Katsunori Tozuka

Alterations of the genes involved in the PI3K and estrogen-receptor pathways influence outcome in human epidermal growth factor receptor 2-positive and hormone receptor-positive breast cancer patients treated with trastuzumab-containing neoadjuvant chemotherapy

BackgroundChemotherapy with trastuzumab is widely used for patients with human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but a significant number of patients with the tumor fail to respond, or relapse. The mechanisms of recurrence and biomarkers that indicate the response to the chemotherapy and outcome are not fully investigated.MethodsGenomic alterations were analyzed using single-nucleotide polymorphism arrays in 46 HER2 immunohistochemistry (IHC) 3+ or 2+/fluorescent in situ hybridization (FISH)+ breast cancers that were treated with neoadjuvant chemotherapy with paclitaxel, cyclophosphamid, epirubicin, fluorouracil, and trastuzumab. Patients were classified into two groups based on presence or absence of alterations of 65 cancer-associated genes, and the two groups were further classified into four groups based on genomic HER2 copy numbers or hormone receptor status (HR+/−). Pathological complete response (pCR) and relapse-free survival (RFS) rates were compared between any two of the groups.Results and discussionThe pCR rate was 54% in 37 patients, and the RFS rate at 3 years was 72% (95% CI, 0.55-0.89) in 42 patients. The analysis disclosed 8 tumors with nonamplified HER2 and 38 tumors with HER2 amplification, indicating the presence of discordance in tumors diagnosed using current HER2 testing. The 8 patients showed more difficulty in achieving pCR (P=0.019), more frequent relapse (P=0.018), and more frequent alterations of genes in the PI3K pathway (P=0.009) than the patients with HER2 amplification. The alterations of the PI3K and estrogen receptor (ER) pathway genes generally indicated worse RFS rates. The prognostic significance of the alterations was shown in patients with a HR+ tumor, but not in patients with a HR- tumor when divided. Alterations of the PI3K and ER pathway genes found in patients with a HR+ tumor with poor outcome suggested that crosstalk between the two pathways may be involved in resistance to the current chemotherapy with trastuzumab.ConclusionsWe recommend FISH analysis as a primary HER2 testing because patients with IHC 2+/3+ and nonamplified HER2 had poor outcome. We also support concurrent use of trastuzumab, lapatinib, and cytotoxic and anti-hormonal agents for patients having HR+ tumors with alterations of the PI3K and ER pathway genes.

True Recurrences and New Primary Tumors Have Different Clinical Features in Invasive Breast Cancer Patients with Ipsilateral Breast Tumor Relapse After Breast-Conserving Treatment

Abstract:  Ipsilateral breast tumor relapse (IBTR) after breast‐conserving treatment (BCT) may represent two distinct types of lesion, including a true recurrence (TR) or a new primary tumor (NPT). The aim of this study was to ascertain the difference between TRs and NPTs and to show the clinical significance of classifying IBTR into these two types of recurrence. Patients (n = 2,075) with unilateral invasive breast cancer who underwent BCT between 1987 and 2005 at Saitama Cancer Center were analyzed. IBTR was classified into TR and NPT, which was based on all clinical and pathological features of both a primary tumor and IBTR that can be evaluated. IBTR‐free survival and the risk factors were analyzed in order to compare the findings for TR and NPT. In addition, the salvage surgical methods for IBTR and overall survival after IBTR were analyzed. Sixty patients with IBTR were classified into 52 with TR and eight with NPT. IBTR‐free survival was significantly shorter in the patients with TR than those with NPT. Young age, tumor size, a positive surgical margin, and omission of radiation therapy (RT) were significant risk factors for TR. Omission of RT was the only significant risk factor for NPT. In 27 patients who underwent a repeat lumpectomy for TR, four had a second IBTR. The overall survival after IBTR was worse in patients with TR than NPT. TR and NPT show quite different clinical features. Classifying IBTR into TR or NPT can therefore help to select the most appropriate treatment for IBTR.

Ipsilateral breast tumor relapse after breast conserving surgery in women with breast cancer.

Ipsilateral breast tumor relapse (IBTR) is a potentially a significant problem after breast conserving surgery (BCS). With a median follow-up period of 64.7 months, IBTR occurred as a first relapse in 67 (3.0%) of a total of 2243 patients and distant recurrence occurred in 167 (7.4%). A positive surgical margin and the omission of radiotherapy (RT) were independently associated with IBTR. The five-year cumulative IBTR rates were 5.1% in patients with positive margins and 2.0% in the patients with negative margins. The five-year cumulative IBTR rates were 1.8% in patients with RT and 8.1% in patients without RT. IBTR was independently associated with distant-recurrence-free survival rates as well as age, nodal metastasis, lymphovascular invasion and progesterone receptor status. The five-year distant-recurrence-free survival rates were 81.9% in patients with IBTR and 93.2% in patients without IBTR. In order to prevent IBTR, a negative margin and the administration of RT are therefore considered to be important in patients who undergo BCS.

Positive sentinel lymph node biopsy predicts the number of metastatic axillary nodes of breast cancer

It remains to be clarified whether a positive sentinel lymph node biopsy (SLNB) can predict the number of metastatic axillary nodes. This study examined a consecutive series of women with unilateral invasive breast cancer who underwent axillary lymph node dissection after an intra-operative positive SLNB. The numbers of positive and negative sentinel lymph nodes (SLNs) were analyzed for a likelihood of pN1a, pN2a, and pN3a diseases as per the UICC TNM classification. Of the 368 study patients, 165 (45%) had one positive SLN and one or more negative SLNs. This result represented the most common combination of positive and negative SLNs. It was also the most predictive indicator (93%) of pN1a disease and the least predictive indicator (7% or 0%) of pN2a or pN3a disease, respectively. The numbers of positive and negative SLNs can predict the number of metastatic axillary nodes in breast cancer patients.

Abstract P3-06-29: Change of circulating tumor cells before and after neoadjuvant chemotherapy in patients with primary breast cancer

Background: Circulating tumor cells (CTCs) in peripheral blood may represent the possible presence of early tumor dissemination. However, relatively few studies were designed to investigate the change of CTC status by adjuvant chemotherapy in operable breast cancer patients. Patients and methods: Peripheral blood (7.5ml) was collected from 95 patients with stage II/III breast cancer before neoadjuvant chemotherapy (NAC). NAC consisted of anthracycline and paclitaxel chemotherapy and additional trastuzumab treatment for patients with HER2-positive tumors. Results: The average age of patients was 52.6 year old (median 52.0). One or more CTCs were detected in 18 (18.9%) of 95 patients. CTCs were detected in 6 (12.0%) of 50 patients with clinical stage II disease and 12 (26.7%) of 45 patients with clinical stage III disease. According to tumor subtypes, CTCs were detected in 5 (17.9%) of 28 patients with hormone receptor (HR)-positive and HER2-negative tumors (L subtype), 3 (12.5%) of 24 patients with HR-positive and HER2-positive tumors (L-H subtype), 4 (22.2%) of 18 patients with HR-negative and HER2-positive tumors (H subtype), and 6 (24.0%) of 25 patients with HR-negative and HER2-negative tumors (TN subtype). After NAC, 17 (94.4%) of 18 patients who were CTC-positive before chemotherapy changed into negative status. 30 (31.6%) of 95 patients had a pathologic complete response (pCR) after NAC. There was no correlation between CTC status before NAC and pathological response. At the median follow up of 27 months, distant metastasis was observed in 9 patients (9.5%). Patients with clinical stage III, TN subtype, or non-pCR had a significantly worse disease-free survival (DFS). However, CTC status before NAC was not a significant prognostic factor. Conclusion: NAC has a significant impact on CTC status irrespective of tumor subtypes. CTC status before NAC was not a significant prognostic factor in this study. The reason of which may be that most of patients showing positive for CTCs before NAC have changed into negative after NAC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-29.

BRCA1 Alterations with Additional Defects in DNA Damage Response Genes May Confer Chemoresistance to BRCA-like Breast Cancers Treated with Neoadjuvant Chemotherapy.

The BRCA‐like phenotype is a feature that some sporadic breast cancers share with those occurring in BRCA1 or BRCA2 mutation carriers. As tumors with the phenotype have defects in the DNA damage response pathway, which may increase sensitivity to drugs such as DNA cross‐linking agents and PARP inhibitors, a method to identify this phenotype is important. The prediction of chemoresistance, which frequently develops in these tumors, is also crucial for improving therapy. We examined genomic aberrations and BRCA1 promoter methylation in tumors of 73 breast cancer (20 HR−/HER2− and 53 HR+/HER2−) patients, who received neoadjuvant chemotherapy with anthracycline, cyclophosphamide, and taxane, using SNP array CGH and quantitative PCR. The methylation and/or loss or uniparental disomy (UPD) of BRCA1 (BRCA1 alterations) and the loss or UPD of BRCA2 (BRCA2 alterations) were detected in 27 (37%) and 21 (29%), respectively, of the 73 tumors. Tumors with BRCA1 or BRCA2 alterations were associated with a higher number of genomic aberrations (P < 0.001 and P < 0.001) and higher percentage of TP53 alterations (P < 0.001 and P < 0.001) than those without. Overall survival (OS) rates were similar between patients with or without BRCA1 or BRCA2 alterations. However, when 27 patients with BRCA1‐altered tumors were classified into those with or without the loss or UPD of PALB2, PAGR1, RAD51B, FANCM, MLL4, or ERCC1/2 in tumors, patients with additional defects in DNA damage response genes had worse OS (P = 0.037, 0.045, 0.038, 0.044, 0.041, or 0.019) than those without. These defects may confer chemoresistance and predict poor outcomes in patients with BRCA1‐altered breast cancer.

Impact of combining the progesterone receptor and preoperative endocrine prognostic index (PEPI) as a prognostic factor after neoadjuvant endocrine therapy using aromatase inhibitors in postmenopausal ER positive and HER2 negative breast cancer

The preoperative endocrine prognostic index (PEPI) predicts survival after neoadjuvant endocrine therapy (NAE) using aromatase inhibitors (AIs) for women with postmenopausal estrogen receptor (ER)-positive breast cancer irrespective of the human epidermal growth factor receptor 2 (HER2) status. Although the progesterone receptor (PgR) is also a prognostic factor for ER-positive breast cancer, the PgR status was not considered a prognostic factor in the original PEPI scoring system. In this study, we investigated the utility of a modified PEPI including the PgR status (PEPI-P) as a prognostic factor after NAE for postmenopausal patients with ER-positive and HER2-negative breast cancer. We enrolled 107 patients with invasive ER-positive and HER2-negative breast cancer treated with exemestane for ≥4 months as NAE. We initially assessed PEPI and compared survival between the groups. Additionally, we obtained an effective cutoff for PgR through survival analysis. Then, we assessed the survival significance of PEPI-P. A PgR staining rate of 50% was the most significant cutoff for predicting recurrence-free survival (RFS) and cancer-specific survival (CSS). PEPI was a significant prognostic factor; moreover, PEPI-P was the most significant prognostic indicator for RFS and CSS. PEPI-P is a potent prognostic indicator of survival after NAE using AIs for postmenopausal patients with ER-positive and HER2-negative breast cancer. This modified PEPI may be useful for therapeutic decision-making regarding postmenopausal ER-positive and HER2-negative breast cancer after NAE.

Abstract P4-14-17: Prognostic value of tumor-infiltrating lymphocytes in residual tumors after neoadjuvant chemotherapy concomitant with trastuzumab for HER2-positive breast cancer

Background: Neoadjuvant chemotherapy (NAC) with taxanes, followed by fluorouracil, epirubicin, and cyclophosphamide (FEC), with concurrent trastuzumab is known to achieve a high pCR rate of more than 60% for HER2-positive breast cancer (BC) as well as good prognoses in those obtaining pCR. On the other hand, the prognostic significance of tumor-infiltrating lymphocytes (TILs) has recently been described in triple-negative BC. However, the prognostic and predictive values of TILs in HER2-positive BC remain unclear. In the present study, we examined the grades of TILs in pre-treatment cancer tissues and residual tumors after NAC with trastuzumab, and also investigated its predictive utility for pCR and prognostic power for HER2-positive BC. Patients and Methods: A total of 128 Japanese women with HER2-positive BC received either paclitaxel or docetaxel followed by FEC, with concomitant trastuzumab. The proportional grades of stromal (Str)-TILs in pre-treatment biopsy specimens and residual tumors after NAC with trastuzumab were determined as follows: low grade (0-10%), intermediate grade (10-40%), and high grade (40-90%), using the criteria of the International Working Group for TILs in BC. Analysis 1: The relationship between the grades of Str-TILs in pre-treatment tumors and pCR rates was investigated. Relapse-free survival (RFS) and cancer-specific survival (CSS) were analyzed for a correlation with pre-treatment Str-TILs. Analysis 2: Alterations in the grade of Str-TILs were examined in the residual tumors of non-pCR patients, and RFS and CSS were analyzed for a correlation with residual Str-TILs. Results: pCR was achieved in 83 out of the 128 patients (pCR rate, 64.8%) who received NAC with trastuzumab, and RFS was significantly better in the pCR group than in the non-pCR group (p = 0.0071). Analysis 1: The patient distribution of the Str-TILs grade in pre-treatment tumors was as follows: high: 24 (18.8%); intermediate: 38 (29.7%); and low: 66 (51.6%). pCR rates correlated with the Str-TILs grade in pre-treatment tumors: 83.3% in the high group, 71.1% in the intermediate group, and 54.5% in the low group (p = 0.026); however, the Str-TILs grade in pre-treatment tumors did not correlate with survival. Analysis 2: In 45 non-pCR patients, the distribution of the Str-TILs grade in residual tumors was as follows: high: 9 (20.0%); intermediate: 8 (17.8%); and low: 28 (62.2%), respectively. In non-pCR patients, the rate of a high Str-TILs grade was greater in residual tumors than in pre-treatment tumors (residual, 20.0%, pre-treatment, 8.9%). RFS was significantly better with a high grade than with a low grade of residual Str-TILs (p = 0.033). Conclusions: The status of TILs in pre-treatment tumors predicted responses to NAC concomitant with trastuzumab in HER2-positive BC. The grade of TILs was higher in residual tumors than in pre-treatment tumors, and, among non-pCR patients, the prognosis of patients with a high residual-TILs grade was better prognosis than that of patients with a low residual-TILs grade. We speculate that an examination of TILs in residual tumors after NAC with trastuzumab may be necessary for selecting patients with a good prognosis from non-pCR patients. Citation Format: Kurozumi S, Inoue K, Matsumoto H, Hayashi Y, Tozuka K, Kubo K, Komatsu K, Takai K, Nagai SE, Oba H, Horiguchi J, Takeyoshi I, Kurosumi M. Prognostic value of tumor-infiltrating lymphocytes in residual tumors after neoadjuvant chemotherapy concomitant with trastuzumab for HER2-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-17.

Abstract P2-02-20: Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability

Background : The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy and a prognostic marker for patients with metastatic breast cancer. One of the leading techniques to detect CTCs uses immune-magnetic separation followed by immunocytochemistry. A microdevice can capture and enumerate CTCs using distinctive physiological difference (size and deformability) between cancer cells and blood cells. This microdevice thus obtains a larger CTC yield than that of affinity based separation which enriches the samples from a particular subgroup of cells based on biomarker (EpCAM) used. In this study, we investigated CTCs in peripheral blood from metastatic breast cancer patients using this microdevice. Patients and methods: We examined blood samples of 9 patients with heavily treated locally recurrent or metastatic breast cancer. Informed consent from these patients was obtained before blood extraction. Blood samples were taken into sodium EDTA tubes after discarding the first 1ml of blood samples. Two ml whole blood were subjected to the microdevice (Clear cell system), and CTCs were trapped in the microwells: Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4,2-diamidino-2-phenylndole dihydrochloride (DAPI) for nuclei. CK8/18- positive, DAPI-positive and CD45-negative cells were defined as CTCs. Three patients were examined using both this microdevice and affinity-based separation with EpCAM, to compare the yield of CTCs. Results: Of the 9 patients: 7 had ER-positive primary tumors, and 6 had PgR-positive ones, HER2 overexpression was detected in 2 primary tumors. CTCs were detected in 8 patients. The single patient in whom CTCs were not detected suffered from local recurrence (axillary lymph node metastasis) only, with no distant metastases. We were also unable to detect CTCs using EpCAM affinity method for this patient. The number of detected CTCs in the other patients ranged from 19/2ml to 156/2ml (mean 90/2ml), and the sizes of CTCs varied from 5 to 16μm. CK8/18-negative and DAPI positive were detected in most patients, and these cells tended to be larger than CK8/18-positive cells, suggesting that epithelial–mesenchymal transition (EMT) might occur in CTCs. The total number of CTCs detected by the microdevice from 2 patients was larger than that of CTCs detected by EpCAM affinity method (107/2ml vs 1/7.5ml, and 19/2ml vs 39/7.5ml). Conclusion: CTCs detected by this microdevice varied in regard to the size of trapped cells and characteristics examined by immunochemistry, suggesting the heterogeneity of CTCs. Further research on this heterogeneity is vital in order to develop personalized treatment for patients with metastatic breast cancer. Citation Format: Tozuka K, Nagai SE, Inoue K, Komatsu K, Matsumoto H, Hayashi Y, Kurozumi S, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-20.

Abstract P2-04-10: Utility of simultaneous HER2 protein and gene assessment for the evaluation of discrepancy and intratumoral heterogeneity of HER2 status and the prediction of prognosis in invasive breast cancer using the gene-protein assay (GPA)

Background: The eligibility of patients for HER2-targeted therapies is determined by the evaluation of HER2 gene amplification and HER2 protein overexpression. The gene-protein assay (GPA, Ventana Medical Systems, Inc., USA) is a new method for simultaneous evaluation of HER2 immunohistochemistry (IHC) and dual in situ hybridization (DISH) using a single tissue section. In this study, we investigated the relationship between HER2 IHC and DISH results evaluated by GPA. In addition, we analyzed the correlation between HER2 status and prognosis of invasive breast cancer patients. Patients and Methods: In this study, invasive carcinoma tissues of 280 consecutive patients treated in Saitama Cancer Center in 2000-2001 (median follow-up: 130 months) were examined. Among HER2 positive initial samplings, no patients originally received adjuvant trastuzumab therapy. However, 76% of HER2 positive cases of recurrence received trastuzumab therapy. GPA was performed on a section of routinely processed primary tumor and the status of HER gene and protein were separately evaluated in whole areas of tumor sections using the following FDA criteria: DISH (negative: HER2/CEN17 Results: The HER2 IHC 3+ group (27.5%), both with or without trastuzumab therapy, had significantly worse survival than HER2 IHC 1+ & 0 group (RFS: P=0.0039; CSS: P=0.0362) and HER2 DISH+ group (27.5%) had significantly worse survival than HER2 DISH- group (RFS: P=0.0056; CSS: P=0.0497). HER2 positive group defined by FDA criteria had significantly worse RFS than HER2 negative group (P=0.0211). HER2 IHC 1+ & 0/DISH+ group had significantly worse RFS than IHC 1+ & 0/DISH- group (P=0.0208). In the HER2 IHC 1+ & 0/ DISH- group, patients with heterogeneity (33 cases) had significantly worse survival than those without heterogeneity (RFS: P=0.0176; CSS: P=0.0199). Conclusions: HER2 GPA technology might be useful for evaluating the discrepancy and heterogeneity of HER2 IHC and DISH results at single cell levels simultaneously and the presence of HER2 tumor cell heterogeneity might be a potent prognostic factor in HER2 negative breast cancer patients. Further clinical research must be conducted for clarification of the relationship between the presence of HER2 intratumoral heterogeneity and the effectiveness of HER2-targeted therapies. Citation Format: Sasagu Kurozumi, Mary Padilla, Masafumi Kurosumi, Hiroshi Matsumoto, Yuji Hayashi, Katsunori Tozuka, Shigenori E Nagai, Kenichi Inoue, Hanako Oba, Jun Horiguchi, Izumi Takeyoshi, Jim Ranger-Moore, Eslie Dennis, Hiroaki Nitta. Utility of simultaneous HER2 protein and gene assessment for the evaluation of discrepancy and intratumoral heterogeneity of HER2 status and the prediction of prognosis in invasive breast cancer using the gene-protein assay (GPA) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-10.