overview breast cancer

This blog aims to give a simplified overview on the latest insights in order to identify the gaps in our knowledge and to provoke discussions on what biomarkers may aid to a further understanding of this large group of diseases.

Subtypes of breast cancer

HR+ (hormone receptors: oestrogen receptor, ER and progesterone receptor, PR)

The HR+ (ER+ and/or PR+), HER2-negative (HER2−) subtype appears to account for the majority of breast cancers (>50%) and it has the best prognosis because of effective targeted hormonal therapies and a more indolent disease phenotype (1. PMID: 22067903). 

There are at least two ER+ subtypes (2. PMID: 11553815):

Luminal/ER+ type A: high expression of ESR1, GATA3, XBP1, TFF3, FOXA1, and SLC39A6.

Luminal/ER+ type B: low to moderate expression of the luminal-specific genes including the ER cluster.

Androgen receptor (AR) is commonly expressed in breast cancers.  In one study among 1467 breast cancers, 78.7% were AR-positive (AR+). Among 1,164 ER-positive cases, 88.0% were AR+. AR positivity was associated with a significant reduction in breast cancer mortality (HR, 0.68; 95% CI, 0.47-0.99) and overall mortality (HR, 0.70; 95% CI, 0.53-0.91) after adjustment for covariates. In contrast, among women with ER-negative tumours (303 cases), 42.9% were AR+. There was a non-significant association between AR status and breast cancer death (HR, 1.59; 95% CI, 0.94-2.68) (3. PMID: 21325075). Compared with AR-negative tumors, AR-positive tumors were associated with a 30% reduction in breast cancer mortality (hazard ratio, 0.68; 95 percent confidence interval, 0.47 to 0.99).Thus AR-status appears to have prognostic value for just the ER-positive patients in this study. However, this observation has not yet been independently confirmed. There remains general controversy over AR status in relation to prognosis, as many other studies show contradictive results, being complicated by heterogeneous populations, by use of different AR antibodies and by using different cut-off values (communications with C. Kaniklidis).

Possible cancer types (Please note that AR+ status in ER-/PR- are not regarded as HR+):

ER+A/PR-/AR-; ER+A/PR-/AR+;  ER+A/PR+/AR-; ER+A/PR+/AR+ (4)

ER+B/PR-/AR-; ER+B/PR-/AR+;   ER+B/PR+/AR-; ER+B/PR+/AR+ (4)

ER-/PR-/AR-; ER-/PR-/AR+;          ER-/PR+/AR-; ER-/PR+/AR+ (2)

(total: 10 HR+ types)

Tumour expression profiling revealed a yet unknown subtype sharing a set of expressed genes with the other luminal subtypes but with an extra unique set of mostly uncharacterized genes. The authors categorized this group as luminal subtype C (2. PMID: 11553815). However, in a later study the same authors do not mention this subtype anymore (4. PMID: 12829800).

ER+ patients are treated with Tamoxifen. This compound is metabolized in the liver by CYP2D6 and by CYP3A4 (PMID: 15159443) into metabolites such as afimoxifene and endoxifen that bind strongly to the Oestrogen Receptor (5. PMID: 15159443), thus preventing further growth of the cancer cells in an oestrogen-dependent manner. There are many alleles in CYP2D6, but the ones tested turn out to be poor pharmaco-dynamic markers (6. PMID: 22395644).  Endoxifen levels show a complex correlation with recurrence of the disease (7. PMID: 23476897). Here is a research field that is moving fast and will reveal more data, including (hopefully) CYP3A4 and afimoxifene, shortly. 

An alternative treatment is the aromatase inhibitors (e.g. Letrozole). They prevent the production of oestrogen by the enzyme aromatase. This inhibition is only effective in post-menopausal patients and has a lower recurrence rate compared to Tamoxifen treatment.  

Patients, whose disease progress regardless treatments with the above mentioned endocrine agents, may be treated with fulvestrant, a selective estrogen receptor down-regulator (SERD). However, some patients remain resistant, also to fulvestrant (8. PMID: 24505287).

Everest Products:  
ER: EB08362, EB11581
PR: EB07207, EB07223
AR: EB06441
GATA3: EB05570
XBP1: EB08557
TFF3: EB12315
FOXA1: EB05999
CYP3A4: EB07194

HER2 amplification

The two HER2+ subtypes (HR+/HER2+ and HR−/HER2+) account for 7% and 14%, respectively. Before targeted therapy, HER2+ tumours portended some of the worst prognoses, but the development of targeted therapies, such as Trastuzumab (Herceptin), has resulted in a marked improvement in outcome (cf 1. PMID: 22067903).  However, HR+/HER+ patients are reported to not respond well to pre-surgery treatment with chemotherapy and HER2-targeted therapies if their cancer had one or more mutations in the PIK3CA gene. Such results were presented at the 2013 San Antonio Breast Cancer Symposium, December 10-14, 2013, but literature remains unclear on this so far (9. PMID: 24471188).

One problem in the studies is the consistent reporting of HR+ within the HER2 context: As we have seen above, the HR+ annotation is not sufficiently defined.  The HR+/HER2+ patients need to be separated into ER+/HER2+ and PR+/HER2+ and the AR-status needs to be defined as well. As long as this does not happen, studies remain unclear on how other genetic factors influence prognosis after therapy.

Possible cancer types:

HR+/HER+ (10); HR+/HER+/PIK3CA (10); HR-/HER+; HR-/HER+/PIK3CA (total:  22 HER+ types)

Co-IP experiments on HER+ cancer cell lines before and after GRB7 expression knock down revealed two HER2-mediated signalling cascades: HER2+ cell proliferation (by HER2-GRB7-SHC-RAS interactions) and  HER2+ cell migration (by alpha5 beta1/alpha4 beta1-FAK-GRB7-VAV2-RAC1 interactions). A coupling of GRB7 with HER2 is required for the proliferative and migratory signals in HER2+ breast tumour cells (PMID: 23593540). However, the role of GRB7 in other types of breast cancer has been confirmed in similar experiments on cell lines (PMID: 24464577), and a clinical link has not been established at this stage.

Everest Products:
HER2: EB11748, EB11749
PIK3CA: EB07512
GRB7: EB05417
VAV2: EB05337

Triple Negative, TN (ER-, PR-, HER-) / Basal-like

The triple-negative (TN) subtype (defined as ER−, PR− and HER2−) comprises 10–30% of all invasive breast cancers (1. PMID: 22067903). However, this estimation varies dramatically depending on race/ethnicity (12. PMID: 16757721, 13. PMID: 19320967). A 2011 analysis reported a frequency of 16% based on 3247 primary human breast cancers from 21 publicly available data sets (14. PMID: 21633166). This frequency was backed up by reported frequency of 17% based on 3744 cases (15. PMID: 18398844) and 16% based on 1726 cases (16. PMID: 19318481). It has been unclear how to distinguish TN from Basal-like cancers for a long time.  One study found all 97 triple-negative tumours they selected to be basal-like (17. PMID: 17910759). Basal-like can be defined as IHC positive for cytokeratines CK5/6 and CK17. However, a larger cohort of 587 TNBC (14. PMID: 21633166) cases revealed 6 subtypes: two basal-like subtypes (BL1 and BL2), immunomodulatory subtype (IM), mesenchymal subtype(M), mesenchymal stem-like subtype(MSL), and luminal androgen receptor subtype (LAR).

BL1 and BL2 subtypes have higher expression of cell cycle and DNA damage response genes.

M and MSL subtypes showed an expression profile specific for epithelial to mesenchymal cell transition.

LAR subtype was characterized by androgen receptor signalling (see also the HR group).

Despite of making considerable progress in cancer research, the challenges for TNBC treatment has remained high in the last decade primarily due to failure in identifying adequate drug targets. Transcriptional Wnt signalling (Wnt/β-catenin pathway)has been reported as a hallmark of TNBC disease associated with specific metastatic pathways (18. PMID: 24209998). According to another report the loss of tumour suppressor gene PTEN is the most common first event associated with basal-like subtype (19. PMID: 22628410). The functional up-regulation of secreted-MMP7, a transcriptional target of Wnt-β-catenin signature pathway in TNBC is associated to the loss of PTEN. Patient data revealed that MMP7 mRNA was high in only a subpopulation of TNBC, and this subpopulation was characterized by a concurrent low expression of PTEN mRNA (PMID: 24143235). Unfortunately, these are all parts of a larger jigsaw puzzle and we are not even close to see the entire picture.

Cancers: TN/BL1; TN/BL2; TN/IM; TN/M; TN/ MSL; TN/LAR; TN/MMP7 (7).

Here are some more jigsaw puzzle pieces who contribute each a little bit to the complex picture we like to see emerging, and it is far from complete.

Everest products:
MMP7: EB06891
PTEN: EB06544, EB07380

Genetic drivers:

BRCA.

A study in an Asian series of TNBC patients (Malaysian Breast Cancer Genetic Study) demonstrated that 27 (24.5%) of 110 patients have germline mutations in BRCA1 (23 of 110) and BRCA2 (four of 110) (21. PMID: 23116406). In the majority of BRCA1 carriers, breast tumours have distinctive morphologic features and immunohistochemical phenotypes characteristic of basal-like breast cancers, including negative expression of the estrogen receptor, high expression of basal markers, such as basal cytokeratins CK5/6 and CK14, and loss of tumour-suppressor PTEN (22. PMID: 14519755, 23. PMID: 16033833, PMID: 18066063) Moreover, molecular gene-expression profiling of BRCA1 tumours showed that the tumours have significant similarities with the basal-like subtype of breast cancer (2. PMID: 11553815). The prevalence of BRCA mutations is higher in patients with a family background and in patients with early onset disease (before 50 years old) (21. PMID: 23116406). This makes BRCA1 an important genetic marker for TNBC, although it covers only about 21% (23/110) of all TNBC.

Cancers: TN/BRCA1; TN/BRCA2 (2)

TP53.

Luminal subtype A contained only 13% TP53-mutated tumours, whereas the HER2+ and basal-like (TNBC) subclasses had 5/7 (71%) and 9/11 (82%), respectively. Also the luminal subtype C showed a high frequency of 80% (8/10) (2. PMID: 11553815) but this subtype has been elusive since this single report. Based on these differences in frequencies, TP53 is generally regarded as one genetic cancer driver out of many and its relation to the different types of breast cancer is poorly understood.

PIK3CA, AKT1, PTEN.

PIK3CA mutations were more common in hormone receptor–positive (34.5%) and HER2-positive (22.7%) than in basal-like tumours (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor–positive cancers (25. PMID: 18676830).

EGFR

Amplification of epidermal growth factor receptor (EGFR) was observed in well-characterised TNBCs (up to 92% out of 138) (27. PMID: 24423920). This amplification was not found to be correlated with any EGFR mutations, but in 26% of these cases mutations were found in PI3K and BRAF. In this study both silver in situ hybridisation (SISH) and IHC were validated as tools to detect EGFR amplification in TNBC patients. With this tool, there is opportunity to investigate EGFR targeting as a therapy, reminiscent to HER2 targeting in HER2 patients. Clearly, the identified PI3K and BRAF mutations make things complicated again in 26% of these cases.

Samples of formalin-fixed paraffin-embedded (FFPE) breast tumour specimens using a custom 512-gene breast cancer bead array-based platform that stained positive for ER, PR and HER2 resulted in DASL mRNA average transcript fold changes in ESR1(=ER), PGR(=PR) and ERBB2(=HER2) (95% CI) of 4.46 (2.01–6.90), 3.41 (1.24–5.58) and 3.59 (1.40–5.77) greater than their respective IHC-negative tumours (P<0.01, Welch's t-test). Taken together, these data indicate concordance of DASL assay intensity with IHC-determined protein expression for these three genes (PMID: 22067903).

Using this technique, an additional 27 other genes were identified to be IHC-indicative where one set of genes points towards HR+ and another set of genes points towards TN (see table), but not at all strictly. One can see that such expression profiles reveal a whole new level of complexity between individual breast cancer cases.

This blog by no means aims to be complete, nor does it have the ambition to serve the experts in breast cancer.

No doubt, a lot of knowledge and data can be added to this overview, but this would be beyond the scope of this writing. I thank both Brian Leyland-Jones and Constantine Kaniklidis for their precious expertise feedback.

Supplementary Table 2 (PMID: 22067903 figure 2). 30 genes indicative of immunohistochemical (IHC) breast cancer subtype in the Montreal cohort of 87 patients identified by Prediction Analysis of Microarrays (PAM).

A total of 87 FFPE tumour specimens came from three major IHC subclasses and were composed of 24 ER−/PR−/HER2− (designated TN); 8 ER−/PR−/HER2+ (designated HER2+); 8 ER+/PR-/HER2+ 11 ER+/PR+/HER2+ 13 ER+/PR−/HER2− and 23 ER+/PR+/HER2− (designated HR+).

Jan Voskuil, February 2014

References in order of appearance

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2. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein  D, Lønning PE, Børresen-Dale AL. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10869-74. PMID: 11553815

3. Hu R, Dawood S, Holmes MD, Collins LC, Schnitt SJ, Cole K, Marotti JD, Hankinson SE, Colditz GA, Tamimi RM. Androgen receptor expression and breast cancer survival in postmenopausal women. Clin Cancer Res. 2011 Apr 1;17(7):1867-74. PMID: 21325075

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8. Knudsen S, Jensen T, Hansen A, Mazin W, Lindemann J, Kuter I, Laing N, Anderson E. Development and validation of a gene expression score that predicts response to fulvestrant in breast cancer patients. PLoS One. 2014 Feb 5;9(2):e87415. PMID: 24505287  9. Arsenic R, Lehmann A, Budczies J, Koch I, Prinzler J, Kleine-Tebbe A, Schewe C, Loibl S, Dietel M, Denkert C. Analysis of PIK3CA mutations in breast cancer subtypes. Appl Immunohistochem Mol Morphol. 2014 Jan;22(1):50-6. PMID: 24471188.

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16. Rakha EA, Elsheikh SE, Aleskandarany MA, Habashi HO, Green AR, Powe DG, El-Sayed ME, Benhasouna A, Brunet JS, Akslen LA, Evans AJ, Blamey R, Reis-Filho JS, Foulkes WD, Ellis IO. Triple-negative breast cancer: distinguishing between basal and nonbasal subtypes. Clin Cancer Res. 2009 Apr 1;15(7):2302-10. doi: 10.1158/1078-0432.CCR-08-2132. Epub 2009 Mar 24. PMID: 19318481.

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18. Dey N, Barwick BG, Moreno CS, Ordanic-Kodani M, Chen Z, Oprea-Ilies G, Tang W, Catzavelos C, Kerstann KF, Sledge GW Jr, Abramovitz M, Bouzyk M, De P, Leyland-Jones BR. Wnt signaling in triple negative breast cancer is associated with metastasis. BMC Cancer. 2013 Nov 10;13(1):537. PMID: 24209998.

19. Martins FC, De S, Almendro V, Gönen M, Park SY, Blum JL, Herlihy W, Ethington  G, Schnitt SJ, Tung N, Garber JE, Fetten K, Michor F, Polyak K. Evolutionary pathways in BRCA1-associated breast tumors. Cancer Discov. 2012 Jun;2(6):503-11. PMID: 22628410.

20. Dey N, Young B, Abramovitz M, Bouzyk M, Barwick B, De P, Leyland-Jones B. Differential activation of Wnt-β-catenin pathway in triple negative breast cancer increases MMP7 in a PTEN dependent manner. PLoS One. 2013 Oct 15;8(10):e77425. PMID: 24143235.

21. Phuah SY, Looi LM, Hassan N, Rhodes A, Dean S, Taib NA, Yip CH, Teo SH. Triple-negative breast cancer and PTEN (phosphatase and tensin homologue)loss are predictors of BRCA1 germline mutations in women with early-onset and familial breast cancer, but not in women with isolated late-onset breast cancer. Breast Cancer Res. 2012 Nov 2;14(6):R142. PMID: 23116406.

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23. Lakhani SR, Reis-Filho JS, Fulford L, Penault-Llorca F, van der Vijver M, Parry S, Bishop T, Benitez J, Rivas C, Bignon YJ, Chang-Claude J, Hamann U, Cornelisse CJ, Devilee P, Beckmann MW, Nestle-Krämling C, Daly PA, Haites N, Varley J, Lalloo F, Evans G, Maugard C, Meijers-Heijboer H, Klijn JG, Olah E, Gusterson BA, Pilotti S, Radice P, Scherneck S, Sobol H, Jacquemier J, Wagner T,  Peto J, Stratton MR, McGuffog L, Easton DF; Breast Cancer Linkage Consortium. Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin Cancer Res. 2005 Jul 15;11(14):5175-80. PMID: 16033833.

24. Saal LH, Gruvberger-Saal SK, Persson C, Lövgren K, Jumppanen M, Staaf J, Jönsson G, Pires MM, Maurer M, Holm K, Koujak S, Subramaniyam S, Vallon-Christersson J, Olsson H, Su T, Memeo L, Ludwig T, Ethier SP, Krogh M, Szabolcs M, Murty VV, Isola J, Hibshoosh H, Parsons R, Borg A. Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair. Nat Genet. 2008 Jan;40(1):102-7. Epub 2007 Dec 9. PMID: 18066063

25. Stemke-Hale K, Gonzalez-Angulo AM, Lluch A, Neve RM, Kuo WL, Davies M, Carey M, Hu Z, Guan Y, Sahin A, Symmans WF, Pusztai L, Nolden LK, Horlings H, Berns K,  Hung MC, van de Vijver MJ, Valero V, Gray JW, Bernards R, Mills GB, Hennessy BT.  An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 2008 Aug 1;68(15):6084-91. PMID: 18676830.

26. Abramovitz M, Barwick BG, Willis S, Young B, Catzavelos C, Li Z, Kodani M, Tang W, Bouzyk M, Moreno CS, Leyland-Jones B. Molecular characterisation of formalin-fixed paraffin-embedded (FFPE) breast tumour specimens using a custom 512-gene breast cancer bead array-based platform. Br J Cancer. 2011 Nov 8;105(10):1574-81. PMID: 22067903

27. Secq V, Villeret J, Fina F, Carmassi M, Carcopino X, Garcia S, Metellus I, Boubli L, Iovanna J, Charpin C. Triple negative breast carcinoma EGFR amplification is not associated with EGFR, Kras or ALK mutations. Br J Cancer. 2014 Feb 18;110(4):1045-1052. PMID: 24423920.