From UMF Carol Davila
Department no.8 – Oncology, Hematology, Radiology

To: Genesis Biopharma Romania
Subject: Implementation of Fluorescence in Situ Hybridization (FISH) in MDS

Project Director: Senior Lecturer Dr Aurelia TATIC
Scientific Officer: Lecturer Cerasela JARDAN
University of Medicine and Pharmacy "Carol Davila", Bucharest
Cytogenetic Laboratory - Center for Hematology and Bone Marrow Transplantation, “Fundeni” Clinical Institute, Bucharest, Romania

With this letter on behalf of UMF Carol Davila,
We would like to address an official request to your company for the financial support of 10000 Eur for this project, related to MDS patients

SCOPE
Implementation of FISH panels in MDS for “Fundeni Clinical Institute”. The results obtained in this project will allow to understand the genetic profile of Myelodysplastic syndromes (MDS) patients from Romania who are addressed to “Fundeni Clinical Institute”.

Background
In the management of myelodysplastic syndromes, the analysis of karyotype by conventional techniques and FISH allows:

1. Establishing the presence of the malignant clone (it must always be taken into account that finding only cells with normal karyotype does not rule out the presence of the malignant clone);
2. Clarifying the diagnosis (detecting a particular malignant clone);
3. Evaluation of prognosis independently or associated with other risk factors;
4. Choice of treatment strategy;
5. Ensuring the monitoring of response to treatment;
6. Investigative researches

Cytogenetic information in myelodysplastic syndromes (MDS) is important in assessing prognosis and in establishing treatment guidelines. Also, the presence of numerical anomalies of both the entire chromosome and some chromosome segments is important in evaluating myelodysplastic syndrome. Recently, Uniparental disomy (UPD) in different loci, has been found in several MDS subjects as well as targeted genes located in regions of the deleted chromosome; these deletions are detectable cytogenetically, leading to a partial or cryptic monosomy.

The cytogenetic exam has been used for over 20 years in the diagnosis and prognosis of myelodysplastic syndrome, being an important parameter in prognostic score systems. It is performed from the bone marrow, at diagnosis, and at least analysis of 20 metaphases is needed, according to the ISCN (International System for Human Cytogenetic Nomenclature).

It also demonstrates the clonallity of  disease and confirms the diagnosis of MDS in cases of persistent cytopenias when dysplastic changes are missing.

The clone is identified based on the presence of two cells with the same additional or structural abnormalities or three cells with the same missing chromosome. MDS most frequently exhibits unbalanced cytogenetic abnormalities. Balanced structural abnormalities (translocations, inversions) are rare. Abnormal karyotype is present in 50% of cases of primary MDS and in over 80% of treatment-related MDS. As a direct consequence a normal karyotype does not rule out the diagnosis of MDS.

Spectral analysis of  the karyotype (chromosome “painting") enables the identification of the balanced or unbalanced cryptic translocations in most MDS patients.

Complex karyotype, defined by the presence of three or more anomalities, is described in 15% of cases of MDS and has a poor prognosis. Certain recurrent chromosomal abnormalities are considered as presumptive for MDS in patients with undetermined persistent cytopenia (Table 1).

In this table are not included some frequently MDS abnormalities like:

del (20q), +8, -Y because these are also described in aplastic anemia,
other cytopenias with good response to immunosuppressive treatment and / or morphological features of MDS with long surveillance period

The karyotype is an important prognostic factor in MDS patients and provides information on the survival of these patients.

Hasse et al showed that median survival of patients with various cytogenetic abnormalities is different, in a group of 2142 SMD patients. The more frequent abnormalities and median survival are listed in Table 2. Favorable prognosis measured as median survival of approximately 3 years was observed in translocations involving chromosome 1q, any anomalies of the chromosome 12, translocations involving chromosome 17q with non-complex karyotype, monosomy  and  trisomy of chromosome 21 and chromosome X losses. Although many cytogenetic abnormalities have been identified, their rarity limits their Prognostic Value.

Morel et al contributors have reported on a group of 408 subjects, that those with normal karyotype had a longer survival time than those with abnormal karyotype, and furthermore subjects with a complex karyotype had a very poor prognosis.

On the other hand, Sole et al presents a very favorable prognostic for del(12p) and for the anomalies of chromosome 1q a very unfavorable prognosis. Trisomy 8, the most common abnormality described in MDS, exhibit a median survival of 1 year and bears a risk of transformation in AML of 34%. Anomalies with intermediar prognosis, defined with median survival between 1 and 3 years, are chromosome 3q rearrangements, translocations involving chromosome 11q23, non-complex karyotype and trisomy 19.

Table no. 1. Recurrent chromosome abnormalities considered as presumptive for MDS in subjects with undetermined persistent cytopenia

Table no. 2. Chromosomal abnormalities depending on MDS subtypes

Table no. 3. Cytogenetic abnormalities and median survival

The most common abnormalities described in MDS are: +8, -5-del (5q), -7-del (7q), del (20q). Cytogenetic analysis remains an important factor in selecting treatment and monitoring response to treatment.

Johansson et all described on a group of 1,663  cases of MDSs, monosomy in 22% and structural abnormalities with partial chromosome loss in 39%.Acquiring new cytogenetic abnormalities is associated with poor prognosis and increased risk of AML transformation.
The International Prognostic Score System (IPSS) and the Revised (IPSS) are the most commonly used clinical risk stratification strategies in MDS.Both models use cytogenetic abnormalities by karyotype analysis in MDS.

Objective
We will develop the methodology of the FISH technique in myelodysplastic syndrome. The results obtained in this project will allow to understand the genetic profile of Myelodysplastic syndromes (MDS) patients from Romania who are addressed to “Fundeni Clinical Institute”.

Methods
The study of leukemic cells by conventional cytogenetics involves a series of steps. Aspiration of bone marrow sample, then processing it by setting up cell cultures with or without synchronization, crop processing including inhibition of colseid division spike, hypotonic solution treatment and fixation, then preparation of GTG banding blades and microscopic analysis.
1. Taking samples.
Harvest 2-5 mL of BM on the heparin syringe or on a transport medium and immediately sent to the laboratory.
2. Estimate the number of cells in the BM sample received:
The leukocytes in the bone marrow should ideally be grown at a concentration of 1 x 10 6 cells / mL. For this, the total number of cells is determined using a hemocytometer.
  3. Overnight Cultures (ONC) - Culture "A":
Colcemid is cultured at the end of the day, the culture is incubated overnight and sacrificed the next morning. Colcemide inhibits the divisional spindle and the longer it is left in the culture, the more metaphases are obtained, but the chromosomes are very short. This type of culture is used because a high mitotic index is obtained and it has been called "hypermetaphase".
4. Cultures with cell cycle synchronization (SYN) - Culture "B" - the divisions are most likely not synchronized, but this effect stems from cell delays in phase S, so the term "blocking" would probably be more correct (9cancymet). This type of culture was introduced to obtain metaphases with long chromosomes due to shorter colchemide exposure (1h); but the number of metaphases is very small or sometimes even metaphase-free. The duration of the mitotic cycle in the leukemic cells is very variable (sometimes considerably long) and exposure to short-acting colcemide, it is possible that the peak of the divisions is not captured and thus the number of metaphases is extremely small or non-existent. Agents used in cell cycle synchronization cultures are: uridine and fluorodeoxyuridine (11) and an excess of thymidine (1).
5. Chromosome Banding Techniques (Giemsa Banding -“GTG” Banding)
The chromosomes obtained after removal from the culture are uniformly colored and have a few markers for their identification.
By chromosome banding, chromosomes with a specific structure of colorful and colorless alternative bands are obtained (these techniques make it possible to accurately identify each chromosome).
Chromosomal bands are specific to each chromosome (cytologic markers of heterogeneous internal chromosome structure). These specific bands make it easy to highlight the numerical or structural anomalies present in karyotype. The most common technique for banding is G-banding using trypsin and Giemsa staining. This method requires digestive control with trypsin and staining with Giemsa; the positive G bands are intensely colored and the negative ones are very poorly colored.
6. Microscopic analysis
Chromosomal analysis is performed using the optical microscope (the essential technique for both diagnosis and basic research in chromosome abnormalities).

FISH technique
FISH probes are prepared for hybridization according to the manufacturer's recommendations.
2. Hybridization: FISH probes are applied to the denatured slide and the creation of a sealed hybridization chamber by placing a slide and sealing it with siliconized rubber. The final volume of the probe is calculated based on the size of the lamella. Hybridization is done in a special device (Hybrite from EuroClone) that provides the required humidity and hybridization program: 75 ° C for 2 min then cooling to 37 ° C for 12-16 h (observing manufacturer's recommendation for specific probes restricted genomic regions).
3. After overnight hybridization, the lamella are removed from the hybridization chamber and the lamella removed. The slide is introduced into the post hybridization solution I (4 X SSC) which is introduced into the bath at 73 ° C for 2 minutes then introduced for a few seconds in the post hybridization solution II (2 x SSC) at room temperature. The slides are dried at room temperature, then a DAPI containing antifouling solution is applied and the lamella is sealed with siliconized rubber. The flasks are then placed in the refrigerator at 4 ° C until they are read.
4. Examination of the slide is done in a dark room using the fluorescence microscope. It uses the 100X lens, digital camera and analysis software (AxioImager, Zeiss, Germany).

Action plan:
 Phase 1 - May 2019 - Mar 2020. Selection and optimization of working protocols
Activities:
1. Establish working protocols
2. Establishment of FISH panels applied to MDS cases with normal karyotype

Phase 2 - Apr- 2020 – May 2020.
1. Statistical analysis of data
2. Dissemination of results through publications, presentations, conferences and the organization of workshops

• Patients’ data will not be provided at the individual level;
• Only aggregated data will be shared.
• The project results will not influence treatment decision for MDS patients.

Resources and Budget
The team will be made up of young researchers, doctors and technicians. In total, the team will have 5 members who will be involved in the project.
The Laboratory of Cytogenetics is part of the Department of Special Analyzes of the “Fundeni” Clinical Institute and has all the infrastructure necessary to support the activity in this project

Infrastructure:

Budget (Euro)

Signatures and stamps:
Project Director: Senior Lecturer Dr Aurelia TATIC

Scientific Officer: Lecturer Cerasela JARDAN

Department director: Prof Dr Daniel CORIU

UMF Carol Davila Stamp