With the improvement of early diagnosis and comprehensive treatment of breast cancer, the incidence of breast cancer is increasing, and although its mortality rate has decreased, it is still one of the most lethal diseases in women. The prognosis of breast cancer with the same pathological type and the same clinical stage still varies greatly even though they undergo the same treatment process, indicating that there is a high degree of heterogeneity. With the progress of molecular biology, medical research has entered the era of molecular level, and the traditional histological classification of pathology can no longer meet the needs of today’s tumor research and diagnosis, and the study of breast cancer pathogenesis, treatment and prognosis at the molecular level is becoming a current research hotspot. The molecular typing of breast cancer based on genetic profile and molecular biological characteristics is a necessary complement to the traditional pathology-based classification of breast cancer.
1. Breast cancer genotyping and immunohistochemical typing
With the progress of molecular biology, gene expression profiling and gene microarray technology are widely used in breast cancer research, and breast cancer is considered as a single disease with multiple subtypes. The epidemiological risk factors, natural disease progression and response to systemic or local therapy vary among different molecular subtypes of breast cancer, and these high heterogeneities suggest the need for individualized treatment of breast cancer with different molecular subtypes. 2000, Perou et al. compared the genetic phenotypes of 8,102 human genes and corresponding cloned cDNA microarrays with breast control tissues based on gene expression profile, breast cancer was classified into four molecular subtypes.
The luminal type (hormone receptor-positive type): the gene expression profile of breast cancer is similar to that of normal ductal luminal epithelial cells and is called the luminal epithelial type, which is generally estrogen receptor (ER) positive. The luminal type is subdivided into two subtypes – luminal A and luminal B.
HER-2 positive (HER-2 overexpressing): HER-2 is overexpressed in the gene profile. luminal B type also has HER-2 overexpression, but the tumor infiltration is much lower than HER-2 positive type.
Basal-like type: The gene expression profile is similar to that of basal cells of the breast, but does not express luminal/ER.
④normal-like: gene expression is similar to normal breast tissue or breast fibroadenoma, and immunophenotype is ER (-), PR (-) and HER-2 (-); meanwhile, basal epithelial molecular markers CK5/6, CK17 and epidermal growth factor receptor (EGFR) are negatively expressed. normal-like type is the least sensitive to chemotherapy.
Due to the high cost of gene microarray typing, most experts at the 2011 St. Gallen International Breast Cancer Congress agreed that instead of gene microarray typing, typing could be based on immunohistochemical detection of ER, PR, HER-2 and Ki-67 results, which are mainly divided into 4 subtypes: luminal A, luminal B, HER-2-positive and triple negative. .
The proportion of these four subtypes in breast cancer is not consistent in each literature, but the proportion of luminal A is the highest. In China, Lian Zhenqiang et al. reported that the molecular typing of 482 breast cancer cases were 46.5% for luminal A, 14.7% for luminal B, 10.4% for HER-2 positive, and 28.8% for triple negative; Carey et al. reported that 51.4% for luminal A, 15.5% for luminal B, 6.7% for HER-2 positive, and 20.2% for triple negative. triple-negative type 20.2%. The distribution of each subtype is not the same among different ethnic groups and ages. The rate of triple-negative breast cancer (TNBC) is significantly higher in African-American women than in white women, and there are more TNBC in young women than in older women. The prognosis of patients is worst for TNBC, followed by HER-2-positive type, and best for luminal A type. Due to the convenience of immunohistochemical typing of breast cancer, it is currently the most widely used in clinical practice and has important significance in clinical individualized treatment. The following section focuses on the immunohistochemical typing of breast cancer.
2.Luminal A type
In addition to high expression of ER and PR, Luminal A type also expresses TF3, GATA3, XBP1, HNF3A, transcription factor FOXA1, ADH1B, etc. It belongs to the subtype of tumors sensitive to endocrine therapy.
2.1 Endocrine therapy for Luminal A breast cancer
Luminal A type has the highest percentage of immunohistochemical staging. Patients with ER (+) or PR (+) breast cancer should be considered for endocrine therapy after surgery, regardless of age, lymph node status, and whether adjuvant or neoadjuvant chemotherapy is administered. Currently, the National Comprehensive Cancer Network (NCCN) guidelines state that an ER or PR positive cell count of ≥1% is considered ER (+) or PR (+) and is indicative of endocrine therapy, and suggest that adjuvant endocrine therapy should be considered in patients with ER (+) invasive breast cancer, regardless of age, lymph node status, or whether adjuvant chemotherapy has been administered. Tamoxifen was once the gold standard of endocrine therapy for ER (+) breast cancer, and 5 years of tamoxifen therapy was the standard of care. However, with the availability of long-term follow-up results from the ATAC, BIG 1-98, and TEAM studies, the status of tamoxifen as the gold standard has been challenged by the results of three studies that demonstrated the superiority of aromatase inhibitors over tamoxifen in the treatment of postmenopausal women. treatment was superior to 5 years. In the MA17 trial, switching to 5 years of aromatase inhibitor therapy at the end of 5 years of tamoxifen therapy in postmenopausal women was superior to 10 years of tamoxifen therapy. An ongoing clinical trial comparing the efficacy of 5 and 10 years of aromatase inhibitors to determine if long-term aromatase inhibitors can benefit patients as much as long-term tamoxifen.
2.2 Adjuvant chemotherapy for Luminal A breast cancer
According to the immunohistochemical staging definition, luminal A breast cancer has a Ki-67 positive cell count of <14%, indicating that this subtype of tumor is very insensitive to chemotherapy and most do not require adjuvant or neoadjuvant chemotherapy. However, some of the luminal A early-stage breast cancers have recurrence and metastasis despite regular endocrine therapy, suggesting that this group of patients may be under-treated, and if adjuvant chemotherapy is administered to all patients, a significant proportion will have the problem of over-chemotherapy. berry et al. analyzed three clinical trials of CALGB 854, 9344, and 741, and ER (+) The IBCSG trial confirmed that patients with axillary lymph node negative luminal A breast cancer in both premenopausal and postmenopausal women treated with standard tamoxifen endocrine therapy plus cytotoxic chemotherapy had limited benefit. The addition of cytotoxic chemotherapy to standard tamoxifen endocrine therapy did not benefit in terms of disease-free survival or overall survival. According to the St. Gallen expert consensus, patients with luminal A breast cancer who have high-risk factors such as large tumor (more than 5 cm), histologic grade 3, more than four lymph node metastases, and the presence of choroidal carcinoma emboli may be considered for standardized adjuvant chemotherapy after surgery and endocrine therapy after chemotherapy. 2013 NCCN guidelines further standardize adjuvant chemotherapy for this subtype in In the adjuvant regimen for hormone receptor positive, HER-2 negative breast cancer, chemotherapy and endocrine therapy are required for those with axillary lymph node metastasis; those without axillary lymph node metastasis and tumor ≤0.5 cm only require endocrine therapy, and those with tumor >0.5 cm undergo 21-gene testing to analyze the risk of recurrence score. Those with risk score <18 were considered low risk of recurrence and required only endocrine therapy; those with risk score 18-30 were considered moderate risk of recurrence and required endocrine therapy ± chemotherapy, and whether they could benefit from chemotherapy was inconclusive; those with risk score ≥31 were considered high risk of recurrence and required chemotherapy + endocrine therapy, and patients could benefit from adjuvant chemotherapy. Endocrine therapy ± chemotherapy can be considered for those who have not undergone 21-gene test analysis. Therefore, it is necessary to select treatment regimens based on genetic and biological characteristics for patients with luminal A. A significant number of patients with luminal A do not require cytotoxic chemotherapy, thus avoiding overtreatment.
2.3 Treatment of recurrent metastatic breast cancer of Luminal A type
The 2013 NCCN guidelines state that endocrine therapy remains the treatment of choice for endocrine-sensitive breast cancer unless life-threatening acute disease or very severe symptomatic disease is present. As luminal A breast cancer is not sensitive to chemotherapy, endocrine therapy with a modified regimen is still preferred after tumor recurrence. In principle, endocrine therapy is still the primary treatment for patients without visceral metastases in the first-line palliative treatment, and if there is rapid progression with visceral metastases, systemic chemotherapy may be preferred and endocrine therapy is maintained after disease control. Regarding the possibility of combined endocrine therapy after recurrent metastasis, the SWOG0226 trial compared the efficacy of fulvestrant combined with anastrozole with that of anastrozole alone in the first-line treatment of advanced recurrent breast cancer, and the results suggested that the combination group could significantly improve disease-free survival and overall survival compared with the single-agent group; however, the FACT study came to a different conclusion, and the difference in efficacy between the 2 groups was not statistically significant. The main reason for this is the different selection of cases in the two groups, as the former group did not have endocrine therapy before treatment, while the latter group included those who relapsed after the application of endocrine therapy. Therefore, combining the two studies, there is no clear evidence to support the combined endocrine therapy for recurrent metastatic breast cancer.
3. Luminal B type
Luminal B type moderately expresses luminal epithelial genes and differs from luminal A type in that it also highly expresses some specific genes, such as ERB-2, GGH, NSEP1, CCNE1, etc. The main immunophenotypic features in clinical practice are ER (+) and/or PR (+), HER-2 (+) or Ki-67 positive cells ≥14%. Among luminal breast cancers, luminal A type is the most common type, and luminal B type accounts for a small proportion, only 5%-10%, mainly because Ki-67 status is not considered, and only ER, PR and HER-2 are classified. In recent years, the proportion of luminal B type has been reported to increase. luminal B type is also HER-2-positive compared with HER-2-positive type, but the tumor infiltration is much lower than HER-2-positive type.
3.1 Chemotherapy and anti-HER-2 targeted therapy for Luminal B breast cancer
In a clinical trial, Hugh et al. found that docetaxel + piroplatin + cyclophosphamide (TAC) regimen could benefit ER-positive patients more than conventional 5-fluorouracil + epi-amycin + cyclophosphamide (FEC) regimen, but only to the extent that Similar results were obtained by Roché et al. in a sequential doxorubicin regimen of cyclophosphamide + epi-adriamycin + 5-fluorouracil (CEF). The 2013 NCCN guidelines recommend endocrine therapy alone for luminal B patients without lymph node metastasis if the tumor diameter is ≤0.5 cm, endocrine therapy + targeted therapy ± chemotherapy for tumor diameter 0.6-1.0 cm, and endocrine therapy + targeted therapy ± chemotherapy for tumor diameter >1.0 cm. Those with lymph node metastasis need to combine endocrine therapy, chemotherapy and targeted therapy regardless of tumor size.
3.2 Endocrine therapy for Luminal B breast cancer
In an in vitro trial, Benz et al. reported that HER-2-positive MCF-7 cells were estrogen-dependent but resistant to triamcinolone, possibly by interfering with tamoxifen in the cellular ER and affecting cellular conduction. The results showed that endocrine therapy with aromatase inhibitor analogs in luminal B breast cancer patients has an efficiency of 88%, and aromatase inhibitors should be preferred in postmenopausal luminal B patients, and premenopausal patients should be treated with aromatase inhibitors on top of sex hormone-releasing hormone analogs. According to the NCCN guidelines, luminal B breast cancer requires endocrine therapy regardless of stage, combined with targeted therapy against HER-2 to achieve the best outcome.
3.3 Neoadjuvant therapy for Luminal B breast cancer
For patients with advanced tumor stage, who need to achieve surgery through down-staging and breast conservation, neoadjuvant chemotherapy is often required to achieve breast conservation. However, clinical studies have confirmed that the overall pCR rate of neoadjuvant chemotherapy for luminal type patients is not high, about 6%-12%, but due to the difference in proliferative characteristics of luminal B breast cancer and luminal A breast cancer, luminal B type may obtain better efficacy through neoadjuvant chemotherapy than luminal A type. In China, Zhou Bo et al. reported that after combined neoadjuvant chemotherapy with paclitaxel and anthracycline, the pCR rate of luminal A was 10.3% and that of luminal B reached 25%, and the effect of neoadjuvant chemotherapy for luminal B was significantly better than that of luminal A. Carey et al. Carey et al. compared neoadjuvant chemotherapy for four subtypes of breast cancer: luminal A, luminal B, HER-2-positive and triple-negative, also suggesting that luminal B is more sensitive than luminal A. The combination of anti-HER-2 targeting therapy with neoadjuvant chemotherapy for HER-2-positive breast cancer in luminal B can further increase the pCR rate substantially, and the difference in efficacy between luminal B and HER-2-positive is not statistically significant. Preliminary results from the NOAH trial suggest that the combination of trastuzumab can increase the pCR rate in HER-2 overexpressed breast cancer from 20% to 39% with chemotherapy alone.
4.HER-2 positive type
HER-2 positive type is characterized by ER (-), PR (-) and HER-2 (+) immunophenotypes, with high expression of HER-2 and HER-2 related genes including GRB7 and TRAP100, and low expression of luminal epithelial genes ER and PR. HER-2-positive breast cancers have active proliferation, high malignancy and poor tumor grading, and were once used as a poor prognostic predictor for breast cancer, but with the advent of anti-HER-2 targeted therapies, the prognosis of HER-2-positive breast cancers has greatly improved and is no longer used as an independent prognostic predictor.
4.1 Adjuvant chemotherapy and targeted therapy for HER-2-positive breast cancer
The results of the CALGB9344 trial suggest that HER-2-positive breast cancer has a significant survival benefit from paclitaxel-based adjuvant chemotherapy. press and other clinical studies suggest that HER-2 overexpression can be used as an adjuvant chemotherapy based on anthracyclines. The clinical study by press et al. suggested that HER-2 overexpression could be a predictor of the efficacy of anthracycline-based adjuvant chemotherapy. Several clinical studies have shown that targeted trastuzumab therapy for HER-2-positive breast cancer significantly reduces the risk of recurrence and metastasis and improves overall survival. The results of the HERA trial, reported at the 2012 SABCS meeting, showed no statistically significant difference between 2 years and 1 year of postoperative trastuzumab targeted therapy, while the results of the PHARE study suggested that 1 year of trastuzumab therapy was superior to 6 months of trastuzumab therapy. The commonly used chemotherapy combined with targeted therapy regimens are paclitaxel combined with trastuzumab (AC-TH) and anthracycline-free doxorubicin + carboplatin + trastuzumab (TCH), and the difference in disease-free survival between these two groups is not statistically significant. Other anti-HER-2 targeting agents such as lapatinib and patuximab have not yet achieved effective positive results in postoperative adjuvant therapy and are not recommended at this time. In the neoadjuvant treatment of HER-2-positive breast cancer, the 2013 ASCO meeting announced the results of the NOAH trial of neoadjuvant therapy, with a pCR rate of 43% in the chemotherapy combined with targeted combination therapy group and 22% in the chemotherapy alone group; the 5-year disease-free survival rate was 57.5% in the combination therapy group and 43.3% in the chemotherapy alone group. baselga et al. published the results of the Neo- ALTTO trial results, the pCR rate was 24.7% in the paclitaxel combined with lapatinib group, 29.5% in the paclitaxel combined with trastuzumab group, and 51.3% in the paclitaxel combined with trastuzumab plus lapatinib group, with a significantly higher pCR rate in the dual-targeted therapy group than in the single-targeted group. Therefore, trastuzumab-containing regimens are recommended in the neoadjuvant treatment of HER-2-positive breast cancer, but due to the synergistic cardiotoxic effects of anthracyclines and trastuzumab, simultaneous use of both should be avoided as much as possible, and if used simultaneously in neoadjuvant treatment, they should be used under close observation, while not combined and used simultaneously for more than 4 cycles.
4.2 Treatment of HER-2-positive advanced breast cancer
HER-2-positive breast cancer suggests a high risk of recurrence and metastasis. Due to the early application of anti-HER-2 drugs, the recurrence and metastasis rate of this type has been greatly reduced and high HER-2 expression is no longer a poor prognostic factor for breast cancer. Several international multicenter clinical trials have confirmed that chemotherapy combined with trastuzumab is now the first-line treatment of choice for patients with HER-2-positive advanced breast cancer who have not been treated with anti-HER-2 therapy, with the results of the H0648g trial confirming that the use of trastuzumab in the first-line treatment of recurrent metastatic HER-2-positive breast cancer significantly prolongs the time to disease progression. The results of the M77001 trial suggested that treatment with doxorubicin combined with trastuzumab was superior to doxorubicin alone; and in the BCIRG 007 trial, the TCH regimen with carboplatin was comparable to the TH regimen without carboplatin in first-line treatment of recurrent metastatic breast cancer, with no statistically significant difference. In the study of second-line treatment regimens for recurrent metastatic breast cancer, the Hermine trial suggested that the mean time to disease progression for patients with recurrent metastatic breast cancer who progressed on first-line treatment with trastuzumab followed by a change in chemotherapy in second-line treatment and continued combination trastuzumab was 10.2 months, which was better than the time to disease progression with chemotherapy-only drugs and discontinuation of trastuzumab, which was 7.1 months. Therefore, anti-HER-2 therapy in the treatment of trastuzumab-resistant breast cancer needs to be applied consistently. In contrast, Bachelot et al. treated most patients with HER-2-positive breast cancer who had previously used trastuzumab and developed brain metastases with a combination of capecitabine and lapatinib and observed 65.9% lesion reduction and clinical partial remission, with no clinical complete remission, and concluded that capecitabine in combination with lapatinib could be a first-line treatment option for patients with HER-2-positive brain metastases. According to the results of current large clinical trials, effective second-line treatment options for HER-2-positive breast cancer include the following categories.
① replacement chemotherapy drugs continued in combination with trastuzumab.
(2) Replacement of chemotherapy drugs combined with other targeted therapies such as patuximab or lapatinib; (3) Dual-targeted therapy combining trastuzumab and patuximab.
5 .TNBC
TNBC immunophenotype is ER (-), PR (-) and HER-2 (-), TNBC accounts for about 10%-20% of female breast cancers. TNBC genotyping is mainly basal-like, with about 85% overlap between the two. The basal-like type originates from the outer ductal myoepithelial cells and has high expression of basal epithelial molecular markers CK5/6, CK17, EGFR, etc., low expression of ER-related genes and HER-2-related genes, mostly with mutations in TP53 (82%) and more often with BRCA1 mutations. The important clinical features of basal-like type are poor prognosis, mostly in young women, and distant metastases mostly in visceral metastases and brain metastases.
TNBC contains a small number of other subtypes, such as normal-like type, which expresses normal adipose tissue genes and has low tumor malignancy, and has a better prognosis, but is not sensitive to chemotherapy. Therefore, the determination of TNBC prognosis needs to identify whether it belongs to the true basal-like type, which requires the assistance of molecular indicators such as CK5/6, CK17 and EGFR.
5.1 TNBC chemotherapy
TNBC is ineffective to endocrine therapy and trastuzumab-targeted therapy due to low expression of ER, PR and HER-2, and is currently a hot spot for research in clinical treatment due to poor prognosis. Currently, chemotherapy is the main systemic treatment modality for TNBC in clinical practice. Several large-scale international multicenter clinical trials have confirmed that paclitaxel combined or sequential anthracycline chemotherapy regimens have shown superiority over conventional anthracycline combination chemotherapy regimens for TNBC. The results of the FinXX study subgroup analysis and NO17629 study presented at the 33rd San Antonio Breast Cancer Forum suggest that capecitabine combined with anthracycline and paclitaxel chemotherapy regimens can improve the overall survival rate of TNBC.
In clinical studies of neoadjuvant chemotherapy for TNBC, it was found that TNBC chemotherapy was more effective than other subtypes, especially the pCR rate was higher than that of non-TNBC, but the overall prognosis was significantly worse than that of non-TNBC, although those who achieved pCR after chemotherapy had a significantly better prognosis than those who did not achieve pCR. Wu et al. reported 249 breast cancer patients treated with neoadjuvant chemotherapy with doxorubicin combined with epoetin, 54 with TNBC and 195 with non-TNBC. The results showed that the pCR rate of TNBC patients was 25.9%, which was significantly higher than that of non-TNBC. After follow-up, the 5-year disease-free survival rate and 5-year overall survival rate of TNBC patients who did not achieve pCR after neoadjuvant chemotherapy were lower than that of non-TNBC patients, while the disease-free survival rate and overall survival rate of those who achieved pCR were significantly higher than that of those who did not achieve pCR. TNBC with pCR and non-TNBC had similar disease-free and overall survival rates. In terms of chemotherapeutic drug selection for TNBC treatment, Silver et al. reported 28 patients with stage II-III TNBC receiving four cycles of single-agent cisplatin neoadjuvant chemotherapy, showing that six patients (22%) achieved pCR and 18 patients (64%) achieved clinical complete or partial remission, indicating that platinum drugs may have an important role in the treatment of TNBC. 2013 The results of GeparSixto-GBG 66, a controlled study of paclitaxel in combination with an anthracycline versus the addition of carboplatin, were presented at the ASCO meeting. pCR rates were 58.7% with the addition of carboplatin and 37.9% without carboplatin, with significantly higher pCR rates with the combination. 2013 SABCS published the CALGB 40603 study of paclitaxel alone versus the combination of carboplatin or bevacizumab. controlled study, the pCR rate was 54% in the combined carboplatin group and 41% in the group without carboplatin, and the difference between the 2 groups was statistically significant. However, due to insufficient follow-up time, there is a lack of evidence-based medical evidence whether the increased pCR rate with the addition of carboplatin translates into a benefit in disease-free survival and overall survival.
5.2 TNBC-targeted therapy
TNBC lacks effective therapies other than chemotherapy due to negative expression of both endocrine therapy and anti-HER-2 target therapy. Currently, researchers have been trying to find other targets for TNBC, among which several receptors highly expressed in TNBC, such as EGFR (also known as HER-1), vascular endothelial growth factor (VEGF) receptor, and polyadenylate diphosphate ribosyltransferase (PARP) inhibitors associated with DNA repair process are being actively investigated in TNBC therapy. Of course these receptors are not factors specifically expressed in TNBC, but have a higher expression rate than other subtypes of breast cancer, and it is hoped that the study of these relevant targeting factors will lead to the exploration of more effective therapeutic approaches against TNBC. Currently, there are clinical trials evaluating the efficacy of EGFR monoclonal antibody (cetuximab) alone or in combination with platinum-based chemotherapy, and the results show that cetuximab in combination with cisplatin has better efficacy in metastatic TNBC. Clinical trials of bevacizumab in combination with chemotherapy have demonstrated significant improvement in progression-free survival in metastatic TNBC. The FDA removed the indication for bevacizumab in metastatic breast cancer due to the lack of benefit on overall survival and a combination of safety and price considerations, but the NCCN guideline panel still recommended retaining bevacizumab based on the primary study endpoint of the E2100 trial and its long-term follow-up results monotherapy in combination with paclitaxel as a treatment option for advanced breast cancer. For adjuvant therapy, Cameron et al. published the results of the BEATRICE study, in which bevacizumab did not differ from placebo in the treatment of TNBC and HER-2-positive breast cancer, and no positive results were obtained, and bevacizumab was not recommended for adjuvant treatment of breast cancer. The role of bevacizumab in neoadjuvant chemotherapy is pending the results of large clinical trials. PARP is a key enzyme for cell proliferation and DNA repair, and PARP inhibitors are sensitive to TNBC with BRCA1 gene mutation. two clinical trials are currently investigating the efficacy and safety of PARP inhibitors.
6. Other typing
With the continuous improvement of gene microarray technology, molecular typing of breast cancer (such as 70 genotyping, 21 genotyping, etc.) and new subtypes have emerged. In recent years, some new subtypes have been discovered, including a new subgroup in the luminal type called luminal C. This subgroup expresses luminal-specific genes in the low and medium, and expresses some new genes of non-luminal-specific genes, and some features are similar to those of the basal-like
The immunophenotype is ER (+), PR (+) and HER-2 (+), also known as triple-positive breast cancer, with poor prognosis. In addition, a subtype of TNBC with low expression of the calaudin protein, called the claudin gene low expression type, was identified as the 6th breast cancer subtype, characterized by low or no expression of epithelial cell adhesion genes (calaudin 3, 4, 7 and E-cadherin), differentiated luminal cell surface markers (EpCAM and MUC1) and epithelial mesenchymal transition markers of immune response and stem cell-like characteristics of tumor cells (CD44+CD24- and high ALDH1) were highly expressed.
Molecular typing studies of breast cancer provide a basis for predicting the risk of breast cancer recurrence and the choice of individualized comprehensive treatment regimens in our clinical practice, resulting in a qualitative leap in the diagnosis and treatment of breast cancer.