- Research
- Open Access
Succinate dehydrogenase expression in breast cancer
- Sewha Kim1,
- Do Hee Kim1,
- Woo-Hee Jung1 and
- Ja Seung Koo1Email author
- Received: 10 June 2013
- Accepted: 20 June 2013
- Published: 3 July 2013
Abstract
The aim of this study was to investigate succinate dehydrogenase (SDH) expression in breast cancer according to breast cancer molecular subtype using immunohistochemistry and to assess the clinical implications of SDH expression. Immunohistochemical staining for ER, PR, HER-2, Ki-67, HIF-1α, SDHA, and SDHB was performed on tissue microarrays of 721 breast cancers. According to the immunohistochemistry results for ER, PR, HER-2, and Ki-67 and fluorescence in situ hybridization (FISH) results for HER-2, breast cancer molecular subtypes were classified into luminal A, luminal B, HER-2, and triple-negative breast cancer (TNBC). HER-2 subtype breast cancers most frequently showed high-level expression of SDHA in tumor cells, while the luminal A subtype most frequently showed low or negative expression of SDHA in tumor cells (P = 0.032). Stromal SDHB expression rate was highest in HER-2 subtype and lowest in TNBC (P < 0.001). SDHA-negative breast cancers were associated with younger age at diagnosis (P = 0.012), and SDHB-negative breast cancers with lower histologic grade (P = 0.044) and lower Ki-67 labeling index (LI) (P = 0.046). Tumor phenotypes according to the SDH status were SDHA(+)/SDHB(+) > SDHA(–)/SDHB(–) > SDHA(–)/SDHB(+) > SDHA(+)/SDHB(–) in order of frequency. The stromal phenotypes were SDHA(–)/SDHB(–) > SDHA(+)/SDHB(+) > SDHA(–)/SDHB(+) > SDHA(+)/SDHB(–). In conclusion, loss of SDHA or SDHB expression was observed in about 3% of breast cancers in this study. Low SDH expression status in breast tumor cells was associated with younger age at diagnosis and low-grade histology.
Keywords
- Breast
- Metabolism
- Mitochondria
- Succinate dehydrogenase
Introduction
Succinate dehydrogenase (SDH) (succinate-coenzyme Q reductase, respiratory Complex II) catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol. Through the coupling of these two reactions in the inner mitochondrial membrane, SDH links glucose oxidation in the TCA cycle with ATP production in the mitochondria (Cardaci & Ciriolo 2012;King et al. 2006;Barletta & Hornick 2012;Van Nederveen et al. 2009). SDH is composed of four subunits; SDHA, SDHB, SDHC and SDHD. The first two are hydrophilic proteins protruding into the mitochondrial matrix, and the second two are hydrophobic proteins that anchor the catalytic core into the inner mitochondrial membrane (Cardaci & Ciriolo 2012; King et al. 2006; Barletta & Hornick 2012; Van Nederveen et al. 2009).
Germline loss-of-function SDH mutations are associated with several tumors such as pheochromocytoma and paraganglioma, gastrointestinal stromal tumor (GIST), and renal cell carcinoma (Hensen & Bayley 2011; Kantorovich et al. 2010; Anderson et al. 2004; Astuti et al. 2001; Miettinen et al. 2011; Badve et al. 2011; Ricketts et al. 2008). Among these, GIST and renal cell carcinoma are reported to display histologic features that distinguish SDH-deficient tumors (Miettinen et al. 2011). Although SDH germline mutation may be accurately evaluated by gene sequencing, a few recent studies have shown very high sensitivity for immunohistochemical detection of SDH mutation in GIST, pheochromocytoma, and paraganglioma (Van Nederveen et al. 2009; Gill et al. 2010a; Miettinen et al. 2013; Janeway et al. 2011).
To date, few studies have investigated SDH expression in breast cancer. We investigated SDH expression status according to breast cancer molecular subtype using immunohistochemical methods and assessed the clinical implications of SDH expression in breast cancer.
Materials and methods
Patient selection
Patients who were diagnosed with invasive ductal carcinoma, not otherwise specified and underwent surgical excision at Severance Hospital between January 2002 and December 2005 were included in the study. Patients who received preoperative hormonal therapy or neoadjuvant chemotherapy were excluded. All hematoxylin and eosin (H&E)-stained slides for each case were retrospectively reviewed by breast pathologists (Koo JS). The histological grade was assessed using the Nottingham grading system.(Elston & Ellis 1991) The clinicopathologic parameters evaluated for each breast cancer included patient age at initial diagnosis, lymph node metastasis, tumor recurrence, distant metastasis, and patient survival. The Institutional Review Board of Yonsei University Severance Hospital approved this study.
Tissue microarray
On H&E-stained slides of tumors, a representative area was selected and a corresponding spot was marked on the surface of the paraffin block. Using a biopsy needle, the selected area was punched out and a 3-mm core of tumor tissue was placed onto a 6 × 5 recipient block. Two tissue cores were extracted from each sample to minimize bias. Each tissue core was assigned a unique microarray location number that was linked to clinicopathologic data in a database.
Immunohistochemistry
Clone, dilution, and source of antibodies used
Antibody | Clone | Dilution | Company |
|---|---|---|---|
HIF-1α | EP1215Y | 1:100 | Biocare, Yorba Linda, CA, USA |
SDHA | 2E3GC12FB2AE2 | 1:100 | Abcam, Cambridge, UK |
SDHB | 21A11AE7 | 1:100 | Abcam, Cambridge, UK |
Tumor phenotype-related | |||
ER | SP1 | 1:100 | Thermo Scientific, CA, USA |
PR | PgR | 1:50 | DAKO, Denmark |
HER-2 | Polyclonal | 1:1500 | DAKO, Denmark |
Ki-67 | MIB-1 | 1:150 | DAKO, Glostrup, Denmark |
Interpretation of immunohistochemical staining
All immunohistochemical markers were assessed by light microscopy. Parameters such as ER, PR, and HER-2 status were obtained from patients’ pathologic reports. A cut-off value of 1% or more positively stained nuclei was used to define ER and PR positivity (Hammond et al. 2010). HER-2 staining was analyzed according to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines using the following categories: 0, no immunostaining; 1+, weak incomplete membranous staining, less than 10% of tumor cells; 2+, complete membranous staining, either uniform or weak in at least 10% of tumor cells; and 3+, uniform intense membranous staining in at least 30% of tumor cells (Wolff AC, et al. 2007). HER-2 immunostaining was recorded as positive when strong (3+) membranous staining was observed, whereas staining categories 0 to 1+ were recorded as negative. Samples showing 2+ HER-2 expression were evaluated for HER-2 amplification by fluorescence in situ hybridization (FISH).
Immunohistochemical evaluation of SDHB expression. (A) negative (B) low positive (C) high positive.
Fish analysis
FISH was performed using a PathVysion HER-2 DNA Probe Kit (Vysis, Downers Grove, IL, USA) according to the manufacturer’s instructions. Invasive tumors were first examined on hematoxylin-eosin-stained slides to confirm histology. HER-2 gene copy number on the slides was evaluated using an epifluorescence microscope (Olympus, Tokyo, Japan) according to the ASCO/CAP guidelines (Wolff AC, et al. 2007). At least 60 tumor cell nuclei in three separate regions were investigated for HER-2 and chromosome 17 signals. An absolute HER-2 gene copy number lower than 4 or an HER-2 gene/chromosome 17 copy number ratio (HER-2/Chr17 ratio) less than 1.8 was considered HER-2-negative. An absolute HER-2 copy number between 4 and 6 or an HER-2/Chr17 ratio between 1.8 and 2.2 was considered HER-2-equivocal. An absolute HER-2 copy number greater than 6 or an HER-2/Chr17 ratio higher than 2.2 was considered HER-2-positive.
Tumor phenotype classification
Breast cancer phenotypes were classified according to the immunohistochemistry results for ER, PR, HER-2, and Ki-67 and the FISH results for HER-2 as follows (Goldhirsch A, et al. 2011): Luminal A subtype as ER- or/and PR-positive and HER-2-negative and Ki-67 LI <14%; Luminal B subtype as ER- or/and PR-positive and HER-2-negative and Ki-67 LI ≥14%; HER-2 positive as ER- or/and PR-positive and HER-2 overexpressed or/and amplified; HER-2-overexpression subtype as ER- and PR-negative and HER-2 overexpressed or/and amplified; and triple-negative breast cancer (TNBC) subtype as ER-, PR-, and HER-2-negative.
Statistical analysis
Data were analyzed using SPSS for Windows, Version 12.0 (SPSS Inc., Chicago, IL, USA). Student’s t and Fisher’s exact tests were used to evaluate continuous and categorical variables, respectively. The significance level was set at 0.05. The time to tumor recurrence and overall survival were evaluated by Kaplan-Meier and log-rank statistics. Multivariate regression analysis was performed using the Cox proportional hazards model.
Results
Patients’ characteristics
Clinicopathologic characteristics according to breast cancer phenotype
Parameters | Total n = 721 (%) | Luminal A n = 302 (%) | Luminal B n = 168 (%) | HER-2 n = 69 (%) | TNBC n = 182 (%) | P-value |
|---|---|---|---|---|---|---|
n = 302 (%) | n = 168 (%) | n = 69 (%) | n = 182 (%) | |||
Age (years, mean ± SD) | 49.6 ± 10.9 | 50.6 ± 10.4 | 48.4 ± 10.0 | 52.4 ± 10.0 | 48.0 ± 12.4 | 0.005 |
Histologic grade | <0.001 | |||||
I | 120 (16.6) | 92 (30.5) | 20 (11.9) | 1 (1.4) | 7 (3.8) | |
II | 364 (50.5) | 182 (60.3) | 91 (54.2) | 36 (52.2) | 55 (30.2) | |
III | 237 (32.9) | 28 (9.3) | 57 (33.9) | 32 (46.4) | 120 (65.9) | |
Tumor stage | 0.007 | |||||
T1 | 350 (48.5) | 167 (55.3) | 85 (50.6) | 30 (43.5) | 68 (37.4) | |
T2 | 356 (49.4) | 127 (42.1) | 81 (48.2) | 38 (55.1) | 110 (60.4) | |
T3 | 15 (2.1) | 8 (2.6) | 2 (1.2) | 1 (1.4) | 4 (2.2) | |
Nodal stage | 0.060 | |||||
N0 | 425 (58.9) | 171 (56.6) | 92 (54.8) | 42 (60.9) | 120 (65.9) | |
N1 | 192 (26.6) | 89 (29.5) | 43 (25.6) | 13 (18.8) | 47 (25.8) | |
N2 | 65 (9.0) | 27 (8.9) | 18 (10.7) | 9 (13.0) | 11 (6.0) | |
N3 | 39 (5.4) | 15 (5.0) | 15 (8.9) | 5 (7.2) | 4 (2.2) | |
Estrogen receptor status | <0.001 | |||||
Negative | 261 (36.2) | 5 (1.7) | 5 (3.0) | 69 (100.0) | 182 (0.0) | |
Positive | 460 (63.8) | 297 (98.3) | 163 (97.0) | 0 (0.0) | 0 (0.0) | |
Progesterone receptor status | <0.001 | |||||
Negative | 346 (48.0) | 49 (16.2) | 46 (27.4) | 69 (100.0) | 182 (100.0) | |
Positive | 375 (52.0) | 253 (83.8) | 122 (72.6) | 0 (0.0) | 0 (0.0) | |
HER-2 status | <0.001 | |||||
Negative | 573 (79.5) | 302 (100.0) | 89 (53.0) | 0 (0.0) | 182 (100.0) | |
Positive | 148 (20.5) | 0 (0.0) | 79 (47.0) | 69 (100.0) | 0 (0.0) | |
Ki-67 LI (%, mean ± SD) | 17.3 ± 18.4 | 4.7 ± 3.7 | 19.6 ± 12.6 | 19.3 ± 12.8 | 35.1 ± 23.0 | <0.001 |
Tumor recurrence | 63 (8.7) | 14 (4.6) | 13 (7.7) | 11 (15.9) | 25 (13.7) | 0.001 |
Patient death | 60 (8.3) | 12 (4.0) | 13 (7.7) | 11 (15.9) | 24 (13.2) | <0.001 |
Duration of clinical follow-up (months, mean ± SD) | 70.0 ± 31.2 | 72.4 ± 29.3 | 70.2 ± 30.0 | 65.2 ± 34.3 | 67.8 ± 34.2 | 0.234 |
HIF-1α and sdh status according to molecular subtype of breast cancer
Expression of HIF-1α and SDH according to tumor phenotype
Parameters | Total n = 721 (%) | Luminal A n = 302 (%) | Luminal B n = 168 (%) | HER-2 n = 69 (%) | TNBC n = 182 (%) | P-value |
|---|---|---|---|---|---|---|
Nuclear HIF-1α | 0.319 | |||||
Negative | 697 (96.7) | 296 (98.0) | 161 (95.8) | 65 (94.2) | 175 (96.2) | |
Positive | 24 (3.3) | 6 (2.0) | 7 (4.2) | 4 (5.8) | 7 (3.8) | |
Cytoplasmic HIF-1α | 0.596 | |||||
Negative | 717 (99.4) | 301 (99.7) | 166 (98.8) | 69 (100.0) | 181 (99.5) | |
Positive | 4 (0.6) | 1 (0.3) | 2 (1.2) | 0 (0.0) | 1 (0.5) | |
Tumoral SDHA | 0.032 | |||||
Negative/Low | 41/319 (49.9) | 21/143 (54.3) | 8/76 (50.0) | 5/19 (34.8) | 7/81 (48.4) | |
High | 361 (50.1) | 138 (45.7) | 84 (50.0) | 45 (65.2) | 94 (51.6) | |
Stromal SDHA | 0.063 | |||||
Negative | 665 (92.2) | 286 (94.7) | 153 (91.1) | 59 (85.5) | 167 (91.8) | |
Positive | 56 (7.8) | 16 (5.3) | 15 (8.9) | 10 (14.5) | 15 (8.2) | |
Tumoral SDHB | 0.291 | |||||
Negative/Low | 24/245 (37.3) | 15/103 (39.1) | 3/56 (35.1) | 3/19 (31.9) | 3/67 (38.5) | |
High | 452 (62.7) | 184 (60.9) | 109 (64.9) | 47 (68.1) | 112 (61.5) | |
Stromal SDHB | <0.001 | |||||
Negative | 641 (88.9) | 277 (91.7) | 142 (84.5) | 51 (73.9) | 171 (94.0) | |
Positive | 80 (11.1) | 25 (8.3) | 26 (15.5) | 18 (26.1) | 11 (6.0) |
Expression of SDHA and SDHB according to the molecular subtype of breast cancer. Tumor cells showed highest SDHA expression in the HER-2 subtype and lowest SDHA expression in the luminal A subtype. Stromal elements expressed SDHA at lower levels than tumor cells.
Expression of SDHA and SDHB according to HIF-1α expression status
Factors | Nuclear HIF-1α | Cytoplasmic HIF-1α | ||||
|---|---|---|---|---|---|---|
Negative n = 697 (%) | Positive n = 24 (%) | P-value | Negative n = 717 (%) | Positive n = 4 (%) | P-value | |
Tumoral SDHA | 0.247 | 0.518 | ||||
Negative | 40 (5.7) | 1 (4.2) | 40 (5.6) | 1 (25.0) | ||
Low | 311 (44.6) | 8 (33.3) | 318 (44.4) | 1 (25.0) | ||
High | 346 (49.6) | 15 (62.5) | 359 (50.1) | 50.0) | ||
Stromal SDHA | 0.423 | 1.000 | ||||
Negative | 644 (92.4) | 21 (87.5) | 661 (92.2) | 4 (100.0) | ||
Positive | 53 (7.6) | 3 (12.5) | 56 (7.8) | 0 (0.0) | ||
Tumoral SDHB | 0.926 | 0.032 | ||||
Negative | 24 (3.4) | 0 (0.0) | 23 (3.2) | 1 (25.0) | ||
Low | 235 (33.7) | 10 (41.7) | 243 (33.9) | 2 (50.0) | ||
High | 438 (62.8) | 14 (58.3) | 451 (62.9) | 1 (25.0) | ||
Stromal SDHB | 0.328 | 1.000 | ||||
Negative | 621 (89.1) | 20 (83.3) | 637 (88.8) | 4 (100.0) | ||
Positive | 76 (10.9) | 4 (16.7) | 80 (11.2) | 0 (0.0) | ||
Correlation between SDHA and SDHB expression.
Clinicopathologic characteristics of breast cancer with SDHA and/or SDHB negativity
Clinicopathologic characteristics of breast cancer with SDHA and/or SDHB negativity in tumor cells
Parameters | SDHA | SDHB | ||||
|---|---|---|---|---|---|---|
Negative n = 41 (%) | Positive n = 680 (%) | P-value | Negative n = 24 (%) | Positive n = 697 (%) | P-value | |
Age (years, mean ± SD) | 45.4 ± 10.3 | 49.9 ± 10.9 | 0.012 | 46.9 ± 9.1 | 49.7 ± 10.9 | 0.212 |
Histologic grade | 0.062 | 0.044 | ||||
I/II | 33 (80.5) | 451 (66.3) | 21 (87.5) | 463 (66.4) | ||
III | 8 (19.5) | 229 (33.7) | 3 (12.5) | 234 (33.6) | ||
ER | 0.405 | 0.286 | ||||
Negative | 12 (29.3) | 249 (36.6) | 6 (25.0) | 255 (36.6) | ||
Positive | 29 (70.7) | 431 (63.4) | 18 (75.0) | 442 (63.4) | ||
PR | 0.748 | 1.000 | ||||
Negative | 21 (51.2) | 325 (47.8) | 11 (45.8) | 335 (48.1) | ||
Positive | 20 (48.8) | 355 (52.2) | 13 (54.2) | 362 (51.9) | ||
HER-2 | 0.693 | 0.800 | ||||
Negative | 34 (82.9) | 539 (79.3) | 20 (83.3) | 553 (79.3) | ||
Positive | 7 (17.1) | 141 (20.7) | 4 (16.7) | 144 (20.7) | ||
Tumor stage | 0.750 | 0.213 | ||||
T1 | 21 (51.2) | 329 (48.4) | 15 (62.5) | 335 (48.1) | ||
T2/T3 | 20 (48.8) | 351 (51.6) | 9 (37.5) | 362 (51.9) | ||
Nodal stage | 0.625 | 0.529 | ||||
N0 | 26 (63.4) | 399 (58.7) | 16 (66.7) | 409 (58.7) | ||
N1/N2/N3 | 15 (36.6) | 281 (41.3) | 8 (33.3) | 288 (41.3) | ||
Molecular subtypes | 0.452 | 0.134 | ||||
Luminal A | 21 (51.2) | 281 (41.3) | 15 (62.5) | 287 (41.2) | ||
Luminal B | 8 (19.5) | 160 (23.5) | 3 (12.5) | 165 (23.7) | ||
HER-2 | 5 (12.2) | 64 (9.4) | 3 (12.5) | 66 (9.5) | ||
TNBC | 7 (17.1) | 175 (25.7) | 3 (12.5) | 179 (25.7) | ||
Ki-67 LI (%, mean ± SD) | 13.0 ± 12.3 | 17.5 ± 18.7 | 0.127 | 9.9 ± 10.0 | 17.5 ± 18.6 | 0.046 |
Tumor recurrence | 4 (9.8) | 59 (8.7) | 0.775 | 1 (4.2) | 62 (8.9) | 0.714 |
Patient death | 4 (9.8) | 56 (8.2) | 0.768 | 1 (4.2) | 59 (8.5) | 0.713 |
Correlations between clinicopathologic parameters and expression of Hif-1α and SDH
Clinicopathologic characteristics breast cancer by expression of HIF-1α and SDH
Parameters | Nuclear HIF-1α | Cytoplasmic HIF-1α | Tumor SDHA | Stromal SDHA | Tumor SDHB | Stromal SDHB | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
- | + | P | - | + | P | -* | + | P | - | + | P | -* | + | P | - | + | P | |
Histologic grade | 0.270 | 0.601 | 0.342 | 0.105 | 0.870 | 0.528 | ||||||||||||
I/II | 465 (66.7) | 19 (79.2) | 482 (67.2) | 2 (50.0) | 248 (68.9) | 236 (65.4) | 452 (68.0) | 32 (57.1) | 182 (67.7) | 302 (66.8) | 433 (67.6) | 51 (63.8) | ||||||
III | 232 (33.3) | 5 (20.8) | 235 (32.8) | 2 (50.0) | 112 (31.2) | 125 (34.6) | 213 (32.0) | 24 (42.9) | 87 (32.3) | 150 (33.2) | 208 (32.4) | 29 (36.3) | ||||||
ER | 0.388 | 1.000 | 0.044 | 0.111 | 0.873 | 0.806 | ||||||||||||
Negative | 250 (35.9) | 11 (45.8) | 260 (36.3) | 1 (25.0) | 117 (32.5) | 144 (39.9) | 235 (35.3) | 26 (46.4) | 96 (35.7) | 165 (36.5) | 231 (36.0) | 30 (37.5) | ||||||
Positive | 447 (64.1) | 13 (54.2) | 457 (63.7) | 3 (75.0) | 243 (67.5) | 217 (60.1) | 430 (64.7) | 30 (53.6) | 173 (64.3) | 287 (63.5) | 410 (64.0) | 50 (62.5) | ||||||
PR | 0.011 | 1.000 | 0.118 | 0.165 | 0.645 | 1.000 | ||||||||||||
Negative | 328 (47.1) | 18 (75.0) | 344 (48.0) | 2 (50.0) | 162 (45.0) | 184 (51.0) | 314 (47.2) | 32 (57.1) | 126 (46.8) | 220 (48.7) | 308 (48.0) | 38 (47.5) | ||||||
Positive | 369 (52.9) | 6 (25.0) | 373 (52.0) | 2 (50.0) | 198 (55.0) | 177 (49.0) | 351 (52.8) | 24 (42.9) | 143 (53.2) | 232 (51.3) | 333 (52.0) | 42 (52.5) | ||||||
HER-2 | 0.304 | 1.000 | 0.021 | 0.083 | 0.057 | <0.001 | ||||||||||||
Negative | 556 (79.8) | 17 (70.8) | 570 (79.5) | 3 (75.0) | 299 (83.1) | 274 (75.9) | 534 (80.3) | 39 (69.6) | 224 (83.3) | 349 (77.2) | 526 (82.1) | 47 (58.8) | ||||||
Positive | 141 (20.2) | 7 (29.2) | 147 (20.5) | 1 (25.0) | 61 (16.9) | 87 (24.1) | 131 (19.7) | 17 (30.4) | 45 (16.7) | 103 (22.8) | 115 (17.9) | 33 (41.3) | ||||||
Tumor stage | 0.304 | 1.000 | 0.031 | 1.000 | 0.011 | 0.237 | ||||||||||||
T1 | 341 (48.9) | 9 (37.5) | 348 (48.5) | 2 (50.0) | 160 (44.4) | 190 (52.6) | 323 (48.6) | 27 (48.2) | 114 (42.4) | 236 (52.2) | 306 (47.7) | 44 (55.0) | ||||||
T2/T3 | 356 (51.1) | 15 (62.5) | 369 (51.5) | 2 (50.0) | 200 (55.6) | 171 (47.4) | 342 (51.4) | 29 (51.8) | 155 (57.6) | 216 (47.8) | 335 (52.3) | 36 (45.0) | ||||||
Nodal stage | 0.208 | 0.648 | 0.198 | 0.575 | 0.639 | 0.149 | ||||||||||||
N0 | 414 (59.4) | 11 (45.8) | 422 (58.9) | 3 (75.0) | 221 (61.4) | 204 (56.5) | 394 (59.2) | 31 (55.4) | 162 (60.2) | 263 (58.2) | 384 (59.9) | 41 (51.3) | ||||||
N1/N2/N3 | 283 (40.6) | 13 (54.2) | 295 (41.1) | 1 (25.0) | 139 (38.6) | 157 (43.5) | 271 (40.8) | 25 (44.6) | 107 (39.8) | 189 (41.8) | 257 (40.1) | 39 (48.8) | ||||||
Clnicopathologic features according the phenotype of SDH expression in breast cancer
Clinicopathologic features according to the SDH expression phenotype in breast cancer
Factors | Tumor phenotype | Stroma phenotype | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
SDHA (+)/ SDHB | SDHA (+)/ SDHB | SDHA (−)/ SDHB | SDHA (-)/ SDHB | P | SDHA (+)/ SDHB | SDHA (+)/ SDHB | SDHA (−)/ SDHB | SDHA (−)/ SDHB | P | |
(+) n = 679 (%) | (−) n = 1 (%) | (+) n = 18 (%) | (−) n = 23 (%) | (+) n = 42 (%) | (−) n = 14 (%) | (+) n = 38 (%) | (−) n = 627 (%) | |||
Histologic grade | 0.038 | 0.150 | ||||||||
I/II | 450 (66.3) | 1 (100.0) | 13 (72.2) | 20 (87.0) | 25 (59.5) | 7 (50.0) | 26 (68.4) | 426 (67.9) | ||
III | 229 (33.7) | 0 (0.0) | 5 (27.8) | 3 (13.0) | 17 (40.5) | 7 (50.0) | 12 (31.6) | 201 (32.1) | ||
Tumor stage | 0.495 | 0.640 | ||||||||
T1 | 329 (48.5) | 0 (0.0) | 6 (33.3) | 15 (65.2) | 20 (47.6) | 7 (50.0) | 24 (63.2) | 299 (47.7) | ||
T2/T3 | 350 (51.5) | 1 (100.0) | 12 (66.7) | 8 (34.8) | 22 (52.4) | 7 (50.0) | 14 (36.8) | 328 (52.3) | ||
Nodal stage | 0.491 | 0.314 | ||||||||
N0 | 398 (58.6) | 1 (100.0) | 11 (61.1) | 15 (65.2) | 21 (50.0) | 10 (71.4) | 20 (52.6) | 374 (59.6) | ||
N1/N2/N3 | 281 (41.4) | 0 (0.0) | 7 (38.9) | 8 (34.8) | 21 (50.0) | 4 (28.6) | 18 (47.4) | 253 (40.4) | ||
ER | 0.284 | 0.205 | ||||||||
Negative | 248 (36.5) | 1 (100.0) | 7 (38.9) | 5 (21.7) | 16 (38.1) | 10 (71.4) | 14 (36.8) | 221 (35.2) | ||
Positive | 431 (63.5) | 0 (0.0) | 11 (61.1) | 18 (78.3) | 26 (61.9) | 4 (28.6) | 24 (63.2) | 406 (64.8) | ||
PR | 0.793 | 0.336 | ||||||||
Negative | 324 (47.7) | 1 (100.0) | 11 (61.1) | 10 (43.5) | 21 (50.0) | 11 (78.6) | 17 (44.7) | 297 (47.4) | ||
Positive | 355 (52.3) | 0 (0.0) | 7 (38.9) | 13 (56.5) | 21 (50.0) | 3 (21.4) | 21 (55.3) | 330 (52.6) | ||
HER-2 | 0.567 | 0.001 | ||||||||
Negative | 539 (79.4) | 0 (0.0) | 14 (77.8) | 20 (87.0) | 26 (61.9) | 13 (92.9) | 21 (55.3) | 513 (81.8) | ||
Positive | 140 (20.6) | 1 (100.0) | 4 (22.2) | 3 (13.0) | 16 (38.1) | 1 (7.1) | 17 (44.7) | 114 (18.2) | ||
Molecular type | 0.057 | <0.001 | ||||||||
Luminal A | 281 (41.4) | 0 (0.0) | 6 (33.3) | 15 (65.2) | 14 (33.3) | 2 (14.3) | 11 (28.9) | 275 (43.9) | ||
Luminal B | 160 (23.6) | 0 (0.0) | 5 (27.8) | 3 (13.0) | 13 (31.0) | 2 (14.3) | 13 (34.2) | 140 (22.3) | ||
HER-2 | 63 (9.3) | 1 (100.0) | 3 (16.7) | 2 (8.7) | 9 (21.4) | 1 (7.1) | 9 (23.7) | 50 (8.0) | ||
TNBC | 175 (25.8) | 0 (0.0) | 4 (22.2) | 3 (13.0) | 6 (14.3) | 9 (64.3) | 5 (13.2) | 162 (25.8) | ||
Ki-67 LI (%, mean ± SD) | 17.5 ± 18.7 | 8.0 ± 0.0 | 16.8 ± 14.0 | 10.0 ± 10.2 | 0.260 | 17.8 ± 15.3 | 24.3 ± 22.8 | 22.1 ± 19.0 | 16.8 ± 18.4 | 0.161 |
Tumor recurrence | 59 (8.7) | 0 (0.0) | 3 (16.7) | 1 (4.3) | 0.955 | 4 (9.5) | 1 (7.1) | 2 (5.3) | 56 (8.9) | 0.877 |
Patient death | 56 (8.2) | 0 (0.0) | 3 (16.7) | 1 (4.3) | 0.968 | 3 (7.1) | 1 (7.1) | 3 (7.9) | 53 (8.5) | 0.727 |
SDHA(+)/SDHB(+) tumors tended to have higher histologic grades than SDHA(+)/SDHB(–) and SDHA(–)/SDHB(-) tumors (P = 0.038). Stromal SDHA(–)/SDHB(+) tumors showed the highest frequency of HER-2 positivity, while stromal SDHA(+)/SDHB(-) tumors showed lowest HER-2 positivity (P = 0.001). Stromal SDHA/SDHB phenotype differed significantly between the molecular subtypes, SDHA(+)/SDHB(+) being most frequent in Luminal A and least in TNBC tumors. The SDHA(+)/SDHB(–) phenotype was most frequent in TNBC and least frequent in HER-2 subtype tumors; SDHA(–)/SDHB(+) was most frequent in Luminal B and least in TNBC; and SDHA(−)/SDHB(–) was most frequent in the Luminal A and least in the HER-2 subtype (P < 0.001).
Prognostic significance of HIF-1α and SDH expression status
Univariate analysis of disease-free survival and overall survival according to breast-tumor expression of hypoxia-related proteins
Parameters | Number of patients/ recurrence/death | Disease-free survival | Overall survival | ||
|---|---|---|---|---|---|
Mean survival (95% CI) months | P-value | Mean survival(95% CI) months | P-value | ||
Nuclear HIF-1α | 0.911 | 0.943 | |||
Negative | 697/61/58 | 126 (122 − 130) | 129 (127 − 132) | ||
Positive | 24/2/2 | 120 (108 − 133) | 131 (122 − 141) | ||
Cytoplasmic HIF-1α | N/A | N/A | |||
Negative | 717/63 | N/A | N/A | ||
Positive | 4/0 | N/A | N/A | ||
Tumoral SDHA | 0.670 | 0.085 | |||
Negative/Low | 360/35/26 | 125 (121 − 129) | 131 (128 − 135) | ||
High | 361/28/34 | 126 (120 − 132) | 126 (121 − 130) | ||
Stromal SDHA | 0.853 | 0.901 | |||
Negative | 665/58/56 | 126 (123 − 130) | 130 (127 − 132) | ||
Positive | 56/5/4 | 115 (101 − 130) | 121 (107 − 134) | ||
Tumoral SDHB | 0.124 | 0.715 | |||
Negative/Low | 269/31/26 | 123 (119 − 128) | 129 (125 − 133) | ||
High | 452/32/34 | 126 (121 − 131) | 129 (125 − 132) | ||
Stromal SDHB | 0.821 | 0.981 | |||
Negative | 641/57/54 | 126 (122 − 130) | 130 (127 − 132) | ||
Positive | 80/6/6 | 119 (110 − 128) | 123 (114 − 131) | ||
Multivariate analysis for breast-cancer survival
Parameters | Disease-free survival | Overall survival | ||||
|---|---|---|---|---|---|---|
Hazard ratio | 95% CI | P-value | Hazard ratio | 95% CI | P-value | |
T stage | 0.003 | 0.008 | ||||
T1 versus T2-3 | 2.535 | 1.376 − 4.670 | 2.218 | 1.229 − 4.001 | ||
N stage | 0.002 | 0.026 | ||||
N0 versus N1-3 | 2.257 | 1.335 − 3.816 | 1.831 | 1.076 − 3.115 | ||
Histologic grade | 0.045 | 0.372 | ||||
I/II versus III | 1.685 | 1.013 − 2.804 | 1.276 | 0.748 − 2.176 | ||
Nuclear HIF-1α | 0.599 | 0.486 | ||||
Negative versus Positive | 0.682 | 0.163 − 2.846 | 0.599 | 0.142 − 2.528 | ||
Tumor SDHA | 0.290 | 0.002 | ||||
Negative/Low versus High | 1.570 | 0.681 − 3.620 | 4.157 | 1.657 − 10.432 | ||
Stromal SDHA | 0.727 | 0.747 | ||||
Negative versus Positive | 1.254 | 0.352 − 4.468 | 0.807 | 0.218 − 2.978 | ||
Tumor SDHB | 0.096 | 0.012 | ||||
Negative/Low versus High | 0.498 | 0.219 − 1.131 | 3.223 | 1.295 − 8.025 | ||
Stromal SDHB | 0.712 | 0.965 | ||||
Negative versus Positive | 0.802 | 0.248 − 2.590 | 0.976 | 0.327 − 2.908 | ||
Discussion
Germline defects in SDH, particularly in SDHA and SDHB, have been detected in several tumor types, including pheochromocytomas (Van Nederveen et al. 2009;Astuti et al. 2001), paragangliomas (Van Nederveen et al. 2009; Astuti et al. 2001; Baysal 2003;Burnichon et al. 2010), GISTs (Gaal et al. 2011;Gill et al. 2010b; Gill et al. 2011a), and renal cell carcinomas (Gill et al. 2011b). Until recently, the accurate detection of SDH mutation depended primarily on direct sequencing and western blotting, methods that a clinical laboratory may find too costly and time-consuming. However, in a study of paraganglioma and pheochromocytoma, Van Nederveen et al. demonstrated the high sensitivity of immunohistochemical methods to detect germline mutations in SDH (Van Nederveen et al. 2009). While the tumors with SDHB, SDHC, or SDHD germline mutations exhibited a loss of SDHB immmunoexpression with intact SDHA expression, tumors with SDHA germline mutations exhibited a loss of expression of both SDHA and SDHB.
Using similar methods in the present study, we found that 23 of 721 breast cancer patients (3.19%) had SDHA mutation (SDHA–/SDHB– expression) and one patient (0.1%) had an SDHB mutation (SHDA+/SDHB– expression; Table 7). As few previous studies have evaluated SDH mutation in breast cancer, these findings provide a starting point for future investigations. However, a previous study reported that SDH germline mutations or variants occur in some patients with Cowden syndrome (CS) who do not present the expected PTEN mutation. Compared with patients positive for germline PTEN mutation, these CS/CS-like individuals develop cancers of the breast, thyroid, and kidney at higher frequencies (Ni et al. 2008). Therefore, some breast cancer patients may be expected to have SDH mutations.
Findings in this study may be limited in that the sensitivity of immunohistochemistry in detecting SDH mutation as compared to direct sequencing has not been tested in breast cancer. We have assumed a degree of reliability in breast cancer similar to that of the detection of SDH germline mutations in paraganglioma and pheochromocytoma (Van Nederveen et al. 2009). To confirm SDH mutation, loss of SDH expression should be tested throughout the entire tumor (Barletta & Hornick 2012), whereas in this study, immunohistochemistry was performed on the tissue microarray only.
In previous studies of pheochromocytoma and GIST, SDH-deficient tumors showed complete loss of cytoplasmic granular expression (Miettinen et al. 2011; Gimenez-Roqueplo et al. 2003). Even though weak, focal or diffuse cytoplasmic staining was observed in a very few tumor cells of SDH-deficient tumor, this finding may be considered negative, because it is clearly distinguishable from the strong speckled expression pattern in surrounding non-neoplastic elements. However, stromal cells did not express SDHA and SDHB in most breast cancers in this study, which made it impossible to compare the staining intensity between tumor cells and internal normal controls (stromal cells). Therefore, expression in tumor cells was interpreted at three grade levels according to the proportion of cells stained: grade (1), negative (complete loss of expression); grade (2), low (expression in less than 50% of cells); and grade (3) high (expression in more than 50% of cells).
Several mechanisms have been proposed to explain the involvement of SDH mutation in tumorigenesis, among which a HIF-1α-pathway-dependent mechanism is the most famous. Loss-of-function mutation of SDH could result in an intracellular SDH accumulation, which in turn inhibits prolyl 4-hydroxylase (PHD), a negative regulator of HIF-1α (Cardaci & Ciriolo 2012; Barletta & Hornick 2012). The impaired PHD activity stabilizes HIF-1α under normoxic condition, which upregulates HIF target gene involved in cell growth stimulation and angiogenesis, thus contributing to tumor progression. As loss-of-function mutations in SDH are predicted to stabilize HIF-1α and upregulate HIF-1 transcriptional activity, we examined the expression of HIF-1α along with SDHA/SDHB. However, we found no close relationship between HIF-1α and SDH expression in these breast cancers.
SDH-deficient GISTs and renal cell carcinomas display characteristic histologic features distinguishable from tumors without SDH mutation (Miettinen et al. 2011; Gill et al. 2011b). Similarly, the SDHA+/SDHB- and SDHA-/SDHB- breast cancers in this study were distinguished by lower histologic grade (Table 7), and in the SDHB-negative tumors, by lower Ki-67 LI (Table 5). The luminal A breast cancer subtype showed the highest frequency of low/negative SDHA expression (P = 0.032, Table 3). Based on these results, SDH mutation in breast cancer is associated with low histologic grade and a less aggressive molecular subtype.
Patients with SDHA- and SDHB-negative breast cancers were also younger than patients with intact SDH expression, although in the case of SDHB, this difference was not statistically significant. In earlier studies of SDH mutations in renal cell carcinomas (Baysal 2003), GISTs (Miettinen et al. 2011), paragangliomas, and pheochromocytomas (Gimenez-Roqueplo et al. 2003), tumors also occurred at younger ages.
Metabolism in many malignant tumors is characterized by the Warburg effect, which is a high level of anaerobic glucose metabolism by glycolysis despite presence of oxygen with relatively low mitochondrial oxidative phosphorylation (OXPHOS) (Warburg 1956). In this study, more than 50% of breast cancers expressed SDHA and SDHB, which are key components of aerobic glucose metabolism through the TCA cycle and mitochondrial electron transport. This finding of high mitochondrial activity in breast cancer agrees with an earlier report that breast cancer cells show high expression of mitochondrial metabolic enzymes such as cytochrome C oxidase, NADH, and SDHB (Whitaker-Menezes et al. 2011).
Expression of Glut-1 and CAIX (indicators of glycolysis) has also been observed in breast cancers, most notably in high-grade tumors such as TNBC or basal-like carcinoma (Choi et al. 2013; Pinheiro et al. 2011). While breast cancers overall may express high levels of activity in both OXPHOS and glycolysis, the predominant mode of energy metabolism may differ according to the tumor type (Moreno-Sanchez et al. 2007: Kallinowski et al. 1989; Liu et al. 2001).
Although most breast cancers in this study were negative for SDH in stromal elements, stromal SDHB expression differed significantly among the molecular subtypes. The HER-2 subtype showed the highest and TNBC showed the lowest frequency of stromal SDHB expression (Table 3). However, in a previous study, stromal cells in breast cancer did not express mitochondrial metabolic enzymes (cytochrome c oxidase, NADH, or SDHB) (Whitaker-Menezes et al. 2011). Further studies will be required to determine the differences in stromal SDHB expression among breast cancer molecular subtypes.
In conclusion, loss of SDHA or SDHB expression was detected in approximately 3% of the breast cancers in this study. Patients with SDH-deficient breast cancers were younger at diagnosis and presented tumors of relatively low-grade histology.
Consent
Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
Declarations
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2012R1A1A1002886).
Authors’ Affiliations
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