SNP/CGH Microarray Based Genomic Testing In Myelodysplastic Syndrome

MDS – An Overview

Describe about the SNP/CGH Microarray Based Genomic Testing in Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a progressive disease characterised by ineffective haematopoiesis, peripheral blood cytopenias, abnormal cellular morphology and variable risk of progression to acute leukaemia. Myelodysplastic syndromes (MDS) are the clonal disorders of an immature hematopoietic progenitor cell. MDS hematopoiesis is categorised by irregular progenitor proliferation and impaired cellular differentiation and maturation. As a result, the bulk of MDS patients have a plodding evolution in the direction of progressive bone marrow failure and leukemic transformation. The hard work made by numerous research assemblies during past few years have assisted to unknot the complex that forms the fundamentals of the pathogenesis of MDS. In this review, we will focus on the major molecular abnormalities and cytogenetic involved in MDS beginning and disease development. Moreover, we will deliberate the impact of the marrow microenvironment on proliferation and survival of hematopoietic progenitors in MDS.

It is one of the commonest haematological disorders worldwide accounting for 1.3% of all cancers in New Zealand [31]. Approximately 86-87% cases of MDS in Australia and New Zealand were reported to be diagnosed in individuals of above 60 years of age [31].   

The MDS pathogenesis is not understood very well but as being a neoplasm, it involves the accumulative acquisition of oncogenic driver mutations. MDS is generally considered a clonal process that is thought to develop from a single transformed hematopoietic progenitor cell [47]. The recent improvement in the detection of recurring chromosomal abnormalities and mutations has provided better understanding of the pathogenesis of MDS.

The investigation gone through the Three decades into the pathophysiology of the myelodysplastic syndromes (MDS) have established the heterogenicity of MDS and emphasised the intricacy in disease biology. Recent improvements in technology have yielded stimulating observations. The aim and objective of this review is to assimilate laboratory and clinical findings into a functioning hypothesis for the advancement of idiopathic MDS, distinguish idiopathic MDS from SAA, and propose latest therapeutic strategies.

Understanding of data from MDS studies still remains challenging. Without a dependable disease marker, here can be a questions about the exactness of an MDS analysis. Supplementary problems ascend the minute patients with incongruent biologies are made compared.

Patients who are possessing MDS may have noticeable single or multiple clonal chromosomal variations at the time of analysis or gain them later through the course of the disease which may pave the way of transformation into acute myeloid leukaemia (AML). Simple chromosome changes may involve a numerical change which may be monosomy or trisomy, a structural abnormality like inversion and interstitial deletion that involve only one chromosome and very rarely a balanced translocation which involve two chromosomes. Complex karyotyping with multiple abnormalities approximately more three can also be detected in advanced cases [29].

Chromosomal Abnormalities Found in MDS

A large MDS series of 1029 patients was studied by Pozdnyakova and his colleagues, found 44% of cases with MDS to have clonal cytogenetic abnormalities evident by standard metaphase karyotyping at diagnosis [33] & [29]. Del(5q) was the most common and seen in 18% of cases followed by complex karyotype in 6% of cases. Trisomy 8 and Del(20q) were present in 4%, and 3%, respectively [29]. Trisomy 8 increases the risk of leukemic transformation which is predominant in male and causes oral ulceration. Loss of the Y chromosome is known to be one of the good prognostic markers in MDS, however, it is generally thought to be an age related phenomenon [46].

The analysis of MDS relies mainly on the detection of clonal genetic abnormalities in the appropriate clinical context as well as the identification of significant morphological dysplasia on peripheral blood film and/or bone marrow. There are certain cytogenetic abnormalities considered as a presumptive evidence of MDS even in the absence of morphological dysplasia. The presence of these specific cytogenetic abnormalities confirms the MDS diagnosis in cases with absent or very little morphologic dysplasia. Similarly, the diagnosis of AML can sometimes be established in the presence of certain cytogenetic abnormalities regardless of blast count. An amazing development in the understanding of the leukemogenesis is made conceivable by the methodological improvements in the cytogenetic field. The cytogenetic irregularities that have usually provided the molecular basis for the finding of the genes that are engaged into the mechanism of the leukemogenesis.  Numerous study illustrates that the cytogenetic turned out is one of the most significant prognostic factor and so it was integrated into the statistical model which aims for the improvement of the forecast the procedures of the individual prognosis.

The identification of clonal chromosomal abnormalities is not only important in establishing the MDS diagnosis but also aids in the classification of MDS, prognostic stratification and treatment planning [33], [14]. The impact of cytogenetics is clearly demonstrated in its role in determining the International Prognostic Scoring System (IPSS).

The conventional metaphase cytogenetics is the standard genetic test routinely performed in evaluating MDS cases in most laboratories. Sometime the traditional cytogenetic analysis and routine chromosome analysis is mentioned to as karyotyping. These kinds of studies are used to identify numerical and structural chromosome irregularities in metaphase cells. Routine chromosome analyses involves sterile viable tissue samples.  The removal of the long arm of the chromosome 5, del (5q) is the most frequently occurring chromosomal abnormalities found in the patient diagnosed with MDS. The lenalidomide developed as the effective targeted therapy for the little and intermediate risk of MDS with a del 5(q) has increased the importance of karyotyping in disease management.  This test has a number of limitations including the low resolution and the need for high quality metaphases and cell division within the abnormal cells. In view of those limitations, new methodology with much higher resolution such as Single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) microarray analysis were incorporated in the evaluation of MDS cases.

Cytogenetic Abnormalities as a Diagnosis Markers

The main principle present behind the microarrays is the hybridization between two strands of DNA , the property by which complementary sequences of the nucleic acid exactly pairs with complementary nucleotide bases by forming hydrogen bonds among each other. A large number of complementary base pairs in a nucleotide sequence means more tight non-covalent bonding among the two strands. Subsequently washing off non-specific bonding sequences, only toughly paired strands will remain hybridized. Fluorescently labeled target sequences which bind to a probe sequence will help to produce a signal which is dependent on the hybridization circumstances (such as temperature), and perform washing after the hybridization. The total strong point of the signal, from a spot (feature), rest on the quantity of target sample binded to the probes that are present on the spot. The Microarrays uses comparative quantitation in which the intensity of a feature is compared to the intensity of the same feature under several different condition, and the distinctiveness of the feature is recognised by its situation.

Both SNP and CGH array are basically high resolution DNA microarray tools that can detect genomic gains or deletions associated with copy number changes down to a level of 5 KB of DNA [28], [30]. SNP arrays however have the additional advantage of its ability to detect copy-neutral loss of heterozygosity (LOH) or uniparental disomy (UPD).   

The basic components of SNP and CGH arrays include:

DNA hybridization of immobilized allele -specific oligonucleotide (ASO) probes with target DNA sequences labelled with florescent dyes [7]. ASO probes are generally designed and selected from a representative pool of healthy individuals [7].

The use of fluorescence microscopy and a solid surface DNA capture system that picks up and interprets hybridization signals [35] [7]. The differences in the fluorescence intensities reflects copy numbers changes in DNA sequences. This indicates the presence of either loss or a gain mutation.

Despite of the high resolution of SNP/CGH microarray analysis, it has important limitations. Those limitations are:

Inability to detect genomic abnormalities that do not result in copy number changes such as balanced translocations.

Inability to detect genomic abnormalities in the settings of small clone sizes.

Mohamedali study showed that the use of SNP microarray analysis in MDS cases resulted in a much higher detection rate of genomic abnormalities up to 75% compared to 50% using conventional metaphase cytogenetics alone. Some of these detected cryptic genomic lesions were shown to have prognostic implications [22].

Conventional Metaphase Cytogenetics as a Genetic Test

Essentially similar results were demonstrated in Tiu’s study of 430 patients with MDS disorders. It concluded that combining both the metaphase cytogenetics and SNP array together led to a higher diagnostic yield of chromosomal defects (74% vs 44%, P < .0001), compared metaphase cytogenetics alone [40, 41 & 42]. It also demonstrated that some of the newly detected SNP array defects contributed to poorer prognosis. Some of these new SNP array detected chromosomal lesions were independent predictors of overall and event-free survival [41].

Both Heinrichs and Volkert studies evaluated the use of SNP/CGH microarray on MDS cases with normal metaphase cytogenetics [16, 43]. In both studies the SNP/CGH microarray detected cryptic genomic lesions in 11-15% of cases respectively [16, 43].  Heinrichs study showed that most of these genetic lesions resulted from segmental uniparental disomies (UPD). The study also showed that UPD affecting chromosome 7q are associated with a progressive course and worse outcome regardless of their low-risk International Prognostic Scoring System score [16]. 

In Volkert study, the majority of CGH array identified genetic lesions were sub-microscopic copy number changes (4% gains and 7% losses) [43]. Compared to sub-microscopic gains, more than half of the sub-microscopic deletions were recurrent and involved the genes TET2, DNMT3A, ETV6, NF1, RUNX1 and STAG2 [43].  The presence of sub-microscopic deletions was associated with lower overall survival rates compared to those without deletions [43].

SNP/ CGH microarray analysis with its superb resolution in detecting genomic abnormalities resulting from copy number changes, is expected to be particularly useful in MDS. This is supported by two factors; firstly, the fact that the vast majority of cytogenetic abnormalities in MDS are characterised by copy number changes and secondly, because balanced translocations are not common in MDS. Overall SNP/CGH microarray analysis appears to be a good complementary method to conventional metaphase cytogenetics analysis in MDS cases, with its own unique prognostic information

The use of SNP/CGH microarray analysis in MDS related disorders has been evaluated in this study by comparing its results with standard metaphase cytogenetics.

AIM:

The overall objective of this study is to demonstrate the ability of SNP/CGH microarray in

1-            Improving the diagnostic yield of suspected MDS cases by detecting clonal genetic lesions in cases with either normal or non-informative conventional metaphase cytogenetics. 

2-            Providing higher resolution genetic testing capable of accurately guiding both prognostic stratification scoring system and therapy. 

3-            Providing early detection of micro genetic abnormalities that can potentially be associated with a progressive course and hence prioritising early bone marrow transplant therapy in transplant eligible patients.

Genomic Micro-Array Studies

4-            Effectively replacing or complementing the conventional metaphase cytogenetic testing in our local practice in evaluating MDS cases.      

We hypothesized that the combination of standard metaphase cytogenetics and SNP/CGH microarray could potentially enhance both the diagnosis and prognostic stratification of MDS related disorders. We also evaluated the possibility that MDS cases with either normal or non-informative metaphase cytogenetics could potentially harbour cryptic chromosomal lesions with possible prognostic implications that can only be detected by SNP/CGH microarray analysis.

A total of 28 cases (14 women and 14 men) with a mean age of 68.4 years (range 22–87 years) were included in the study. Out of those 28 cases, A total 17/28 cases including 15 cases with normal metaphase cytogenetics and 2 cases with failed metaphase cytogenetics were randomly selected for the study (Table 1a). A total of 11/28 cases with previously detected genetic abnormalities by metaphase cytogenetics were preselected for the study. Those 11 cases have wide range of chromosomal abnormalities ranging from simple numerical changes (ie, monosomy or trisomy), structural chromosomal abnormalities such as interstitial deletion, translocation involving two chromosomes and complex karyotypes with multiple abnormalities.

The 28 cases included in the study are 18 cases of confirmed MDS, five suspected cases of MDS with borderline morphologic dysplasia, three cases of AML with myelodysplasia related changes, one case of Chronic myelomonocytic leukaemia (CMML) and one case of Myelodysplastic syndrome / Myeloproliferative neoplasm overlap unclassifiable (MDS/MPN u) (Table 1b). 

According to the bone marrow morphological assessment and the WHO classification, the cohort comprised of the following MDS subtypes: refractory cytopenia with multilineage dysplasia  (RCMD, n=7), refractory cytopenia with multilineage dysplasia with ring sideroblasts (RCMD-RS, n=3),  refractory anaemia with ring sideroblasts (RARS, n=4), refractory anaemia (RA, n=1), refractory anaemia with excess blasts ( RAEB-1, n=1; RAEB-2:n=1).

All cases had their bone marrow samples submitted to our regional laboratory within the period from March 2014 to April 2016 for evaluation of cytopenias and/or clinical suspicion of MDS related disorders.  

The cytomorphological examination and assessment was performed in Auckland regional laboratory and MDS diagnosis reported according the world health organisation (WHO) Classification criteria 2008. Samples were also referred to IGENZ laboratory for further cytogenetic testing.  These laboratories are IANZ accredited.

Metaphase cytogenetic analysis was carried out on marrow samples according to standard methods.  Chromosome preparations were G-banded using trypsin and Giemsa (GTG) and karyotypes were reported according to the International System for Human Cytogenetic Nomenclature (ISCN). 

Principles of Microarrays

SNP/CGH microarray analysis was performed using an Agilent 8 x 60K SUREPRINT G3 custom CGH/SNP array platform according to the manufacturer instructions and with a practical resolution of ~180kb. Data was analysed using Agilent Cytogenomic Software v3.0 using human genomic build 19. The sample was hybridised against a commercially produced reference DNA obtained from Agilent.

The research project has been registered and approved by the local Middlemore hospital research office and adhered to the national HDEC guidelines where formal ethics approval was deemed not required. The study cohort was subdivided in to two main groups (Group A & Group B) and one small group (Group C).    

Group A included the randomly selected 15 cases with normal karyotype by conventional metaphase cytogenetics analysis. Group B included the preselected eleven cases with wide range of chromosomal abnormalities detected by metaphase cytogenetics. Group C however is comprised of two cases with failed conventional metaphase cytogenetics.

The detection rate of genomic abnormalities was compared between metaphase cytogenetics analysis and SNP/CGH microarray analysis across the three groups.  

Further analysis including IPSS scores, survival data and disease outcome was performed on the 18 cases with confirmed MDS diagnosis. The standard metaphase cytogenetics based IPSS scores were compared to the SNP/CGH microarray based IPSS scores. (Table 2).

Table 1a. Result showing the Standard metaphase cytogenetic and SNP/CGH microarray analysis of  28 patients

Case #	AGE	Gender	Diagnosis	Metaphase cytogenetics	SNP/CGH microarray
1	70	Male	AML-with-MDS-related-changes	Normal	Normal
2	73	Male	AML-with-MDS-related-changes	Normal	Normal
3	78	Female	RARS	Normal	Normal
4	80	Female	RCMD	-20	dic (17;20)
5	72	Male	RAEB-2	-7	-7
6	52	Female	MDS/MPN-unclassifiable	Complex	Complex
7	76	Male	RCMD-RS	Del(20q)+t(4;7)	Del(20q)
8	78	Female	RCMD	Del(20q)	Del(20q)
9	82	Male	Possible MDS	-y	-y
10	58	Female	RCMD+RS	Normal	Normal
11	67	Female	Possible MDS	Normal	Normal
12	83	Female	RCMD	Normal	Normal
13	87	Female	RCMD and Myeloma	Normal	Del(20q)
14	73	Female	RARS	Normal	Normal
15	22	Male	RCMD	Normal	Normal
16	53	Female	Hypoplastic MDS and Myeloma	Normal	Del(13q)
17	71	Male	Possible MDS	+20	Normal
18	72	Male	AML-with-MDS-related-changes	Complex	Complex
19	72	Male	RARS	karyotype-failed	-y
20	34	Female	Possible MDS	karyotype-failed	Normal
21	74	Male	RAEB-1	Complex	Complex+additional
22	77	Male	RARS	Normal	Complex
23	78	Female	RCMD	Del(20q)	Del(20q)
24	56	Female	RA	Normal	Normal
25	79	Male	Possible MDS	Normal	Del(5q13.2)
26	75	Male	CMML2	Normal	Del(17q11.2)
27	65	Male	RCMD+RS	Normal	Normal
28	57	Female	RCMD	Del(7q)	Complex (including Del(7q)

RCMD = refractory cytopenia with multilineage dysplasia, RCMD-RS = refractory cytopenia with multilineage dysplasia with ring sideroblasts. RARS = refractory anaemia with ring sideroblasts, RA = refractory anaemia, RAEB-1 or 2 = refractory anaemia with excess blasts 1 or 2.    

The SNP/CGH microarray analysis results were compared with metaphase cytogenetics in the three groups.

Table1a(i). Results  which are considered  in Group A

Case #	AGE	Gender	Diagnosis	Metaphase cytogenetics	SNP/CGH microarray
1	70	Male	AML-with-MDS-related-changes	Normal	Normal
2	73	Male	AML-with-MDS-related-changes	Normal	Normal
3	78	Female	RARS	Normal	Normal
10	58	Female	RCMD+RS	Normal	Normal
11	67	Female	Possible MDS	Normal	Normal
12	83	Female	RCMD	Normal	Normal
13	87	Female	RCMD and Myeloma	Normal	Del(20q)
14	73	Female	RARS	Normal	Normal
15	22	Male	RCMD	Normal	Normal
16	53	Female	Hypoplastic MDS and Myeloma	Normal	Del(13q)
22	77	Male	RARS	Normal	Complex
24	56	Female	RA	Normal	Normal
25	79	Male	Possible MDS	Normal	Del(5q13.2)
26	75	Male	CMML2	Normal	Del(17q11.2)
27	65	Male	RCMD+RS	Normal	Normal

In group A there are 15 cases with normal metaphase cytogenetics but SNP/CGH microarray analysis was able to detect a wide spectrum of cryptic genomic abnormalities in 5 cases out of 15 cases that I  one third of cases. Among those five cases, there is only one case with a very little  morphological dysplasia and the rest of the four cases had a confirmed MDS diagnosis with significant morphological dysplasia.       

Those five cases with only positive SNP/CGH microarray findings are:

1) CMML case where SNP/CGH microarray analysis detected a 521 kb loss in the long arm of chromosome 17 [Del(17q11.2)].  

2) A case of suspected MDS with borderline dysplasia. The SNP/CGH microarray detected Del(5q13.2) characterised by a 2.2 Mb deletion in the long arm of chromosome 5. 

SNP and CGH Arrays

3) A case of confirmed MDS diagnosis WHO subtype RARS, the SNP/CGH microarray detected complex cytogenetics abnormalities (5p+, 19q+, 21q+) in the form of three small chromosomal gains in 5p13.3-p15.1, 19q13.2-q13.3 and 21q21.1-q21.2. 

4) A case of Hypoplastic MDS and myeloma, the SNP/CGH microarray detected Del(13q) characterised by 30Mb deletion in the long arm of chromosome 13 at band q21.31 – q31.3.

5) A case with established MDS and RCMD WHO subtype, the SNP/CGH array identified the presence of Del(20q).      

Table1a(ii). Results  which are considered  in Group B

Case #	AGE	Gender	Diagnosis	Metaphase cytogenetics	SNP/CGH microarray
4	80	Female	RCMD	-20	dic (17;20)
5	72	Male	RAEB-2	-7	-7
6	52	Female	MDS/MPN-unclassifiable	Complex	Complex
7	76	Male	RCMD-RS	Del(20q)+t(4;7)	Del(20q)
8	78	Female	RCMD	Del(20q)	Del(20q)
9	82	Male	Possible MDS	-y	-y
17	71	Male	Possible MDS	+20	Normal
18	72	Male	AML-with-MDS-related-changes	Complex	Complex
21	74	Male	RAEB-1	Complex	Complex+additional
23	78	Female	RCMD	Del(20q)	Del(20q)
28	57	Female	RCMD	Del(7q)	Complex (including Del(7q)

In group B there were 11 cases with already established chromosomal abnormalities by metaphase cytogenetics. However,  the SNP/CGH microarray showed a wide spectrum of findings that correlated quite well with most of the already established metaphase cytogenetics results. However, the microarray managed to reveal additional significant cryptic genomic abnormalities in three out of the eleven cases (27.2%) that were not apparent by metaphase cytogenetics. Some of those additional SNP/CGH array abnormalities have shown significant prognostic implication.  On the other hand, metaphase cytogenetics were superior to SNP/CGH microarray in two out of the eleven cases (18.2%), where the SNP/CGH microarray failed to detect some of the already established chromosomal abnormalities by metaphase cytogenetics. 

The three cases where the SNP/CGH microarray detected additional genetic abnormalities are: 

80-year-old female with confirmed diagnosis of MDS WHO subtype refractory cytopenia with multiline age dysplasia (RCMD) and presented with moderate anaemia and thrombocytopenia. Conventional metaphase cytogenetics identified -20 as a sole cytogenetic abnormality. The SNP/CGH microarray analysis however revealed a serious cryptic genomic abnormality, dic(17;20) with both 17p and 20q deletions.

74-year-old male with a confirmed diagnosis of MDS WHO subtype refractory anaemia with excess blast -1 (RAEB-1). Metaphase cytogenetics analysis established a complex karyotype (+19, Del(20q), +21). The SNP/CGH microarray however confirmed the complex abnormalities with an additional abnormality of +8.

A 57-year-old female with established diagnosis of MDS, WHO subtype RCMD. She presented with moderate anaemia and neutropenia. Metaphase cytogenetics analysis revealed Del (7q) only. The SNP/CGH microarray analysis however confirmed the presence of Del(7q) and showed two additional cryptic genomic lesions; Del(7p) and gain of 21. This confirmed the presence of complex cytogenetics. 

The two cases with higher detection of chromosomal lesions by metaphase cytogenetics than SNP/CGH microarray analysis are:

71-year-old male with suspected diagnosis of MDS and borderline dysplasia. Initial conventional metaphase cytogenetic revealed the presence of trisomy 20. The SNP/CGH microarray analysis failed to detect the trisomy 20.

Components of SNP and CGH Arrays

76-Year-old male with confirmed diagnosis of MDS WHO subtype refractory cytopenia with multilineage dysplasia and ring sideroblast (RCMD+RS). Conventional metaphase cytogenetics identified the presence of two clonal abnormalities; t(4;7) and Del(20q). The SNP+CGH microarray analysis was able to accurately detect the presence of Del(20q) but failed to identify the presence of t(4;7).

Table1a(iii). Results  which are considered  in Group C

Case #	AGE	Gender	Diagnosis	Metaphase cytogenetics	SNP/CGH microarray
19	72	Male	RARS	karyotype-failed	-y
20	34	Female	Possible MDS	karyotype-failed	Normal

The group C consist of two cases with failed metaphase cytogenetics analysis and  the SNP/CGH microarray detected –Y chromosome in the first case of 72-year-old male with established morphologic diagnosis of MDS and RARS subtype. The second case is a suspected MDS case and the SNP/CGH microarray did not reveal any genomic abnormality.

Table 1b. Frequency and Percentage of  diseases
	Frequency	Percent
	AML-with-MDS-related-changes	3	10.7
CMML2	1	3.6
Hypoplastic MDS	1	3.6
MDS/MPN-unclassifiable	1	3.6
Possible MDS	5	17.9
RA	1	3.6
RAEB-1	1	3.6
RAEB-2	1	3.6
RARS	4	14.3
RCMD	7	25.0
RCMD+RS	3	10.7
Total	28	100.0

AML= acute myeloid leukaemia, CMML2 = chronic myelomonocytic leukaemia2, MDS, MDS = myelodysplastic syndrome, RCMD = refractory cytopenia with multilineage dysplasia, RCMD-RS = refractory cytopenia with multilineage dysplasia with ring sideroblasts. RARS = refractory anaemia with ring sideroblasts, RA = refractory anaemia, RAEB-1 or 2 = refractory anaemia with excess blasts 1 or 2.  

Table 2. Result showing IPSS score analysis of 18 cases

Diagnosis (MDS subtype)	Metaphase cytogenetics	Microarray	Metaphase cytogenetics based IPSS score	Microarray based IPSS score	Patient outcome.	Survival in months	Time to transformation to AML.
RARS	Normal	Normal	0.0 (Low)	0.0 (Low)	Alive	23M	No transformation
RCMD	-20	Dic(17;20)	0.5 (INT-1)	0.5 (INT-1)	Dead	14M	11 M
RAEB-2	-7	-7	2.5 (High)	2.5 (High)	Dead	28M	2M
RCMD+rs	Del(20q), t(4;7)	Del(20q)	0.5 (INT-1)	0.0 (Low)	Alive	29M	No transformation
RCMD	Del(20q)	Del(20q)	0.0 (Low)	0.0 (low)	Alive	20M	No Transformation
RCMD+rs	Normal	Normal	0.0 (Low)	0.0 (Low)	Alive	5M	No Transformation
RCMD	Normal	Normal	0.0 (Low)	0.0 (Low)	Alive	22M	No Transformation
RCMD	Normal	Del(20q)	0.0 (Low)	0.0 (Low)	Alive	34M	No Transformation
RARS	Normal	Normal	0.0 (Low)	0.0 (Low)	Alive	15M	No Transformation
RCMD	Normal	Normal	0.0 (Low)	0.0 (Low)	Alive	24M	No Transformation
Hypoplastic MDS	Normal	Del(13q)	0.0 (Low)	0.0 (Low)	Alive	24M	No Transformation
RARS	Failed	-Y	–	0.0 (Low)	Alive	9M	No Transformation
RAEB-1	Complex	Complex + Additional	1.5 (INT-2)	1.5 (INT-2)	Alive	31M	No Transformation
RARS	Normal	Complex (undetermined clinical significance)	0.0 (Low)	0.0 (Low)	Alive	29M	No Transformation
RCMD	Del(20q)	Del(20q)	0.0 (Low)	0.0 (Low)	Alive	20M	No Transformation
RA	Normal	Normal	0.0 (Low)	0.0 (Low)	Alive	276M	No Transformation
RCMD+rs	Normal	Normal	0.0 (Low)	0.0 (Low)	Alive	3M	No Transformation
RCMD	Del(7q)	Complex	1.5 (INT-2)	1.5 (INT-2)	Alive	2M	No Transformation.

AML= acute myeloid leukaemia, MDS = myelodysplastic syndrome, RCMD = refractory cytopenia with multilineage dysplasia, RCMD-RS = refractory cytopenia with multilineage dysplasia with ring sideroblasts. RARS = refractory anaemia with ring sideroblasts, RA = refractory anaemia, RAEB-1 or 2 = refractory anaemia with excess blasts 1 or 2. M= month, IPSS= international prognostic scoring system, INT-1 or 2 Intermediate 1 or 2.   

The IPSS score analysis on the 18 cases with confirmed MDS cases diagnosis showed that the microarray based IPSS scores correlated quite well with conventional metaphase cytogenetics based IPSS scores.

In a total of 7/18 cases, the SNP/CGH microarray detected more genomic abnormalities than metaphase cytogenetics. In all of these 7/18 cases with newly microarray detected additional genomic abnormalities, there has been no change in the IPSS score following the microarray compared to the conventional metaphase cytogenetics based IPSS score.

On the other hand the metaphase cytogenetics was superior to microarray in one case with confirmed MDS diagnosis (1/18), where the Array failed to identify the balanced translocation t(4;7). This resulted in the case being inappropriately categorised into a lower risk category with the microarray based IPSS score of 0.0, compared to standard metaphase cytogenetics based IPSS score of 0.5 consistent with Intermediate-1 risk category.   

The 18 cases with confirmed MDS diagnosis have a median survival 22.5 months from time of diagnosis (range 2-276 months). Two cases progressed into AML and died, while the rest of the 16 cases are still alive with no transformation to AML .

The application of SNP/CGH microarray analysis has the potential to greatly enhance the diagnostic and prognostic stratification processes for MDS cases especially when conventional metaphase cytogenetics is not informative.  

In group A 15 cases were found with normal metaphase cytogenetics, the SNP/CGH microarray analysis was able to detect cryptic chromosomal lesions in 5 cases put of 15 that means 33.3% of cases were having normal standard metaphase cytogenetics. This can be attributed to the higher resolution of SNP/CGH microarray analysis and the ability to reliably detect sub-microscopic chromosomal lesions.   

Relatively similar results have been demonstrated in other studies such as in Volkert study where the microarray identified copy number changes in 11% of total 520 MDS cases with confirmed normal karyotyping by metaphase cytogenetics [43]. Similarly, Mohamedali showed SNP array detected 10% cytogenetically cryptic deletions and 8% gains in low-risk MDS cases [22].

Our result of 33.3% is slightly higher than those studies, this can be attributed to sample size differences and also to patient selection. 

Those microarray identified chromosomal abnormalities are; Del(20q), Del(5q13.2), Del(17q11.2), complex (5p+, 19q+, 21q+) and Del(13q).   

Some of them helped determining the exact underlying gene mutation as Del(17q11.2) in the CMML-2 case where a 521 kb loss was identified in the long arm of chromosome 17. This abnormality had been previously described by Kolquist and thought to be encompassing NF1 tumour suppressor gene which has a possible role as a negative regulator of the RAS pathway [18]. This gene has also been shown to have some prognostic implications as per Frank’s study showing relatively increased incidence of NF1 mutation as the MDS disease progresses to acute leukaemia [34]. This was particularly relevant in our case as the patient had a relatively high risk CMML-2 with high blast count.   

The detection of Del(5q13.2) in a suspected case of MDS with borderline dysplasia also supports the hypothesis of enhanced diagnostic yield of SNP/CGH microarray analysis in suspected MDS cases with normal metaphase cytogenetics. This chromosomal lesion is different from the common 5q- and has been reported to be related to the gene RAD17 which have been previously identified in MDS by Starczynowski and others [36].  This clonal abnormality however will support the diagnosis of either MDS or clonal cytopenia of undetermined significance in the right clinical context.  

The detection of Del(20q) by the microarray in the case with established MDS diagnosis, will help in supporting the MDS diagnosis, given that it is very well known to be MDS related.       

The new identified microarray complex cytogenetics abnormalities; (5p+, 19q+, 21q+) and Del(13q) are not known to be specifically MDS related lesion. However, they may well indicate clonality and further support the MDS diagnosis in the right clinical context. 

Overall, it is worth highlighting the diagnostic yield of the SNP/CGH microarray as it helped in supporting the diagnosis of either MDS or Clonal Cytopenia of Undetermined Significance (CCUS) in 5/15 cases (33.3%) by detecting either known MDS cytogenetic abnormalities or clonal lesions.    

In group C (the two cases with failed metaphase cytogenetics analysis), SNP/CGH microarray however was normal in the case with inconclusive MDS diagnosis and only detected –Y in the other established case of MDS. Given that – Y may not be considered as a clonal abnormality in the elderly the utility of SNP/CGH array has not been accurately analysed in this context. 

A study by Arenillas showed that SNP array was able to detect cytogenetic abnormalities in 50% patients with failed metaphase cytogenetics. It also demonstrated some prognostic implications related to copy number alterations detected by the array [2].

In the Group B results it was found that 11 cases with already established chromosomal abnormalities by metaphase cytogenetics. The SNP/CGH microarray analysis showed good correlation characterised by the detection of majority of chromosomal abnormalities identified by standard metaphase cytogenetics. It also managed to reveal additional cryptic genomic lesion in 3 cases out of 11 cases which is 27.2% of the total cases. Some of those novel SNP/CGH array additionally identified genomic lesions s were noted to have major prognostic implications. 

This has been demonstrated clearly in the case of the 80-year-old lady with an established diagnosis of MDS RCMD subtype. Her conventional metaphase cytogenetics identified monosomy 20 as a sole cytogenetic abnormality and as a result of that her case had been categorised into the intermediate-1 risk category according to standard IPSS scoring system. Subsequent the SNP/CGH microarray analysis however revealed a seriously hidden cryptic genomic abnormality characterised by unbalanced dicentric (17;20), resulting in loss of 17p (including TP53) and 20q.  Although dic(17;20) is a rare abnormality it has been  reported in MDS cases by Tirado and Campbell and has been shown to be associated with disease progression and worse outcome [39].  

Moreover, the SNP/CGH microarray in this case has also suggested the presence of TP53 mutation which is one of the known mutations associated with MDS and AML. The presence of this mutation has been shown to be of great clinical significance and associated with poor survival even after bone marrow transplant [4,5,6 &11]. Some studies also showed that TP53 mutations have a major and unique prognostic value independent of the current IPSS score system for patients with MDS. [4,6 &17]. This was consistent with our patient’s progressive course with early transformation to acute leukaemia in 11 months’ time.  The Array results however did not lead to change in the IPSS score for this patient. This will support the hypothesis that large-scale genetic and molecular profiling is needed for further sub-classification and prognostic stratification in MDS patients [15].

The other two cases with the SNP/CGH microarray showing additional clonal chromosomal lesions, have already confirmed MDS diagnosis at the high risk category based on the metaphase cytogenetics based IPSS score. The additional clonal abnormalities identified by the SNP/CGH microarray however in those cases did not indicate significant change in the disease outcome or prognostic risk stratification.   

The combined SNP+CGH microarray analysis however did not identify a couple of chromosomal lesions previously detected by metaphase cytogenetics analysis in 2/11 (18%) of cases with abnormal karyotype.  In one case due to low level of the abnormal clone of 10%, the SNP/CGH microarray was unable to detect the presence of trisomy 20. This is one of the known limitation of SNP/CGH microarray analysis in detecting low clone size less than 30%. Given that microarray was reported as normal in this particular case, this could potentially lead to a reduction on its diagnostic yield. The array result in this case did not result in change in the IPSS score, given that trisomy 20 is one of the low risk clonal genetic abnormality.  In the second case the SNP/CGH microarray analysis failed to detect the presence of a likely balanced translocation t(4;7), however it managed to detect the presence of Del(20q). This can be explained by the known limitation of the SNP/CGH microarray analysis in detecting balanced translocations. This could potentially lead to inappropriately stratifying the patient into a lower risk group, if karyotyping has not been performed.   

The IPSS score analysis on the 18 cases with confirmed MDS cases showed that the Array based IPSS scores correlated quite well with the conventional karyotyping based IPSS score (Table 3).

There has been no significant change in IPSS score following the SNP/CGH microarray in the 7/18 cases with higher detection of genomic abnormalities by the SNP/CGH microarray compared to conventional metaphase cytogenetics.

This could possibly be explained by three factors:

1) The fact that some of these SNP/CGH microarray detected genomic lesions were of not known to be MDS related such as Del(13q) and (5p+, 19q+, 21q+). As a result of that those newly detected genomic lesions are not included in the standard IPSS score assessment.

2) Despite of the SNP/CGH microarray ability to detect certain poor prognostic genetic mutations, most of these mutations act as an independent risk factor regardless of the IPSS score.

3) Those Array identified good prognostic chromosomal abnormalities (e.g. Del(20q) and -Y) were mostly detected in the low risk group with already established low standard metaphase cytogenetics based IPSS score. On the other hand, the SNP/CGH microarray identified poor prognostic genomic abnormalities were also mostly detected in high risk group with already high IPSS score based on conventional metaphase cytogenetics. This has resulted in the IPSS score to essentially remain unchanged following the SNP/CGH microarray analysis results.

Overall in this small study the conventional metaphase cytogenetic appears to be slightly better than the microarray analysis in guiding the IPSS scoring system. This was particularly demonstrated in the case with the balanced translocation t(4;7) missed by the SNP/CGH microarray.      

There is no enough follow up period to reliably assess the presence of survival differences among the group with new or additional microarray identified genomic abnormalities. However, only one case with unique cryptic genomic abnormalities detected only by the array demonstrated progressive course with early transformation to AML and death. This case showed that certain genomic abnormalities (Del(17p) /TP53 gene mutation) have extremely poor prognosis independent of the IPSS score. This is consistent with other studies by Bejar and Horiike [4,5, 6 & 17].

Conclusion:

This pilot study has demonstrated that the combination of SNP/CGH microarray and conventional G banding analysis enhances the diagnostic yield and provides additional prognostic information in the assessment of MDS cases.  

The SNP/CGH microarray analysis was able to detect some cryptic genomic lesions which are of utmost clinical and prognostic significance. It was also able to identify the presence of some of the common genetic mutations with some of these lesions acting as an independent risk factor regardless of the IPSS /IPSS-R scoring system.  This will open the door for the creation of a new risk stratification system that includes all these relevant clonal genomic abnormalities.    

The SNP/CGH microarray analysis is relatively more expensive and costs $750 NZD pert test compared to $560 NZD for conventional metaphase cytogenetics. While our data showed that microarray analysis has a higher detection rate of genomic abnormalities with relatively good correlation with metaphase cytogenetics, however in view of its inherent limitation to detect small clone size and balanced translocation, it is recommended to be used in combination with standard metaphase karyotyping.

We conclude that SNP/CGH microarray is relevant option that can be easily performed in routine diagnostics in MDS in combination with standard G banding analysis. However given the significant cost implications associated with that, this option could be of particular relevance in certain cases such as:

Diagnostically uncertain cases with normal standard metaphase cytogenetics.

Transplant eligible patients where it is important to confirm the diagnosis and accurately determine the disease prognosis in order to make appropriate recommendations about the merits of a proceeding to stem cell transplant.

Research situations.

Further prospective studies with a larger sample size and adequate follow up period would be recommended to reliably assess both the diagnostic and prognostic utilities of SNP/CGH microarray analysis in MDS related disorders.  

• Irene Zeng, Biostatistician at Middlemore hospital
• Daniel Ng, IGENZ laboratory.
• Dr Amanda Dixon-McIver, IGENZ laboratory
• Dr Ross Henderson, North Shore Hospital.

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