The role of the P-53 gene and the P-53 protein in non-Hodgkin malignant lymphomas

ISRCTN	ISRCTN12539707
DOI	https://doi.org/10.1186/ISRCTN12539707
Secondary identifying numbers	UEFISCDI ID (UEF-iD): U-1900-063Y4656

Submission date: 08/11/2022
Registration date: 12/11/2022
Last edited: 03/05/2023

Recruitment status: No longer recruiting
Overall study status: Completed
Condition category: Cancer

Prospectively registered

Protocol

Statistical analysis plan

Results

Individual participant data

Plain English summary of protocol

Background and study aims
P-53 gene mutations are the most common genetic abnormalities of cancer. They have been extensively studied in various mature B-cell malignancies, including chronic lymphocytic leukemia (CLL). In recent years, more attention has been paid to the importance of the p53-expressed protein in CLL, and a combination with low survival and non-response to classical conventional chemotherapy, due to mutations in the p53 gene, with progression to Richter Syndrome. Identifying different p53 gene mutations is very important because these mutations have an impact on the patient's clinical course in CLL.

Who can participate?
Patients with CLL-B who were hospitalized in the Hematology departments of the Targu Mures Oncology Institute and Cluj-Napoca between November 2016 and September 2019

What does the study involve?
Participants undergo a complete physical examination and laboratory blood tests. p53 protein levels are measured at a single timepoint.

What are the possible benefits and risks of participating?
Not provided at time of registration

Where is the study run from?
Titu Maiorescu University (Romania)

When is the study starting and how long is it expected to run for?
October 2016 to January 2020

Who is funding the study?
Investigator initiated and funded

Who is the main contact?
Dr Aurelian Udristioiu, aurelianu2007@yahoo.com

Study website

Contact information

Dr Aurelian Udristioiu
Principal Investigator

Tiru Maiorescu University of Bucharest
Faculty of Medicine, Molecular Biology
Bucharest
040051
Romania

ORCID ID	0000-0001-8375-8287
Phone	+40 (0)723565637
Email	aurelianu2007@yahoo.com

Study information

Study design	Observational cohort study
Primary study design	Observational
Secondary study design	Cohort study
Study setting(s)	Other
Study type	Diagnostic
Scientific title	The role of the P-53 gene and the P-53 Protein in the oncogenesis of non-Hodgkin malignant lymphomas
Study objectives	p53 gene mutations are the most common genetic abnormalities of cancer. They have been extensively studied in various mature B-cell malignancies, including chronic lymphocytic leukemia (CLL). In recent years, more attention has been paid to the importance of the p53-expressed protein in CLL, and a combination with low survival and non-response to classical conventional chemotherapy, due to mutations in the p53 gene, with progression to Richter Syndrome. Identifying different p53 gene mutations is very important because these mutations have an impact on patients' clinical course in CLL with the p53 protein mutant isoform.
Ethics approval(s)	Ethics approval not required
Health condition(s) or problem(s) studied	Non-Hodgkin malignant lymphomas
Intervention	Complete physical examination: In patients diagnosed with CLL-B, symptoms such as frequent cough, night sweats, and retrosternal pain were evaluated. Clinical examination and ultrasound revealed lymphadenopathy and/or splenomegaly, with an enlarged spleen. Laboratory examinations: Hemoleukogram with 5 Diff and cytological examination of the blood smear on the peripheral blood in the May Grunwald-Geimsa staining, and bone marrow puncture, BM with medullary forcing. The cases were classified as CLL with >5000 lymphocytes in absolute value, present at the cytological examination of the blood smear, from the peripheral blood or LLC with less than 10% prolymphocytes based on the peripheral blood smears May-Grunwald Giemsa, stained. Immunophenotyping: The diagnosis of CLL was confirmed by immune phenotyping. All samples that entered the study were lymphocytes with positive CD19⁺, CD20⁺⁻, CD5⁺ and CD23⁺ cell receptors. The CD38+ receptor was considered positive if the distinct lymphocytes of the population showed a higher intensity of staining than the granulocytes in the sample and was associated with the presence of protein ZAP-70. A sandwich ELISA colorimetric quantitative method was used for direct detection of the p53 isoform protein, the product of gene p53: Specificity: human p53 protein (aa20-25); Format: Purified product: Monoclonal antibody clone: Isotype DO-1: IgG2a. The antibody is suitable for the techniques: ICC / IF and ELISA. The research antibody PAb 1620 has been reported to be specific for the conformation of the normal p-53 protein, and PAb 240 antibodies bind specifically to denatured p-53 protein. Compatible sample types: cell culture supernatants, plasma, serum; solid support: 96-well microplate; firm: Ray Biotech Life, Inc. Plasma is collected from patient samples using vacutainers with EDTA or heparin as an anticoagulant by centrifuging the samples for 15 minutes at 4500 rpm (280 G). After the blood sample has been centrifuged and its plasma has been separated from red blood cells, the plasma is fractionated into four distinct fractions placed on a layer of white blood cells (lymphocytes). With a pipette, a quantity of 100 µl is extracted from the lymphocyte ring. The extracted lymphocytes are introduced into 25 ml cuvettes with a 3 ml wash buffer medium for washing the lymphocytes. Washing is done three times, once after 10 minutes at 1500 revolutions/minute and twice for 10 minutes at 1000 revolutions/minute. Lysis of washed lymphocytes is done with a Mini Wave Smart Laboratory microwave. In the case of small-volume samples, a preliminary step dilution, such as 1: 5 or 1:10, can be performed using PBS buffer (0.02 mol / L pH 7.0-7.2) as the diluent. The final dilution should always be done using the same buffer used to dilute the Standards. This analysis is based on the sandwich ELISA principle. Each well of the microtiter plate was pre-coated with a specific target capture antibody. Standards or samples are added to the wells and the target antigen, in this case, the p53 protein, binds to the capture antibody. Summary of test procedure: 1. Prepare all reagents, samples and standards: add 100 μl of sample, standard or blank to each well and incubate for 2.5 hours at room temperature or overnight at 4 °C 2. Aspirate the volume of liquid initially added and wash three times 3. Add 100 μl of biotinylated detection antibody (biotin detection antibody) and incubate for 1 hour at room temperature 4. Vacuum and wash three times 5. Add 100 μl of HRP-streptavidin conjugate and incubate for 45 minutes at room temperature 6. Add 100 μl of TMB substrate and incubate for 30 minutes at 37 °C 7. Add 50 μl of stop solution 8. Read immediately at 450 nm wavelength A series of dilutions of the positive control standard must be performed in duplicate or triplicate, the last well in each series being the negative control sign. The tests should also be performed in duplicate or in triplicate. Unknown samples should function as dilution series to identify the optimal dilution that produces an OD value in the OD range of the standard control dilution series. Data analysis: Prepare a standard curve from the serial dilution data with concentration on the x-axis (logarithmic scale) from the absorption on the Y-axis (linear). Peroxidase (HRP) and alkaline phosphatase (ALP) are the two most widely used enzymes for detection in ELISA tests. Measure the yellow color of nitrophenol at 405 nm after 15-30 minutes of incubation at room temperature and stop the reaction by adding an equal volume of 0.75 M NaOH.
Intervention type	Genetic
Primary outcome measure	P-53 isoform protein concentration measured using sandwich ELISA colorimetric quantitative method at a single timepoint
Secondary outcome measures	There are no secondary outcome measures
Overall study start date	15/10/2016
Completion date	15/01/2020

Eligibility

Participant type(s)	Patient
Age group	Senior
Sex	Both
Target number of participants	20
Total final enrolment	20
Key inclusion criteria	1. Patients diagnosed with CLL-B who were hospitalized in the Hematology departments of the Targu Mures Oncology Institute and Cluj-Napoca between November 2016 and September 2019 2. CLL with> 5000 lymphocytes in absolute value, present at the cytological examination of the blood smear, from the peripheral blood or LLC with less than 10% prolymphocytes based on the peripheral blood smears May-Grunwald Giemsa, stained
Key exclusion criteria	Does not meet inclusion criteria
Date of first enrolment	01/11/2016
Date of final enrolment	01/09/2019

Locations

Countries of recruitment

Romania

Study participating centre

Titu Maiorescu University of Bucharest

Faculty of Medicine
Damboviciului Street, NoL 22
Bucharest
040051
Romania

Sponsor information

Titu Maiorescu University
University/education

Faculty of Medicine
Damboviciului Stree, No: 22
Bucharest
040051
Romania

Phone	+40 (0)723326663
Email	manole.cojocaru@yahoo.com
Website	http://www.brainmap.ro
"ROR"	https://ror.org/0367qb939

Funders

Funder type

Other

Investigator initiated and funded

No information available

Results and Publications

Intention to publish date	11/11/2022
Individual participant data (IPD) Intention to share	Yes
IPD sharing plan summary	Published as a supplement to the results publication
Publication and dissemination plan	Planned publication in a high-impact peer-reviewed journal
IPD sharing plan	The datasets generated and/or analysed during the current study will be published as a supplement to the results publication

Study outputs

Output type	Details	Date created	Date added	Peer reviewed?	Patient-facing?
Other publications	Review	01/10/2018	03/05/2023	Yes	No
Other unpublished results			03/05/2023	No	No
Results article			03/05/2023	Yes	No

Additional files

ISRCTN12539707 Results report.pdf

Editorial Notes

03/05/2023: Publication reference added and results report file uploaded.
14/11/2022: Internal review.
11/11/2022: Trial's existence confirmed by Universitatea "Titu Maiorescu" din Bucuresti