10q	NA	Wild type	p.CYs275Tyra	Meth	p.Trp274*,b	nd	Wild type	Unmeth
Case 2, PT	10q	NA	Wild type	Wild type	Unmeth	c.209 + 1G>Tc	Wild type	Wild type	Meth
Case 2, AC	Wild type	NA	Wild type	Wild type	Unmeth	Wild type	Wild type	Wild type	Meth
Case 3, PT	nd	NA	Wild type	Wild type	Meth	Wild type	nd	Wild type	Unmeth

Notes:

a COSMIC ID.10893

b COSMIC ID.5158

c splice variant.

Abbreviations: AC, adherent cells; COSMIC, Catalogue of Somatic Mutations in Cancer; LOH, loss of heterozygosity; Meth, methylated; NA, not amplified; nd, not determined due to lack of constitutional DNA; NS, neurospheres; PT, primary tumor; Unmeth, unmethylated.

Figure 4 Immunofluorescence.

Notes: Case 1: (A) neurospheres, Musashi-1 expression; 40×; (B) id, nestin expression; 40×; (C) adherent cells, GFAP expression; 40× (confocal microscopy). Case 2 (first intervention): (D) adherent cells, GFAP expression; 40×. DAPI counterstaining was used for all.
Abbreviations: DAPI, 4′,6-diamidino-2′-phenylindole dihydrochloride; id, neurospheres.