Survival is associated with complete response on MRI after neoadjuvant chemotherapy in ER-positive HER2-negative breast cancer.

BACKGROUND: Pathological complete remission (pCR) of estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer is rarely achieved after neoadjuvant chemotherapy (NAC). In addition, the prognostic value of pCR for this breast cancer subtype is limited. We explored whether response evaluation by magnetic resonance imaging (MRI) is associated with recurrence-free survival after NAC in ER-positive/HER2-negative breast cancer. METHODS: MRI examinations were performed in 272 women with ER-positive/HER2-negative breast cancer before, during and after NAC. MRI interpretation included lesion morphology at baseline, changes in morphology and size, and contrast uptake kinetics. These MRI features, clinical characteristics and final pathology were correlated with recurrence-free survival. RESULTS: The median follow up time was 41 months. There were 35 women with events, including 19 breast-cancer-related deaths. On multivariable analysis, age younger than 50 years (hazard ratio (HR) = 2.55, 95 % confidence interval (CI) 1.3, 5.02, p = 0.007), radiological complete response after NAC (HR = 14.11, CI 1.81, 1818; p = 0.006) and smaller diameters of washout/plateau enhancement at MRI after NAC (HR = 1.02, CI 1.00, 1.04, p = 0.036) were independently associated with best recurrence-free survival. Pathological response was not significant; HR = 2.12, CI 0.86, 4.64, p = 0.096. CONCLUSIONS: MRI after NAC in ER-positive/HER2-negative tumors may be predictive of recurrence-free survival. A radiological complete response at MRI after NAC is associated with an excellent prognosis.

Next generation sequencing of triple negative breast cancer to find predictors for chemotherapy response.

INTRODUCTION: In triple negative breast cancers (TNBC) the initial response to chemotherapy is often favorable, but relapse and chemotherapy resistance frequently occur in advanced disease. Hence there is an urgent need for targeted treatments in this breast cancer subtype. In the current study we deep sequenced DNA of tumors prior to chemotherapy to search for predictors of response or resistance. METHODS: Next generation sequencing (NGS) was performed for 1,977 genes involved in tumorigenesis. DNA from 56 pre-treatment TNBC-biopsies was sequenced, as well as matched normal DNA. Following their tumor biopsy, patients started neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. We studied associations between genetic alterations and three clinical variables: chemotherapy response, relapse-free survival and BRCA proficiency. RESULTS: The mutations observed were diverse and few recurrent mutations were detected. Most mutations were in TP53, TTN, and PIK3CA (55 %, 14 %, and 9 %, respectively). The mutation rates were similar between responders and non-responders (average mutation rate 9 vs 8 mutations). No recurrent mutations were associated with chemotherapy response or relapse. Interestingly, PIK3CA mutations were exclusively observed in patients proficient for BRCA1. Samples with a relapse had a higher copy number alteration rate, and amplifications of TTK and TP53BP2 were associated with a poor chemotherapy response. CONCLUSIONS: In this homogenous cohort of TNBCs few recurrent mutations were found. However, PIK3CA mutations were associated with BRCA proficiency, which can have clinical consequences in the near future.

Identifying subgroup markers in heterogeneous populations.

Traditional methods that aim to identify biomarkers that distinguish between two groups, like Significance Analysis of Microarrays or the t-test, perform optimally when such biomarkers show homogeneous behavior within each group and differential behavior between the groups. However, in many applications, this is not the case. Instead, a subgroup of samples in one group shows differential behavior with respect to all other samples. To successfully detect markers showing such imbalanced patterns of differential signal, a different approach is required. We propose a novel method, specifically designed for the Detection of Imbalanced Differential Signal (DIDS). We use an artificial dataset and a human breast cancer dataset to measure its performance and compare it with three traditional methods and four approaches that take imbalanced signal into account. Supported by extensive experimental results, we show that DIDS outperforms all other approaches in terms of power and positive predictive value. In a mouse breast cancer dataset, DIDS is the only approach that detects a functionally validated marker of chemotherapy resistance. DIDS can be applied to any continuous value data, including gene expression data, and in any context where imbalanced differential signal is manifested.

Genomic patterns resembling BRCA1- and BRCA2-mutated breast cancers predict benefit of intensified carboplatin-based chemotherapy.

INTRODUCTION: BRCA-mutated breast cancer cells lack the DNA-repair mechanism homologous recombination that is required for error-free DNA double-strand break (DSB) repair. Homologous recombination deficiency (HRD) may cause hypersensitivity to DNA DSB-inducing agents, such as bifunctional alkylating agents and platinum salts. HRD can be caused by BRCA mutations, and by other mechanisms. To identify HRD, studies have focused on triple-negative (TN) breast cancers as these resemble BRCA1-mutated breast cancer closely and might also share this hypersensitivity. However, ways to identify HRD in non-BRCA-mutated, estrogen receptor (ER)-positive breast cancers have remained elusive. The current study provides evidence that genomic patterns resembling BRCA1- or BRCA2-mutated breast cancers can identify breast cancer patients with TN as well as ER-positive, HER2-negative tumors that are sensitive to intensified, DSB-inducing chemotherapy. METHODS: Array comparative genomic hybridization (aCGH) was used to classify breast cancers. Patients with tumors with similar aCGH patterns as BRCA1- and/or BRCA2-mutated breast cancers were defined as having a BRCA-likeCGH status, others as non-BCRA-likeCGH. Stage-III patients (n = 249) had participated in a randomized controlled trial of adjuvant high-dose (HD) cyclophosphamide-thiotepa-carboplatin (CTC) versus 5-fluorouracil-epirubicin-cyclophosphamide (FE90C) chemotherapy. RESULTS: Among patients with BRCA-likeCGH tumors (81/249, 32%), a significant benefit of HD-CTC compared to FE90C was observed regarding overall survival (adjusted hazard ratio 0.19, 95% CI: 0.08 to 0.48) that was not seen for patients with non-BRCA-likeCGH tumors (adjusted hazard ratio 0.90, 95% CI: 0.53 to 1.54) (P = 0.004). Half of all BRCA-likeCGH tumors were ER-positive. CONCLUSIONS: Distinct aCGH patterns differentiated between HER2-negative patients with a markedly improved outcome after adjuvant treatment with an intensified DNA-DSB-inducing regimen (BRCA-likeCGH patients) and those without benefit (non-BRCA-likeCGH patients).

Sequential (18)F-FDG PET/CT for early prediction of complete pathological response in breast and axilla during neoadjuvant chemotherapy.

PURPOSE: To investigate the value of response monitoring in both the primary tumour and axillary nodes on sequential PET/CT scans during neoadjuvant chemotherapy (NAC) for predicting complete pathological response (pCR), taking the breast cancer subtype into account. METHODS: In 107 consecutive patients 290 PET/CT scans were performed at baseline (PET/CT1, 107 patients), after 2 - 3 weeks of chemotherapy (PET/CT2, 85 patients), and after 6 - 8 weeks (PET/CT3, 98 patients). The relative changes in SUVmax (from baseline) of the tumour and the lymph nodes and in both combined (after logistic regression), and the changes in the highest SUVmax between scans (either tumour or lymph node) were determined and their associations with pCR of the tumour and lymph nodes after completion of NAC were assessed using receiver operating characteristic (ROC) analysis. RESULTS: A pCR was seen in 17 HER2-positive tumours (65 %), 1 ER-positive/HER2-negative tumour (2 %), and 16 triple-negative tumours (52 %). The areas under the ROC curves (ROC-AUC) for the prediction of pCR in HER2-positive tumours after 3 weeks were 0.61 for the relative change in tumours, 0.67 for the combined change in tumour and nodes, and 0.72 for the changes in the highest SUVmax between scans. After 8 weeks equivalent values were 0.59, 0.42 and 0.64, respectively. In triple-negative tumours the ROC-AUCs were 0.76, 0.84 and 0.76 after 2 weeks, and 0.87, 0.93 and 0.88 after 6 weeks, respectively. CONCLUSION: In triple-negative tumours a PET/CT scan after 6 weeks (three cycles) appears to be optimally predictive of pCR. In HER2-positive tumours neither a PET/CT scan after 3 weeks nor after 8 weeks seems to be useful. The changes in SUVmax of both the tumour and axillary nodes combined correlates best with pCR.

Combined use of ¹8F-FDG PET/CT and MRI for response monitoring of breast cancer during neoadjuvant chemotherapy.

PURPOSE: To explore the potential complementary value of PET/CT and dynamic contrast-enhanced MRI in predicting pathological response to neoadjuvant chemotherapy (NAC) of breast cancer and the dependency on breast cancer subtype. METHODS: We performed (18)F-FDG PET/CT and MRI examinations before and during NAC. The imaging features evaluated on both examinations included baseline and changes in (18)F-FDG maximum standardized uptake value (SUVmax) on PET/CT, and tumour morphology and contrast uptake kinetics on MRI. The outcome measure was a (near) pathological complete response ((near-)pCR) after surgery. Receiver operating characteristic curves with area under the curve (AUC) were used to evaluate the relationships between patient, tumour and imaging characteristics and tumour responses. RESULTS: Of 93 patients, 43 achieved a (near-)pCR. The responses varied among the different breast cancer subtypes. On univariate analysis the following variables were significantly associated with (near-)pCR: age (p = 0.033), breast cancer subtype (p < 0.001), relative change in SUVmax on PET/CT (p < 0.001) and relative change in largest tumour diameter on MRI (p < 0.001). The AUC for the relative reduction in SUVmax on PET/CT was 0.78 (95% CI 0.68-0.88), and for the relative reduction in tumour diameter at late enhancement on MRI was 0.79 (95% CI 0.70-0.89). The AUC increased to 0.90 (95% CI 0.83-0.96) in the final multivariate model with PET/CT, MRI and breast cancer subtype combined (p = 0.012). CONCLUSION: PET/CT and MRI showed comparable value for monitoring response during NAC. Combined use of PET/CT and MRI had complementary potential. Research with more patients is required to further elucidate the dependency on breast cancer subtype.

Pre-chemotherapy 18F-FDG PET/CT upstages nodal stage in stage II-III breast cancer patients treated with neoadjuvant chemotherapy.

In breast cancer patients treated with neoadjuvant chemotherapy (NAC) the number of tumor-positive nodes can no longer reliably be determined. Furthermore, ultrasound (US) seems suboptimal for the detection of N3-disease. Therefore we assessed the proportion of breast cancer patients treated with NAC in which pre-chemotherapy 18F-FDG PET/CT detected ≥4 axillary nodes or occult N3-disease, upstaging nodal status and changing risk estimation for locoregional recurrence (LRR). Conventional regional staging consisted of US with fine needle aspiration and/or sentinel lymph node biopsy. Patients were classified as low-risk (cT2N0), intermediate-risk (cT0N1, cT1N1, cT2N1, cT3N0), or high-risk (cT3N1, cT4, cN2-3) for LRR. The presence and number of FDG-avid nodes were evaluated and the proportion of patients that would be upstaged by PET/CT, based on detection of ≥4 FDG-avid axillary nodes defined as cN2(4+) or occult N3-disease, was calculated. In total, 87 of 278 patients were considered high-risk based on conventional staging. PET/CT detected occult N3-disease in 5 (11 %) of 47 low-risk patients. In 144 intermediate-risk patients, PET/CT detected ≥4 FDG-avid nodes in 24 (17 %) patients and occult N3-disease in 22 (15 %) patients, thereby finally upstaging 38 (26 %) of intermediate-risk patients. Of 43 (23 %) upstaged patients, 18 were ypN0, 12 were ypN1, and 13 were ypN2-3. Pre-chemotherapy PET/CT is valuable for selection of breast cancer patients at high risk for LRR. In our population, 23 % of patients treated with NAC were upstaged to the high-risk group based on PET/CT information, potentially benefiting from regional radiotherapy.

SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2-negative breast cancer.

Response rates to chemotherapy remain highly variable in breast cancer patients. We set out to identify genes associated with chemotherapy resistance. We analyzed what is currently the largest single-institute set of gene expression profiles derived from breast cancers prior to a single neoadjuvant chemotherapy regimen (dose-dense doxorubicin and cyclophosphamide). We collected, gene expression-profiled, and analyzed 178 HER2-negative breast tumor biopsies ("NKI dataset"). We employed a recently developed approach for detecting imbalanced differential signal (DIDS) to identify markers of resistance to treatment. In contrast to traditional methods, DIDS is able to identify markers that show aberrant expression in only a small subgroup of the non-responder samples. We found a number of markers of resistance to anthracycline-based chemotherapy. We validated our findings in three external datasets, totaling 456 HER2-negative samples. Since these external sets included patients who received differing treatment regimens, the validated markers represent markers of general chemotherapy resistance. There was a highly significant overlap in the markers identified in the NKI dataset and the other three datasets. Five resistance markers, SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B, were identified in three of the four datasets (p value overlap < 1 × 10(-6)). These five genes identified resistant tumors that could not have been identified by merely taking ER status or proliferation into account. The identification of these genes might lead to a better understanding of the mechanisms involved in (clinically) observed chemotherapy resistance and could possibly assist in the recognition of breast cancers in which chemotherapy does not contribute to response or survival.

Early assessment of axillary response with ¹8F-FDG PET/CT during neoadjuvant chemotherapy in stage II-III breast cancer: implications for surgical management of the axilla.

BACKGROUND: If all initially node-positive patients undergo axillary lymph node dissection (ALND) after neoadjuvant chemotherapy (NAC), overtreatment may occur in patients with complete response. Positron emission tomography-computed tomography (PET/CT) during NAC may predict axillary response and select patients appropriate for less invasive treatment after NAC. We evaluated the value of sequential (18)F fluorodeoxyglucose (FDG) PET/CTs during NAC for axillary response monitoring in stage II-III breast cancer. METHODS: A total of 219 PET/CTs were performed in 80 patients with cytology-proven, node-positive disease at baseline (PET/CT1, n = 80) and twice during NAC (PET/CT2 n = 62, PET/CT3, n = 77). The relative changes in maximum standardized uptake value (SUVmax) of axillary nodes were examined for their ability to assess pathological response. All patients underwent ALND after chemotherapy, and complete axillary response (pCR), defined as absence of isolated tumor cells and of micro- and macrometastases, served as the reference standard. RESULTS: A total of 32 (40 %) patients experienced axillary pCR. The relative decrease in SUVmax was significantly higher in patients with pCR than in those without, both on PET/CT2 (p < 0.001) and PET/CT3 (p = 0.025). The area under the receiver operating characteristic curve values for PET/CT2 and PET/CT3 were 0.80 (95 % confidence interval 0.68-0.92) and 0.65 (95 % confidence interval 0.52-0.79), respectively. A relative decrease of ≥60 % on PET/CT2 had an excellent specificity (35 of 37, 95 %), a high positive predictive value (12 of 14, 86 %), and a sensitivity of 48 %-that is, it accurately identified histologic pCR in 12 of 25 patients with disease that responded to therapy. CONCLUSIONS: (18)F-FDG PET/CT early during NAC is useful for axillary response monitoring in cytology-proven node-positive breast cancer because it identifies pathological response, thus permitting ALND to be spared.

Paclitaxel, carboplatin, and trastuzumab in a neo-adjuvant regimen for HER2-positive breast cancer.

To evaluate a nonanthracycline-containing regimen consisting of 24 weekly administrations of paclitaxel, carboplatin, and trastuzumab as neo-adjuvant therapy for human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Patients with stage II or III breast cancer, including inflammatory disease, with HER2 overexpression (immunohistochemistry and/or fluorescent in situ hybridization) were treated with 24 weekly administrations of paclitaxel 70 mg/m(2) , carboplatin AUC = 3 mg/mL/minute, and trastuzumab 2 mg/kg (loading dose 4 mg/kg). In cycles 7, 8, 15, 16, 23, and 24, only trastuzumab was given. The primary end point was pathologic complete response (pCR) in both breast and axilla. Of 61 evaluable patients, 61% had stage II disease and 75% were node-positive. The median NRI (Neoadjuvant Response Index, a measure of the degree of downstaging by chemotherapy) of all patients was 0.86. Twenty-seven (44%) had a NRI of 1.0, which corresponds to pCR in breast and lymph nodes. The most commonly reported grade 3/4 toxicities were neutropenia (72%) and thrombocytopenia (36%). Dose reduction was necessary in 51% of the patients. A weekly carboplatin-paclitaxel-trastuzumab neo-adjuvant regimen is highly active in HER2-positive breast cancer with an acceptable toxicity profile. A multicenter phase 2 trial has recently reached its accrual target and will serve as a basis for a subsequent randomized phase 3 study comparing this regimen to a similar regimen preceded by anthracyclines.

Locoregional lymph node involvement on 18F-FDG PET/CT in breast cancer patients scheduled for neoadjuvant chemotherapy.

The optimal method for locoregional staging in patients treated with neoadjuvant chemotherapy (NAC), usually ultrasound (US) and pre- or post-chemotherapy sentinel lymph node biopsy (SLNB), remains subject of debate. The aim of this study was to assess the value of 18F-FDG PET/CT for detecting locoregional lymph node metastases in primary breast cancer patients scheduled for NAC. 311 breast cancer patients, scheduled for NAC, underwent PET/CT of the thorax in prone position with hanging breasts. A panel of four experienced reviewers examined PET/CT images, blinded for other diagnostic procedures. FDG uptake in locoregional nodes was determined qualitatively using a 4-point scale (0 = negative, 1 = questionable, 2 = moderately intense, and 3 = very intense). Results were compared with pathology obtained by US-guided fine needle aspiration or SLNB prior to NAC. All FDG-avid extra-axillary nodes were considered metastatic, based on the previously reported high positive predictive value of the technique. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of FDG-avid nodes for the detection of axillary metastases (score 2 or 3) were 82, 92, 98, 53, and 84 %, respectively. Of 28 patients with questionable axillary FDG uptake (score 1), 23 (82 %) were node-positive. Occult lymph node metastases in the internal mammary chain and periclavicular area were detected in 26 (8 %) and 32 (10 %) patients, respectively, resulting in changed regional radiotherapy planning in 50 (16 %) patients. In breast cancer patients scheduled for NAC, PET/CT renders pre-chemotherapy SLNB unnecessary in case of an FDG-avid axillary node, enables axillary response monitoring during or after NAC, and leads to changes in radiotherapy for a substantial number of patients because of detection of occult N3-disease. Based on these results, we recommend a PET/CT as a standard staging procedure in breast cancer patients scheduled for NAC.

18F-FDG PET/CT as a staging procedure in primary stage II and III breast cancer: comparison with conventional imaging techniques.

The aim of the present study was to investigate if 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) outperforms conventional imaging techniques for excluding distant metastases prior to neoadjuvant chemotherapy (NAC) treatment in patients with stage II and III breast cancer. Second, we assessed the clinical importance of false positive findings. One hundred and fifty four patients with stage II or III breast cancer, scheduled to receive NAC, underwent an 18F-FDG PET/CT scan and conventional imaging, consisting of bone scintigraphy, ultrasound of the liver, and chest radiography. Suspect additional lesions at staging examination were confirmed by biopsy and histopathology and/or additional imaging. Metastases that were detected within 6 months after the PET/CT scan were considered evidence of occult metastasis, missed by staging examination. Forty-two additional distant lesions were seen in 25 patients with PET/CT and could be confirmed in 20 (13%) of 154 patients. PET/CT was false positive for 8 additional lesions (19%) and misclassified the presence of metastatic disease in 5 (3%) of 154 patients. In 16 (80%) of 20 patients, additional lesions were exclusively seen with PET/CT, leading to a change in treatment in 13 (8%) of 154 patients. In 129 patients with a negative staging PET/CT, no metastases developed during the follow-up of 9.0 months. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of PET/CT in the detection of additional distant lesions in patients with stage II or III breast cancer are 100, 96, 80, 100, and 97%, respectively. FDG PET/CT is superior to conventional imaging techniques in the detection of distant metastases in patients with untreated stage II or III breast cancer and is associated with a low false positive rate. PET/CT may be of additional value in the staging of breast cancer prior to NAC.

Lobular histology and response to neoadjuvant chemotherapy in invasive breast cancer.

Invasive lobular carcinoma (ILC) has been reported to be less responsive to neoadjuvant chemotherapy (NAC) than invasive ductal carcinoma (IDC). We sought to determine whether ILC histology indeed predicts poor response to NAC by analyzing tumor characteristics such as protein expression, gene expression, and imaging features, and by comparing NAC response rates to those seen in IDC after adjustment for these factors. We combined datasets from two large prospective NAC trials, including in total 676 patients, of which 75 were of lobular histology. Eligible patients had tumors ≥3 cm in diameter or pathologic documentation of positive nodes, and underwent serial biopsies, expression microarray analysis, and MRI imaging. We compared pathologic complete response (pCR) rates and breast conservation surgery (BCS) rates between ILC and IDC, adjusted for clinicopathologic factors. On univariate analysis, ILCs were significantly less likely to have a pCR after NAC than IDCs (11 vs. 25 %, p = 0.01). However, the known differences in tumor characteristics between the two histologic types, including hormone receptor (HR) status, HER2 status, histological grade, and p53 expression, accounted for this difference with the lowest pCR rates among HR+/HER2- tumors in both ILC and IDC (7 and 5 %, respectively). ILC which were HR- and/or HER2+ had a pCR rate of 25 %. Expression subtyping, particularly the NKI 70-gene signature, was correlated with pCR, although the small numbers of ILC in each group precluded significant associations. BCS rate did not differ between IDC and ILC after adjusting for molecular characteristics. We conclude that ILC represents a heterogeneous group of tumors which are less responsive to NAC than IDC. However, this difference is explained by differences in molecular characteristics, particularly HR and HER2, and independent of lobular histology.

Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness.

INTRODUCTION: Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-like(aCGH) profile is also present in about half of all triple-negative sporadic breast cancers and is predictive for benefit from intensified alkylating chemotherapy. As aCGH is a rather complex method, we translated the BRCA1(aCGH) profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-like(aCGH) profile. METHODS: The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-like(aCGH) breast cancers and 45 non-BRCA1-like(aCGH) breast cancers, which had previously been analyzed by aCGH. The BRCA1-like(aCGH) group consisted of germline BRCA1-mutated cases and sporadic tumours with low BRCA1 gene expression and/or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validation was performed on an independent test set of 40 BRCA1-like(aCGH) breast cancers and 32 non-BRCA1-like(aCGH) breast cancer tumours. In addition, we validated the set prospectively on 69 new triple-negative tumours. RESULTS: BRCAness in the training set of 84 tumours could accurately be predicted by prediction analysis of microarrays (PAM) (accuracy 94%). Application of this classifier on the independent validation set correctly predicted BRCA-like status of 62 out of 72 breast tumours (86%). Sensitivity and specificity were 85% and 87%, respectively. When the MLPA-test was subsequently applied to 46 breast tumour samples from a randomized clinical trial, the same survival benefit for BRCA1-like tumours associated with intensified alkylating chemotherapy was shown as was previously reported using the aCGH assay. CONCLUSIONS: Since the MLPA assay can identify BRCA1-deficient breast cancer patients, this method could be applied both for clinical genetic testing and as a predictor of treatment benefit. BRCA1-like tumours are highly sensitive to chemotherapy with DNA damaging agents, and most likely to poly ADP ribose polymerase (PARP)-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues.

Concordance of clinical and molecular breast cancer subtyping in the context of preoperative chemotherapy response.

ER, PR and HER2 status in breast cancer are important markers for the selection of drug therapy. By immunohistochemistry (IHC), three major breast cancer subtypes can be distinguished: Triple negative (TN(IHC)), HER2+(IHC) and Luminal(IHC) (ER+(IHC)/HER2-(IHC)). By using the intrinsic gene set defined by Hu et al. five molecular subtypes (Basal(mRNA), HER2+(mRNA), Luminal A(mRNA), Luminal B(mRNA) and Normal-like(mRNA)) can be defined. We studied the concordance between analogous subtypes and their prediction of response to neoadjuvant chemotherapy. We classified 195 breast tumors by both IHC and mRNA expression analysis of patients who received neoadjuvant treatment at the Netherlands Cancer institute for Stage II-III breast cancer between 2000 and 2007. The pathological complete remission (pCR) rate was used to assess chemotherapy response. The IHC and molecular subtypes showed high concordance with the exception of the HER2+(IHC) group. 60% of the HER2+(IHC) tumors were not classified as HER2+(mRNA). The HER2+(IHC)/Luminal A or B(mRNA) group had a low response rate to a trastuzumab-chemotherapy combination with a pCR rate of 8%, while the HER2+(mRNA) group had a pCR rate of 54%. The Luminal A(mRNA) and Luminal B(mRNA) groups showed similar degrees of response to chemotherapy. Neither the PR status nor the endocrine responsiveness index subdivided the ER+(IHC) tumors accurately into Luminal A(mRNA) and Luminal B(mRNA) groups. Molecular subtyping suggests the existence of a HER2+(IHC)/Luminal(mRNA) group that responds poorly to trastuzumab-based chemotherapy. For Luminal(IHC) and triple negative(IHC) tumors, further subdivision into molecular subgroups does not offer a clear advantage in treatment selection.

The 70-gene signature as a response predictor for neoadjuvant chemotherapy in breast cancer.

The 70-gene signature (MammaPrint) is a prognostic tool used to guide adjuvant treatment decisions. The aim of this study was to assess its value to predict chemosensitivity in the neoadjuvant setting. We obtained the 70-gene profile of stage II-III patients prior to neoadjuvant chemotherapy and classified the prognosis-signatures. Pathological complete remission (pCR) was used to measure chemosensitivity. Among 167 patients, 144 (86%) were having a poor and 23 (14%) a good prognosis-signature. None of the good prognosis-signature patients achieved a pCR (0/23), whereas 29/144 patients (20%) in the poor prognosis-signature group did (P = 0.015). All triple-negative tumors (n = 38) had a poor prognosis-signature. Within the non triple-negative subgroup, the response of the primary tumor remained associated with the classification of the prognosis-signature (P = 0.023). A pCR is unlikely to be achieved in tumors that have a good prognosis-signature. Tumors with a poor prognosis-signature are more sensitive to chemotherapy.

Polymorphisms of drug-metabolizing enzymes (GST, CYP2B6 and CYP3A) affect the pharmacokinetics of thiotepa and tepa.

AIMS: Thiotepa is widely used in high-dose chemotherapy. Previous studies have shown relations between exposure and severe organ toxicity. Thiotepa is metabolized by cytochrome P450 and glutathione S-transferase enzymes. Polymorphisms of these enzymes may affect elimination of thiotepa and tepa, its main metabolite. The purpose of this study was to evaluate effects of known allelic variants in CYP2B6, CYP3A4, CYP3A5, GSTA1 and GSTP1 genes on pharmacokinetics of thiotepa and tepa. METHODS: White patients (n = 124) received a high-dose regimen consisting of cyclophosphamide, thiotepa and carboplatin as intravenous infusions. Genomic DNA was analysed using polymerase chain reaction and sequencing. Plasma concentrations of thiotepa and tepa were determined using validated GC and LC-MS/MS methods. Relations between allelic variants and elimination pharmacokinetic parameters were evaluated using nonlinear mixed effects modelling (nonmem). RESULTS: The polymorphisms CYP2B6 C1459T, CYP3A4*1B, CYP3A5*3, GSTA1 (C-69T, G-52A) and GSTP1 C341T had a significant effect on clearance of thiotepa or tepa. Although significant, most effects were generally not large. Clearance of thiotepa and tepa was predominantly affected by GSTP1 C341T polymorphism, which had a frequency of 9.3%. This polymorphism increased non-inducible thiotepa clearance by 52% [95% confidence interval (CI) 41, 64, P < 0.001] and decreased tepa clearance by 32% (95% CI 29, 35, P < 0.001) in heterozygous patients, which resulted in an increase in combined exposure to thiotepa and tepa of 45% in homozygous patients. CONCLUSIONS: This study indicates that the presently evaluated variant alleles explain only a small part of the substantial interindividual variability in thiotepa and tepa pharmacokinetics. Patients homozygous for the GSTP1 C341T allele may have enhanced exposure to thiotepa and tepa.

Pharmacokinetics of cyclophosphamide and thiotepa in a conventional fractionated high-dose regimen compared with a novel simplified unfractionated regimen.

High-dose alkylating chemotherapy with cyclophosphamide (4000 or 6000 mg/m2) and thiotepa (320 or 480 mg/m2) has commonly been administered in a fractionated regimen over 4 days. A simplified unfractionated regimen would be preferable, especially because cyclophosphamide and thiotepa have been shown to influence the metabolism of each other. However, altering a dose regimen can have a profound effect on the pharmacokinetics of the compounds involved. The aim of this study was to investigate the effect of altering the fractionated administration schedule of the CTC regimen on cyclophosphamide and thiotepa pharmacokinetics. Plasma samples were collected from 124 patients who received a fractionated tiny CTC or CTC regimen of cyclophosphamide (1000 or 1500 mg m(-2) d(-1)), thiotepa (40 or 60 mg/m2 twice daily), and carboplatin (267 or 400 mg m(-2) day(-1)) for 4 days, and 16 patients who received an unfractionated mini CTC regimen of cyclophosphamide (3000 mg/m2 at day 1), carboplatin (400 mg/m2 at days 1 and 2), and thiotepa (250 mg/m2 at day 2). Plasma concentrations of cyclophosphamide and 4-hydroxycyclophosphamide were determined using high-performance liquid chromatography coupled with tandem mass spectrometric detection; plasma concentrations of thiotepa and tepa were determined using gas chromatography. Pharmacokinetics of cyclophosphamide and thiotepa were assessed using nonlinear mixed-effect modeling. The study showed that alteration of a fractionated high-dose regimen into a simplified unfractionated regimen resulted in saturation of thiotepa elimination, with a Vmax of 212 (+/-58) micromol/h and a Km of 13.7 (+/-5.9) microM. This resulted in an increased dose-corrected exposure to thiotepa (13%) and decreased dose-corrected exposure to its metabolite tepa (21%). Elimination of cyclophosphamide was not shown to be saturable. Dose-corrected exposures to cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide were comparable in both regimens. Because the simplified unfractionated mini CTC regimen was more patient-friendly and because overall dose-corrected exposures to cyclophosphamide and thiotepa were not affected to a relevant extent, our data suggest that this unfractionated regimen can be used safely in future studies.

Haptoglobin phenotype is not a predictor of recurrence free survival in high-risk primary breast cancer patients.

BACKGROUND: Better breast cancer prognostication may improve selection of patients for adjuvant therapy. We conducted a retrospective follow-up study in which we investigated sera of high-risk primary breast cancer patients, to search for proteins predictive of recurrence free survival. METHODS: Two sample sets of high-risk primary breast cancer patients participating in a randomised national trial investigating the effectiveness of high-dose chemotherapy were analysed. Sera in set I (n = 63) were analysed by surface enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF MS) for biomarker finding. Initial results were validated by analysis of sample set II (n = 371), using one-dimensional gel-electrophoresis. RESULTS: In sample set I, the expression of a peak at mass-to-charge ratio 9198 (relative intensity 20), identified as haptoglobin (Hp) alpha-1 chain, was strongly associated with recurrence free survival (global Log-rank test; p = 0.0014). Haptoglobin is present in three distinct phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2), of which only individuals with phenotype Hp 1-1 or Hp 2-1 express the haptoglobin alpha-1 chain. As the expression of the haptoglobin alpha-1 chain, determined by SELDI-TOF MS, corresponds to the phenotype, initial results were validated by haptoglobin phenotyping of the independent sample set II by native one-dimensional gel-electrophoresis. With the Hp 1-1 phenotype as the reference category, the univariate hazard ratio for recurrence was 0.87 (95% CI: 0.56 - 1.34, p = 0.5221) and 1.03 (95% CI: 0.65 - 1.64, p = 0.8966) for the Hp 2-1 and Hp 2-2 phenotypes, respectively, in sample set II. CONCLUSION: In contrast to our initial results, the haptoglobin phenotype was not identified as a predictor of recurrence free survival in high-risk primary breast cancer in our validation set. Our initial observation in the discovery set was probably the result of a type I error (i.e. false positive). This study illustrates the importance of validation in obtaining the true clinical applicability of a potential biomarker.

Dynamic contrast-enhanced MRI for prediction of breast cancer response to neoadjuvant chemotherapy: initial results.

OBJECTIVE: The aim of this study was to establish changes in contrast-enhanced MRI of breast cancer during neoadjuvant chemotherapy that are indicative of pathology outcome. MATERIALS AND METHODS: In 54 patients with breast cancer, dynamic contrast-enhanced MRI was performed before chemotherapy and after two chemotherapy cycles. Imaging was correlated with final histopathology. Multivariate analysis with cross-validation was performed on MRI features describing kinetics and morphology of contrast uptake in the early and late phases of enhancement. Receiver operating characteristic (ROC) analysis was used to develop a guideline that switches patients at high risk for incomplete remission to a different chemotherapy regimen while maintaining first-line therapy in 95% of patients who are not at risk (i.e., high specificity). RESULTS: Change in largest diameter of late enhancement during chemotherapy was the single most predictive MRI characteristic for tumor response in multivariate analysis (A(z) [area under the ROC curve] = 0.73, p < 0.00001). Insufficient (< 25%) decrease in largest diameter of late enhancement during chemotherapy was most indicative of residual tumor at final pathology. Using this criterion, the fraction of unfavorable responders indicated by MRI was 41% (22/54). Approximately half (44%, 14/32) of the patients who showed favorable response at MRI achieved complete remission at pathology. Conversely, 95% (21/22) of patients who showed unfavorable response at MRI had residual tumor at pathology. CONCLUSION: Reduction of less than 25% in largest diameter of late enhancement during neoadjuvant chemotherapy shows the potential to predict residual tumor after therapy with high specificity.