Evaluation of HIF-1α and VEGF-A expression in radiation-induced cystitis: A case-control study

Mattos, Ricardo Castellani de; Guimarães, Isabella dos Santos; Thiago, Leandro de Souza; Melo, Andreia Cristina de

doi:10.1590/S1677-5538.IBJU.2020.0054

Ricardo Castellani de Mattos Correspondence address: Ricardo Castellani de Mattos, MD, Divisão de Pesquisa Clínica e Desenvolvimento Tecnológico, Instituto Nacional de Câncer - INCA, Rua André Cavalcanti, 37, 3° andar, Rio de Janeiro, RJ, Brasil. Telephone: + 55 21 3207-6570 E-mail: ricardocastellanidemattos@gmail.com

Divisão de Pesquisa Clínica, Instituto Nacional do Câncer - INCA, Rio de Janeiro, RJ, Brasil.

Isabella dos Santos Guimarães

Divisão de Pesquisa Clínica, Instituto Nacional do Câncer - INCA, Rio de Janeiro, RJ, Brasil.

Leandro de Souza Thiago

Divisão de Pesquisa Clínica, Instituto Nacional do Câncer - INCA, Rio de Janeiro, RJ, Brasil.

http://orcid.org/0000-0002-4003-3400

Andreia Cristina de Melo

Divisão de Pesquisa Clínica, Instituto Nacional do Câncer - INCA, Rio de Janeiro, RJ, Brasil.

Correspondence address: Ricardo Castellani de Mattos, MD, Divisão de Pesquisa Clínica e Desenvolvimento Tecnológico, Instituto Nacional de Câncer - INCA, Rua André Cavalcanti, 37, 3° andar, Rio de Janeiro, RJ, Brasil. Telephone: + 55 21 3207-6570 E-mail: ricardocastellanidemattos@gmail.com

CONFLICT OF INTEREST

None declared.

Figures (3)
Tables (2)

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Figure 1
Aspect of the bladder mucosa of irradiated patients with varying degrees of neovascularization comparing with normal bladder mucosa. Images were obtained during cystoscopy examinations of patients included in the study and correspond to the endoscopic findings of RTOG/EORTC late morbidity scale: A and B - normal bladder mucosa, C and D -Grade 1: slight epithelial atrophy, minor telangiectasia, E and F - Grade 2: generalized telangiectasia, G and H – Grade 3: severe generalized telangiectasia (often with petechiae).

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Figure 2
Graphics of digital PCR in drops. The graphs indicate the fluorescence intensity (Ch1 Amplitude and Ch2 Amplitude for FAM or HEX, respectively) of the drops analyzed (events number). The drops are termed "positive" or "negative" based on their fluorescence amplitudes. Positive events are marked in blue (HIF-1α and VEGF-A) and green (GAPDH), while negative events in gray (drops without the DNA molecule of interest). All analyzes were performed using QuantaSoft software (Biorad). During drop formation cDNA molecules are randomly distributed in the droplets. Due to the high number of independent events, the Poisson algorithm is used to determine absolute quantitation of copy number independent of a standard curve, allowing accurate quantification of gene expression. (A) Expression of HIF1α in a control group patient (left) and in an irradiated patient (right). (B) Expression of VEGFA in a control group patient (left) and in an irradiated patient (right). (C) Expression of GAPDH in a control group patient (left) and in an irradiated patient (right).

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Figure 3
Droplet digital PCR expression analysis of HIF-1α and VEGF-A mRNA. (A) and (B) Analysis of HIF-1α and VEGF-A mRNA in non-irradiated patients (control) and patients previously submitted to pelvic radiotherapy (cystitis). (C) and (D) Analysis of HIF-1α and VEGF-A mRNA expression in patients previously submitted to pelvic radiotherapy according to the late RTOG/EORTC morbidity score. (E) and (F) Analysis of HIF-1α and VEGF-A mRNA in bladder tissue samples and washing bladder samples of non-irradiated patients. The targets mRNA expression were calculated relative to a stable expressed reference gene (GAPDH). Results were expressed as median and comparison between groups was performed by Mann–Whitney U test. RTOG/EORTC: grade 1 - mild epithelial atrophy, small telangiectasia (microscopic hematuria); grade 2 -moderate frequency, generalized telangiectasia, and intermittent macroscopic hematuria.

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Table 1
Clinical characteristics of patients.

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Table 2
Characteristics of the radiotherapy regimen employed, bladder toxicity score and radiotherapy-related complications.

		Irradiated - n (%)	Control - n (%)
Age (years, median)		48, 31 - 66	44.5, 33 - 66
	brown	6 (42.9)	5 (41.7)
Color/race	white	7 (50.0)	4 (33.3)
	black	1 (7.1)	3 (25.0)
Histopathology	squamous cell carcinoma	12 (85.7)	11 (91.7)
Histopathology	adenocarcinoma	2 (14.3)	1 (8.3)
	I	0	1 (8.3)
Grade	II	13 (92.9)	7 (58.3)
Grade	III	1 (7.1)	3 (25)
	Not informed	0	1 (8.3)
	IB1	2 (14.3)	1 (8.3)
	IB2	1 (7.1)	1 (8.3)
FIGO stage at diagnosis^* * FIGO: International Federation of Gynecology and Obstetrics	IIA	1 (7.1)	1 (8.3)
	IIB	9 (64.3)	8 (66.7)
	IIIA	0	1 (8.3)
	IIIB	1 (7.1)	0
Total		14	12

Patient ID	External Radiotherapy				Bladder	Dosec	Brachytherapy		Total Dosec	Proctopathye	RTOG/EORTC			LENT/SOMA				CTCAE	Sediment^f f urinary sediment; N: normal examination.
	Equipmenta	Techiniqueb	Dosec	td	Average	Maximum	Insertion	Dosec				S	O	M	A	Maximum	Total
101	LA	2D	4500	51	-	-	3	2400	6900	N	1	2	1	0	0	2	3	4	mild hemoglobinuria. moderate leukocyturia
102	cobalt	3D	4500	49	4806	4960	-	-	-	Grade 1	2	2		0	0	2	3	5	N
103	LA	2D	4500	49	-	-	3	2400	6900	N	1	0	1	0	0	1	1	0	N
104	LA	2D	4500	49	-	-	3	2400	6900	S	1	3	1	0	0	3	4	2	mild leukocyturia
105	LA	3D	4500	54	4730	4840	3	2400	6900	S	1	0	1	0	0	1	1	1	N
106	LA	3D	4500	54	4590	4680	4	2400	6900	N	1	0	1	0	0	1	1	2	moderate leukocyturia
107	LA	3D	4500	49	4700	4890	4	2800	7300	Grade 2	1	0	1	0	0	1	1	1	mild leukocyturia. mild proteinuria
108	LA	3D	4500	19	4384	4860	3	2400	6900	N	3	3	3	2	0	3	8	6	very intense hemoglobinuria, mild proteinuria
109	LA/cobalt	2D	5040	52	-	-	3	2400	7440	N	3	1	2	1	2	2	6	3	N
110	LA	3D	5040	49	5510	5580	4	2800	7840	N	1	2	1	1	0	2	4	4	mild proteinuria
111	LA	3D	4500	40	4610	4690	3	2400	6900	S	2	0	2	0	0	2	2	2	mild hemoglobinuria. mild proteinuria. intense leukocyturia
112	LA	3D	4500	69	4798	4850	4	2400	6900	S (diarrhea)	2	2	2	0	0	2	4	3	N
114	LA	2D	4500	24	-	-	3	2400	6900	N	2	2	2	1	0	2	5	3	intense hemoglobinuria. moderate proteinuria
115	cobalt	3D	4500	36	4820	4960	3	2400	6900	S	3	3	2	2	0	3	7	6	Not available
Mean			4557	46				2461	7044		1.64						3.57	3

[1] Correspondence address: Ricardo Castellani de Mattos, MD, Divisão de Pesquisa Clínica e Desenvolvimento Tecnológico, Instituto Nacional de Câncer - INCA, Rua André Cavalcanti, 37, 3° andar, Rio de Janeiro, RJ, Brasil. Telephone: + 55 21 3207-6570 E-mail: ricardocastellanidemattos@gmail.com

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Evaluation of HIF-1α and VEGF-A expression in radiation-induced cystitis: A case-control study

ABSTRACT