Cytogenetic Analysis
Cytogenetic analysis was performed on 150 breast cancer patients (147 females and 3 males) and 150 age and gender matched controls. Out of 150 patients, 20 (13.3%), 74 (49.3%), 37 (24.7%) and 14 (9.3%) were diagnosed with stage I, Stage II, stage III and stage IV breast carcinoma, respectively. Exact stage of 5 (3.3%) patients could not be determined. Majority of the patients (89.3%) had IDC of breast. The chromosomal aberrations were counted as in metaphases with: only structural aberrations, with only numerical aberrations and metaphases with both structural and numerical aberrations. The difference in the frequencies of chromosomal aberrations amongst patients and controls was statistically significant (Table 1). The aberrations were higher in patients as compared to controls: mean (%) aberrant metaphases (22.6±12.3 vs 12.5±4.6, p<0.0001), mean (%) metaphases with structural aberrations (11.7±10.8 vs 4.5±3.1, p<0.0001), mean (%) metaphases with numerical aberrations (9.5±6.7 vs 6.2±3.5; p<0.0001). However, mean (%) metaphases with both structural and numerical aberrations were similar in both the groups (2.6±2.0 vs 2.6±1.1; p=1.00).
The stage-wise comparison of cytogenetic profile of breast cancer patients with controls has been shown in Table 2. The chromatid type aberrations observed in patients included premature cetromeric division, chromatid break and gap while the chromosome type aberrations included polyploidy, chromosomal gap, pulverizatrion, telomeric associations, chromosomal break, endoreduplication, robertsonian translocations, acentric fragments, ring chromosomes, deletions. Association between the acrocentric chromosome 13, 14, 15, 21 and 22 were scored separately in all metaphases. Acrocentric associations and telomeric bridges were also scored but not counted in the total aberrations. Telomeric associations were commonly seen in acrocentric chromosomes. Apart from acrocentric chromosomes, chromosome 1, 2, 16, 18, 20 and X were also frequently involved in telomeric associations. Breaks and gaps were the most frequent structural chromosomal aberration observed in various regions of different chromosomes. The chromosomes frequently involved in aberrations like loss, gain, deletion, addition and translocations have been shown in Table 3.
Table 1. Cytogenetic profile of breast cancer patients and controls
|
Patients
|
Controls
|
p-value
|
No. of subjects
|
150
|
150
|
|
Age (Mean±SD)
|
50.2±11.5
|
49.2±14.6
|
0.51
|
Mean (%) aberrant metaphases
|
22.6±12.3
|
12.5±4.6
|
<0.0001
|
Mean (%) metaphases with structural aberrations
|
11.7±10.8
|
4.5±3.1
|
<0.0001
|
Mean (%) metaphases with numerical aberrations
|
9.5±6.7
|
6.2±3.5
|
<0.0001
|
Mean (%) metaphases with both structural and numerical aberrations
|
2.6±2.0
|
2.6±1.1
|
1.00
|
Mean(%) metaphases with acrocentric associations
|
27.6±14.7
|
28.9±14.6
|
0.32
|
Significant p-value (<0.05), calculated by t-test, are shown in bold
Chromosomal aberrations present in 2% or more that 2% of metaphases in an individual were considered as clonal anomalies. Both structural and numerical clonal chromosomal anomalies were observed in 28 breast cancer patients. Clonal structural chromosomal anomalies observed in 5 cases were: [(46,XX,add(1)(pter→q21::?::q21→qter)], [45,XX,del(2)(pter→q11.2::21.2 →qter)], [46,XX,i(21)(q10;q10)], [45,XX,t(1;5)(5pter→5q23::1q25→qter)], [46,XX,?add(1) (q?21)]. Noticeably, chromosome 1 was found to be involved in clonal anomalies in three of the cases. Clonal numerical chromosomal anomalies were observed in 23 cases. The most frequent was loss of chromosome X which was observed in 10 cases. Other clonal numerical anomalies included: loss of chromosome 7, 9, 16 and 22 in two cases each; and loss of chromosome 2, 8, 11, 13, 14, 17 and 20 in one case each.
The control subjects had predominantly normal karyotype and the chromosomal aberrations found were lesser in frequency as compared to cases. Moreover, no specific or recurring anomaly was observed in controls. Frequency of non-clonal chromosomal aberrations observed in control were: telomeric association 26.3%; robertsonian translocation 14.3%; premature centromeric division 9.7%; break 9.2%; deletion 8.9%; acentric fragments 8.2%; marker chromosome 5.7%; triradial 3.9%; gap 3.2%; translocation 2.3%; endoreduplication 2.0%; dicentric 2.0%; double minute 0.9%; polyploidy 0.9%; addition 0.7%; ring chromosome 0.5%; fragile site 0.5%; duplication 0.5%; and inversion 0.4%.
Table 2. Comparison of cytogenetic profile of Breast cancer patients stage-wise and matched controls
S.No.
|
Patient group and controls
|
Mean (%) TAM
|
Mean (%) MSA
|
Mean (%) MNA
|
Mean (%) M(SA+NA)#
|
Mean MAA (%)
|
1
|
Stage I Cases (n=20)
Controls (n=20)
p-value
|
20.6±7.6
10.3±3.0
<0.0001
|
9.3±5.8
4.7±3.1
0.0034
|
9.2±5.0
4.5±3.2
0.0011
|
3.0±2.3
1.2±1.1
0.0031
|
28.6±13.5
30.2±11.8
0.069
|
2
|
Stage II Cases (n=74)*
Controls (n=74)
p-value
|
12.0±9.2
12.9±5.0
0.4609
|
13.1±9.2
4.7±3.2
<0.0001
|
9.4±6.2
6.3±3.4
0.0002
|
2.2±1.5
2.0±1.4
0.4031
|
28.3±15.0
29.6±15.0
0.5989
|
3
|
Stage III Cases(n=37)
Control (n=37)
p-value
|
21.7±12.1
11.9±4.1
<0.0001
|
10.6±7.9
4.1±2.8
0.0015
|
9.4±7.5
6.3±3.4
0.0250
|
3.5±2.8
1.5±1.2
0.0002
|
26.0±13.2
18.9±17.9
0.0561
|
4
|
Stage IV Cases (n=14)
Controls (n=14)
p-value
|
22.2±9.7
13.8±5.1
0.0081
|
10.9±7.2
5.1±3.5
0.0117
|
10.2±9.2
6.8±3.9
0.2142
|
2.1±1.0
1.9±1.8
0.7192
|
31.6±19.3
26.1±18.5
0.4484
|
5
|
Indeterminate Stage
Cases (n=5)
Controls (n=5)
p-value
|
16.7±2.4
13.0±5.5
0.2053
|
4.7±3.3
3.7±2.2
0.5883
|
11.5±3.3
7.7±4.8
0.1828
|
1.0±0.0
1.6±1.5
0.3972
|
24.6±8.1
26.0±14.0
0.8514
|
Significant p-value (<0.05), calculated by t-test, are shown in bold
TAM: total aberrant metaphases; MSA: metaphases with structural aberrations; MNA: metaphases with numerical aberrations; M(SA+NA): metaphases with structural and numerical aberrations; MAA: metaphases with acrocentric associations.
*One of the subjects with Stage II breast cancer had more than 90% frequency of structural aberration as it was a clonal chromosomal anomaly. Therefore, it was not included in the calculations. Similarly, one of the subjects with Indeterminate stage had a very higher frequency of structural aberrations, thus, it was not included in the calculations.
# The zero values were omitted during the calculation of Average and Standard Deviation due to the presence of high number of zero values in Mean(%) M(SA+NA).
To identify the genes harbored by the chromosomal regions showing increased aberration frequency in present study sample, data was retrieved from Atlas of Genetics and Cytogenetics in Oncology and Hematology [49] and Genatlas database [50] (Table 4).
In-silico analysis: Functional Interaction analysis revealed the involvement of various genes (linker genes) that are linked to the query genes (observed to be harboured by the chromosomal region frequently involved in anomalies in the present study) through different networks (Figure 1). Pathway enrichment for invasive ductal breast carcinoma was performed to identify the genes invloved in IDC as majority of the patients in the present study sample (89.3%) had IDC of breast (Figure 2). Linker genes that were involved in IDC were SMAD4, EP300, PIK3CA, TP53, HIF1A and AKT1.
We analyzed pathways on a set of genes that are not linked together by checking ‘show genes not linked to others’ in FI Network Construction Parameters. Pathway Enrichment analysis revealed that genes HDAC3, NCOA1, NLRC4, COL1A1, RARA, WWTR1, and BRCA1 are enriched in the RNA Polymerase II Transcription pathway (Figure 3).
Table 3. Comparison of frequency of chromosomes involved in various aberrations in the breast cancer patients and controls
Type of aberration
|
Cases
|
Controls
|
p-value
|
Chromosomes/
chromosome arms involved
|
Frequency
|
Chromosomes/ chromosome
arms involved
|
Frequency
|
Loss
|
5, 8,16, 17, 18, 19, 20, 21, 22, X
|
78.8±21.20
|
8, 9, 15, 17, 19, 20, 22, X
|
57.25±9.23
|
0.0169
|
Gain
|
2, 3, 8, 9, X
|
8.8±4.6
|
3, 4, 6, 16, 21
|
9.8±2.28
|
0.674
|
Break
|
1p, 1q, 2p, 2q, 3q, 4p, 4q, 7q, 9q, 17q
|
12.43±5.29
|
1q, 2q, 3p, 3q, 4q, 16q
|
4.4±1.95
|
0.0065
|
Gap
|
1p, 1q, 2p, 3p, 3q, 4q, 5q, 6q, 9q, 11q
|
4.25±2.19
|
1q, 2q, 5q, 14q, 17q
|
1.8±0.84
|
0.0334
|
Deletion
|
1p, 1q, 2q, 3q, 4q, 5q, Xq
|
7.2±2.6
|
1p, 1q, 5p, 6q, Xp
|
4.2±1.1
|
0.0264
|
Addition
|
1q, 9q
|
|
9q
|
NC
|
NC
|
Translocations
|
1, 5, 8, 10, 12, X
|
4.83±1.47
|
2, 4, 16
|
2.66±0.57
|
0.0475
|
Robertsonian Translocation
|
15, 21
|
26.5±7.78
|
13, 21, 22
|
26.33±3.79
|
0.9749
|
Telomeric Associations
|
1, 2, 3, 12,16, 18, 19, 20
|
9.83±3.37
|
7,19, X
|
14.33±4.04
|
0.1182
|
Acrocentric chromosomes:13, 14, 15, 21, 22
|
101±14.73
|
Acrocentric chromosomes: 13, 14, 15, 21, 22
|
44.8±11.19
|
0.0008
|
Triradials
|
15, 21, 22
|
17±5.29
|
14, 15, 21, 22
|
9.75±2.36
|
0.0552
|
Significant p-value (<0.05), calculated by t-test, are shown in bold
Table 4: Genes harboured by the chromosomal regions recurring in anomalies in present study sample
Chromosomal region
|
Genes*
|
1p32
|
RNF11
|
1q21
|
ARNT, SHC1, PIP5K1A, S100A10, BCL9, MAD1L1, PDE4DIP
|
2p21
|
EML4
|
2p22
|
NLRC4, MSN, BIRC6, STRN, EIF2AK2
|
2p23
|
NCOA1, ALK
|
3p21
|
LIMD1, MAP4, RHOA, PFKFB4, MST1, SEMA3F, SETD2, PBRM1, BAP1, PBRM1, PRKCD
|
3q25
|
WWTR1, SIAH2, MLF1, RARRES1
|
4q12
|
FIP1L1
|
4q31
|
INPP4B, NR3C2
|
5q31
|
AFF4, SLIT3, VDAC1, ANKHD1, HDAC3, ARHGAP26
|
6q13
|
SMAP1
|
6q25
|
RGS17, AKAP12, LATS1
|
6q27
|
FGFR10P, THBS2
|
7q22
|
CUX1
|
10q21
|
CCDC6, RHOBTB1, ARID5B
|
11q23
|
SDHD, ARHGEF12
|
15q22
|
PCLAF, DAPK2
|
17q21
|
GSDMB, RARA, CDC6, STAT3, GAST, ACLY, BRCA1, ETV4, NMT1, KPNB1, IGF2BP1, NGFR, XYLT2, PPP1R9B, COL1A1
|
*Source: Atlas of Genetics and Cytogenetics in Oncology and Hematology [49] and Genatlas database [50]