HER2 Positivity Rate Research Articles

Cancer stem cell markers in locally advanced breast cancer.

1075 Background: the expressions of CD44/CD24, CXCR4 and ABCG2 have been reported as potential breast cancer stem-like cell (CSLC) markers. The association between the quantity of CSLCs and the response to neoadjuvant chemotherapy (NACT) remains unclear. Methods: we prospectively analyzed the expression of CD44/CD24, CXCR4 and ABCG2 in 20 patients with locally advanced or metastatic (LAMBC) invasive ductal carcinomas of the breast subjected to NACT. The mammosphere assay (Mammocult) was studied in 10 samples. Patients’ mean age was 55.6 ± 8.2 yo. According to clinical stage (CS), 5 patients were IIb, 4 - IIIa, 8 - IIIb and 3 - IV. The mean clinical tumor diameter was 6.3 ± 2.8cm. The ER, PgR and HER2 positive expression rates were 50%, 45% and 50%, respectively. Ten patients were treated with EC-T, eight were treated with EC-TH (HER2+) and two were treated with FEC75 combination as NACT. The median percentage CD44+/CD24-, CXCR4+, ABCG2+ and ESA+ cells within Lin- cells were determined by flow cytometry in fresh sampled tumors after tissue digestion. The relationship between flow cytometry analyses and clinical and pathological response to therapy was analyzed. Results: complete clinical response (cCR) and complete pathological response (pCR) was observed in 9 (45%) and 5 (25%) patients. We did not observe a significant association between pCR and ER, PgR or HER2 expression. We observed and association between the pCR with percentage of ABCG2+ cells within the tumor and with the number of mammospheres. No correlation between pCR and CD44+/CD24- cell population within the tumor was observed. The median percentage of ESA+/Lin-/ABCG2+ cells within the tumor in pCR patients was 0.6% and 3.5% in patients with no pCR (p= 0.02). The median number of sphere formation was 5/100 cells and 0.9/1000 cells in pCR and non-pCR patients, respectively (p= 0.02). Interestingly, there was a positive correlation between ABCG2 expression and the number of mammosfere formation (r= 0.66; p= 0.03). This correlation was not significant comparing to CD44+/CD24- cells or CXCR4. Conclusions: the percentage of ABCG2+ cancer cells within the tumor and the number of mammosphere formation are predictive factors for pCR in LAMBC patients subjected to NACT. ABCG2 is a potential marker for CSLCs.

Neoadjuvant chemotherapy in locally advanced breast cancer.

e11034 Background: Neoadjuvant chemotherapy(NAC)is one of the treatment options for locally advanced breast cancer. In this study, we evaluated the efficacy and safety of 4 cycles of NAC doxorubicine,docetaxel and cyclophosphamide(TAC),correlation between the response to NAC and molecular classification sub-groups and between the pCR and the time to progression(TTP). Methods: This is a prospective study from January 2005 to December 2008.110 pts with locally advanced breast cancer stage III.All pts have received 4 cycles of NAC based on docetaxel 75 mg/m², doxorubicine 50 mg/m² and cyclophosphamide 500 mg/m² every 3 weeks, followed by surgery.101 pts were assessed, since 9 of them have progressed on treatment and came out of the study. Pts were stratified according to age, menopausal status, histopathological analysis (luminal tumors(ER-positive and HER2-negative), triple-negative tumors (TN)and HER2-positive tumors), response to the treatment and survival. The median follow up of patients was 39 months. The statistical study was done using SPSS 17. Results: The median age was 41(23–65).30% of pts were younger than 35 and 80% were premenopausal. 55% luminal tumors(56 pts), 33% HER2 positive(33 pts) and 12 % TN(12 pts).CRR was estimated at 89%(37% of CR and 63% of PR).There were 23, 7% of pCR according to Chevallier’s classification. In luminal, TN and HER2-positive pCR rates were 16%(9 of 56), 66,6%(8 of 12), and 21,2%(7 of 33) respectively. Multivariate analysis showed that the ER status was the only significant predictor of pCR(P = 0.025).HER2 status was not significantly associated with pCR(P= 0,423).TTP was 50 months. In luminal tumors, TN and HER2-positive tumors the TTP was respectively 59, 52 and 49 months. There was not a significant difference in TTP between the pCR(51 months) and the non-pCR group(44 months)(CI 95, p= 0.109).Grade III/IV toxicity included neutropenia(22%), febrile neutropenia(6,5%), mucositis(13%), and diarrhea(4%). Conclusions: Breast cancer occurs in young women in Algeria. In this study, neoadjuvant TAC was effective and well tolerated. The ER status was the only significant predictor of pCR. The molecular classification group with the highest percentage of pCR was the TN group. pCR was not associated with a better prognosis.

HER2 status in locally advanced rectal cancer and metachronous metastases: Opportunity for a new therapeutic approach?

3600 Background: Even though the implementation of multimodal treatment strategies including neoadjuvant radiochemotherapy (RCT) has led to improved survival distant metastases are still limiting the prognosis of rectal cancer patients. In this context, we investigated the HER-2 status in rectal cancer patients, UICC stages II and III. Our aim was to assess the HER-2 positivity rate in primary tumors and metachronous metastases. Methods: In this study 264 rectal cancer patients (192 male, 72 female; median age 64 years) from phase-II/-III-trials of the German Rectal Cancer Study Group (CAO/ARO/AIO-94 and 04) were included. HER-2 status was determined pretherapeutically in tumor biopsies as well as resection specimens and metachronous metastases (n=27) using immunohistochemistry (IHC0 to IHC3+) scoring and S-ISH-amplification detection. Tumors with IHC3+ or S-ISH ratio ?2.0 were classified as HER-2 positive; results were correlated with clinicopathological parameters and long-term survival. Results: A positive HER-2 status was found in 12.4% of pre-treatment biopsies, in 29.3% of the resection specimens and 22.2% (n=6) of metastases. With a median follow-up of 46.5 months patients with HER-2-positivity showed better disease free survival (p=0.06) and cancer-specific survival (CSS, p=0.05). The 5-years survival rate was 96.4% (HER-2-positive) versus 79.5% (HER-2-negative). In multivariate analyses HER-2 status was as an independent (p=0.0053) predictor for CSS along with (y)pN-status (p<0.0001) and R-status (p=0.023). Conclusions: HER-2 amplification is detectable in a significant proportion (about 30%) of primary tumors of patients with advanced rectal cancer. Furthermore HER-2 amplification was detectable in 22% of resected metachronous metastases during follow-up. Therefore HER-2 represents a promising target and should be further assessed within prospective clinical trials.

Abstract 685: ALDH1 and hypoxia inducible factors in locally advanced breast cancer patients subjected to neoadjuvant chemotherapy

Abstract The presence of stem-like cells within malignant tumors has been reported as a prognostic factor in patients with solid tumors. Recent studies suggest that hypoxia promotes tumor stemness. This mechanism can be regulated by hypoxia inducible factors such as HIF1A and HIF2A. In breast carcinomas, the aldehyde dehydrogenase 1 (ALDH1) was identified as a potential stem-like cell marker and prognostic factor. However, the association of HIFs and ALDH1 expressions in breast cancer patients subjected to neoadjuvant treatment has not been studied yet. Methods: we retrospectively analyzed the expression of ALDH1, HIF1A and HIF2A proteins in 80 tumors from patients with histologically confirmed stage II and III breast carcinomas treated with neoadjuvant chemotherapy in the Hospital das Clínicas of the University of São Paulo from 2000 to 2005 [stage IIa: 12 patients (14%); stage IIb: 30 patients (35.5%); stage IIIa: 15 patients (17.5%) and stage IIIb: 28 patients (33%)]. The patients’ mean age was 48.9 ± 11.7 years old with a mean follow-up of 100 months. The ER, PgR and HER2 positive expression rates were 63%, 43% and 32%, respectively. The immunohistochemistry for ALDH1, HIF1A and HIF2A was performed in tumors samples before and after neoadjuvant treatment. We analyzed the influence of clinical and pathological features on clinical and pathological response, disease free survival and overall survival. Results: the objective clinical response (partial and complete response) to neoadjuvant therapy was 80% with 12% of complete pathological response. Among all clinical and pathological parameters, only HIF1A expression was associated with pathological response (21% of complete pathological response in HIF1A positive vs 5% in HIF1A negative; p= 0.04). There was a positive association between ALDH1 and HIF1A expression before and after chemotherapy (p= 0.038 and p= 0.03, respectively). The ALDH1 expression was associated with HIF2A expression in tumors after neoadjuvant treatment (p= 0.01). In univariate analysis, the prognostic was influenced by age (p= 0.01), pathological response (p= 0.01), metastasis to axillary lymph nodes after neoadjuvant chemotherapy (p= 0.006), HER2 overexpression (p= 0.03) and by the presence of ALDH1+ cells within the primary tumor after neoadjuvant chemotherapy (p= 0.01). In multivariate analysis, only the presence of ALDH1+ cells after chemotherapy and age were associated with reduced overall survival (p= 0.04 and p= 0.01, respectively). Conclusion: the presence of ALDH1 positive cells within the residual tumor after neoadjuvant chemotherapy is associated with and increase in HIF2A expression and poor prognostic in patients with locally advanced breast cancer. There is a association between ALDH1 expression in tumor cells and the expression of HIF1A in locally advanced breast carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 685. doi:1538-7445.AM2012-685

The impact of HER2 positivity on survival in metastatic gastric and GEJ cancer.

17 Background: HER2 positive (+) gastric (GC) and GEJ adenocarcinoma (AC) has been associated with a worse survival outcome. This study examines the survival rate of patients (pts) with metastatic HER2 (+) GC and GEJ AC in the province of BC. Methods: Formalin fixed embedded tissue from pts with resected GC or GEJ AC from 2004−2011 were identified retrospectively through the BC Cancer Agency registry and prospectively for pts with a new diagnosis of advanced disease. Biopsies and resection samples were analyzed via previously validated methods. IHC scores of 3 were considered (+), 2 were equivocal and 0/1 were negative (−). 10% cut-off was used to determine (+) samples. Equivocal staining was considered (+) via FISH or SISH with a ratio of > 2.2 considered amplified. Pt characteristics were abstracted to an anonymized database. Kaplan meier curves were calculated to evaluate overall survival from time of diagnosis or relapse to death for pts with Her 2 (+) or (-) disease. Results: 164 of 294 pts were identified with metastatic or relapsed GC (49%) or GEJ (51%) AC without traztumumab treatment. 21 (13%) of pts were HER2 (+) either by IHC, FISH or SISH. See Table for pt characteristics. The HER2 (+) group was older (median age 68 yrs vs 60), more GEJ (62% vs 49% p=1.23), and a higher number of pts that did not receive treatment (52% vs. 27% p=5.09). Median survival was not significantly different among HER2 (+) and (−) disease (4.0 mo vs. 7.3 mo, p=0.089). Conclusions: The rate of HER2 positivity is similar to that seen in trial data. HER2 positivity trends to a worse prognosis but was not significant in this study. [Table: see text]

Researchers find discordance between standard human epidermal growth factor receptor 2 (HER2) testing and HER2 status reported on Oncotype DX

Researchers find discordance between standard human epidermal growth factor receptor 2 (HER2) testing and HER2 status reported on Onco<i>type</i> DX

Increased concentrations of growth factors and activation of the EGFR system in breast cancer

In this study the total and phosphorylated amount of epidermal growth factor receptor 1 (EGFR) and 2 (HER2) were measured together with EGFR ligands in tissue samples of breast cancer patients in order to investigate interrelations and possible prognostic values. Samples of malignant and non-cancer autologous reference tissue were collected from 415 breast cancer patients. The tissue samples were cut and either paraffin-embedded or homogenized in a lysis buffer to extract the proteins. HER2 was measured using both immunohistochemistry (IHC)/fluorescence in situ hybridization (FISH) and ADVIA Centaur. Phosphorylated HER2 and EGFR (pHER2, pEGFR), total EGFR and the ligands: epidermal growth factor (EGF), transforming growth factor-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC) and epiregulin (EREG) were measured using the Luminex. The HER2 positivity rate was determined to be 25.2% by the Centaur method vs. 15.8% by IHC and FISH. HER2, HB-EGF, TGFα and AREG were upregulated in cancer tissue as compared with autologous reference tissue while EGFR, pEGFR and EGF were downregulated (p<10-6). pEGFR in autologous reference tissue was negatively correlated to the number of positive lymph nodes and to the tumor size (p=0.0007 and p=0.001, respectively) and furthermore, decreased in the group of mastectomy operated patients as compared with the lumpectomy group (p<10-6). HB-EGF in cancer tissue was positively associated with high grade tumors (p<10-6) and pHER2, HB-EGF and BTC were associated with poor disease free survival (p=0.017, p=0.012 and p=0.0026, respectively). Our study demonstrated a profound activation of the EGFR system. HB-EGF was increased by factor 10 in cancer tissue and related to the biological aggressiveness of the tumors, and pHER2, HB-EGF and BTC were associated with poor clinical outcome.

HER-2/neu Assessment for Gastric Carcinoma: Validation of Scoring System

Gastric cancer is the second leading cause of cancer mortality in the world. Amplification of HER-2/neu oncogene has become an important biomarker for identifying patients who respond to HER-2 targeting therapy. A number of studies have analyzed HER-2/neu overexpression in gastric carcinoma, and the rate of HER2 positivity is variable, ranging from 6% to 35%. In our study HER-2/neu expression was assessed on 73 samples of primary gastric cancer, using immunohistochemistry. For 19 patients preoperative biopsy samples and resected specimens were available. Additionally, internal ring study was performed to estimate intraobserver variability of IHC scoring among pathologists at our department. HER-2/neu overexpression was found in 10 (13.6%) of the tested samples, and it was more common in intestinal (22.5%) than the diffuse type (3.7%). Not one of the 6 analyzed mixed type tumors showed HER-2/neu expression. For the paired samples (preoperative biopsy samples and resected specimens) the concordance rate for HER-2/neu expression was 94.7%. According to high concordance rate in paired samples we consider it appropriate to evaluate HER2 expression on biopsy specimens, especially in unresectable cases, and to re-evaluate it on resected specimens if available, due to high heterogeneity of a gastric cancer.

Serum HER2 levels determined by two methods in patients with metastatic breast cancer

The role and the optimal measurement method of serum HER2 levels are not defined in patients with metastatic breast cancer (MBC). We prospectively assessed the prognostic value of serum HER2 levels in MBC using two methods, enzyme immunoassay (EIA) and chemiluminescence immunoassay (CLIA). We collected blood samples from patients with MBC at baseline and at subsequent 3- to 4-week intervals up to 12 weeks. Samples were divided, and serum HER2 levels were determined using EIA and CLIA. We also determined whether serum HER2 levels had decreased by ≥20% at first follow-up. These results were evaluated against overall survival, progression-free survival, and tumor response. We obtained 196 samples from 52 patients. In 59 samples from patients who received trastuzumab, serum HER2 positivity rates were significantly lower for EIA (n = 22) than for CLIA (n = 33, P = 0.042); in 137 samples from patients who did not receive trastuzumab, there was no significant difference in rates of serum HER2 positivity for CLIA (n = 83) and EIA (n = 80). Serum HER2 level at baseline, the level at first follow-up, and a decrease of ≥20% between baseline and first follow-up were not associated with overall survival, progression-free survival, and tumor response. Chemiluminescence immunoassay was a more sensitive method than EIA for measuring serum HER2 levels in patients who received trastuzumab. However, because serum HER2 levels did not correlate with patient outcome, we do not currently recommend measuring serum HER2 levels by either method for prognostic evaluation in patients with MBC.

Human epidermal growth factor receptor 2 (HER2) positivity shown by fluorescence in-situ hybridization in patients with advanced gastric cancer: A multicenter study in Taiwan.

e14576 Background: Based on the results of ToGA (Trastuzumab for Gastric Cancer) study, trastuzumab in combination with chemotherapy can be considered as a new standard option for patients with human epidermal growth factor receptor 2 (HER2)-positive advanced gastric or gastro-esophageal junction (GEJ) cancer (Lancet 2010;376:687-97). In ToGA study, overall HER2-positivity rate was 22.1%; however, exceptionally low HER2-positivity rate was observed in some countries, including Taiwan (only 3%; ASCO-GI 2008:A11). The underlying factors contributing to low HER2-positivity rate in Taiwan remain obscured. Re-evaluation of HER2-positivity for Taiwanese GC patients in a larger sample size is highly warranted. Methods: We tested HER2-positivity by gene amplification using fluorescence in-situ hybridization (FISH) in formalin-fixed paraffin-embedded gastric or GEJ cancer samples. The testing was centralized to a single laboratory (Quest, UK). The PathVysion HER2 DNA probe Kit (Vysis, Abbott Molecular Inc., Germany) was used, which is FDA approved to detect the HER2 gene amplification. Results of hybridization on enumeration of 20 interphase nuclei from tumor cells are reported as the ratio of average HER2 copy number to that of CEP17, a specific region of the Chromosome 17’s centromere. Specimens with amplification showed a HER2 and CEP17 signal ratio of >/= 2.0. If the final ratio is calculated by the primary observer as near/at the cut-off point (1.8-2.2), an additional 20 cells is counted. The second observer will count an additional 20 cells. Results: Between Sep. 2007 and Nov. 2010, 180 patients (M:109, F:71) with gastric or GEJ adenocarcinoma from 8 medical centers in Taiwan were enrolled and evaluated by FISH testing. Among them, 47 patients (M:34, F:13) showed a HER2/CEP17 signal ratio of >/= 2.0, the overall HER2-positivity for Taiwanese GC patients was 26.1% (19.7%-32.5%, 95% CI). Conclusions: More than a quarter of Taiwanese advanced GC patients showed HER2-positivity by FISH, comparable with that of ToGA study. Novel HER2-targeted agents are justified for future active investigation in Taiwanese GC patients.

Therapeutic response to induction chemotherapy and prognosis of patients with inflammatory breast cancer in respect to HER2 and hormonal status.

e11517 Background: IBC presents the most aggressive form of BC with high incidence of HER2 positivity (+) and low rate of complete remission to induction chemotherapy (iCT). It is initially inopera...

Expression of human epidermal growth factor receptor 2 in gastroesophageal cancer with different scoring systems and its relationship with clinicopathological features

To compare the expression of human epidermal growth factor receptor 2 (HER2) under different scoring systems in gastroesophageal cancer and its relationship with clinicopathological features. Clinicopathological data were retrospectively reviewed of 127 patients with gastroesophageal cancer between January and December 2009 in the Department of Surgery at the Cancer Hospital of Peking University. Tumor specimens were collected. The expression of HER2 protein was detected by immunohistochemistry. HER2 positive rate(++/+++) in gastroesophageal cancer was 15.0%(19/127) with both new and traditional immunohistochemical scoring systems, while HER2 strong positive rate(+++) was 10.2%(13/127) and 6.3%(8/127), respectively(P=0.26). Univariate analysis showed that the expression rate of HER2 protein was associated with differentiation and Lauren classification of the tumor. Multivariable analysis showed that TNM staging and tumor differentiation were independently associated with the expression of HER2 protein(both P<0.05). There is no change in HER2 positive rate in gastroesophageal cancer between the new and traditional immunohistochemical scoring systems. Expression of HER2 is associated with clinicopathological features in the gastroesophageal cancer.

Incidence and distribution of HER2-positive gastric and gastroesophageal junction (GEJ) adenocarinomas in British Columbia (BC).

91 Background: HER2-positive rates have been reported as 20% of GC and 34% in GEJ adenocarcinoma in a recent global phase III trial with trastuzumab and chemotherapy. This study examines the incidence and distribution HER2-positive GC and GEJ adenocarcinomas in the province of BC. Methods: Formalin fixed embedded tissue from patients (pts) with resected gastric or GEJ adenocarcinoma from 2004-2007 were identified retrospectively through the BC Cancer Agency registry and prospectively for pts with a new diagnosis of advanced disease. Biopsies and resection samples were analyzed via previously validated methods IHC (Ventana 4B5 antibody), FISH (Eurovision probes) and SISH (Ventana probes). IHC scores of 3 were considered positive, 2 were cequivocal and 0 or 1 were negative. A 10% cut-off was used to determine positive samples. Equivocal staining was considered positive via FISH or SISH. A ratio of &gt; 2.0 was considered amplified for FISH and SISH. P values were calculated using a logistic regression model with HER2 positive as the endpoint. Results: Of the 87 samples, 64 (74%) were gastric and 23 (26%) GEJ. Overall, HER2 was positive 20% (IHC), 18% (FISH) and 18% (SISH). 13% of cases tested IHC 3+ while 24% tested IHC 2+. In equivocal IHC 2+ cases, 7% were considered positive by FISH. Positivity rates were higher for GEJ (26%, 22% and 17%) vs. gastric (8%, 11% and 9%) via IHC (p = 0.02), but did not significantly differ by FISH (p = 0.19), or SISH (p = 0.30) respectively. The majority of positive cases were intestinal type vs. other (diffuse type or mixed) 25% vs. 0% IHC p = 0.0002, 25% vs. 3% FISH p = 0.001, 20% vs. 3% SISH p = 0.006. The positivity rates were similar with biopsy vs resection specimens (12% vs. 16% IHC p = 0.64, 12% vs. 21% FISH p = 0.30, 12% vs. 10%, SISH p = 0.88). Conclusions: The rates of HER2-positive disease in a Western population based study were similar to the ToGA study. HER 2 positivity was associated with cancers arising in the GEJ and of intestinal type. The frequency of positivity was similar for testing by biopsy versus resection specimens. This study is ongoing and updated results will be reported. No significant financial relationships to disclose.

Adjuvant S‐1 Chemotherapy for Gastric Cancer and Peritoneal Wash

We appreciate the interest of Drs. Spiliotis and Zoras [1] in our article and their knowledge and faith in ACT-GC, the Japanese randomized trial that explored S-1 as a postoperative adjuvant chemotherapy for gastric cancer [2], despite the fact that the drug is unavailable in Europe. Their remark that a definitive conclusion about the efficacy of S-1 monotherapy for CEA mRNA-positive patients cannot be reached from such a small-scale study is quite a relevant observation. Actually, in clinical practice, we treat stage II/III gastric cancer patients using S-1 monotherapy regardless of the CEA mRNA status. Our study was started when the ACTS-GC was still ongoing. Phase III trials for postoperative chemotherapy had a long history of negative studies in Japan [3, 4] and elsewhere. Reasons for these failures could be the lack of effective cytotoxic agents, inclusion of an excessive number of patients who had no residual disease and therefore had been cured by surgery [3], and insufficient power in the trial design [4]. One reason for our conducting the RT-PCR trial was to find the potential danger of the ACTS-GC failing to show a benefit of adjuvant chemotherapy and join the long list of negative trials. Since RT-PCR could identify patients with residual disease with more sensitivity than the conventional staging scheme, our intention in that event was to urge our colleagues to perform RT-PCR for all stage II/III patients and prescribe S-1 for the CEA mRNA (?) population. ATCS-GC turned out to be a success while our phase II failed to prove our hypothesis. Ironically, our study demonstrated that the rate of peritoneal relapse among CEA mRNA-positive patients is much higher than in the ACTS-GC study. The main relevance of our article is that detection of CEA mRNA remains useful for identifying patients at high risk of peritoneal relapse at a time when S-1 therapy has become standard. The opinion of Spiliotis and Zoras that the combination of radiotherapy with S-1 as the next crucial step does not apply in Japan where local control has been achieved by lymph node dissection [2, 5]. The efficacy of trastuzumab for treating unresectable gastric cancer has been confirmed by the ToGA trial [6] and its use as an adjuvant therapy appears promising. However, lessons learned from colorectal cancer do not guarantee that molecular targeting agents that are effective in the advanced setting will function similarly in the adjuvant setting. In addition, the rate of HER2 positivity is about 20% in stomach cancer and is higher in intestinal-type cancer than in diffuse-type cancer, which commonly disseminates intraperitoneally [6]. Therefore, trastuzumab might not contribute greatly to the prevention of peritoneal dissemination. Peritoneal carcinomatosis represents the most common route of tumor dissemination in gastric cancer patients and the prevention of peritoneal carcinomatosis is a crucial part of the therapeutic strategy. There is a promising treatment option of intraperitoneal chemotherapy that will soon develop into a clinical trial [7]. At the same time, intraperitoneal chemotherapy is a technically demanding procedure compared to systemic chemotherapy, given the need for catheter placement and management of particular kinds of adverse events. Thus, we hope that our genetic detection of peritoneal micrometastasis will prove helpful in facilitating the proper selection of patients who are likely to benefit from intraperitoneal chemotherapy. S. Ito (&) Department of Gastroenterological Surgery, Aichi Cancer Center, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan e-mail: seito@aichi-cc.jp

HER2 status of circulating tumor cells in patients with metastatic breast cancer: a prospective, multicenter trial

There is a growing body of evidence that HER2 status can change during disease recurrence or progression in breast cancer patients. In this context, re-evaluation of HER2 status by assessment of HER2 expression on circulating tumor cells (CTCs) is a strategy with potential clinical application. The aim of this trial was to determine the HER2 status of CTCs in metastatic breast cancer patients comparing two CTC assays. A total of 254 patients with metastatic breast cancer from nine German university breast cancer centers were enrolled in this prospective study. HER2 status of CTCs was assessed using both the FDA-approved CellSearch® assay and AdnaTest BreastCancer™. Using the CellSearch assay, 122 of 245 (50%) patients had ≥5 CTCs, and HER2-positive CTCs were observed in 50 (41%) of these patients. Ninety of 229 (39%) patients were CTC positive using AdnaTest BreastCancer, and HER2 positivity rate was 47% (42 of 90). The rate of breast cancer patients with HER2-negative primary tumors but HER2-positive CTCs was 32% (25 of 78) and 49% (28 of 57) using the CellSearch assay and AdnaTest BreastCancer, respectively. Considering only those patients who had CTCs on both tests (n = 62), concordant results regarding HER2 positivity were obtained in 50% of the patients (31/62) (P = 0.96, κ = -0.006). HER2-positive CTCs can be detected in a relevant number of patients with HER2 negative primary tumors. Therefore, it will be mandatory to correlate the assay-dependent HER2 status of CTCs to the clinical response on HER2-targeted therapies.

Confirmation of a low HER2 positivity rate of breast carcinomas - limitations of immunohistochemistry and in situ hybridization

BackgroundAccurate assessment of the human epidermal growth factor receptor 2 (HER2) of invasive breast cancer is essential to treatment decisions since the advent of targeted therapy with the humanized monoclonal antibody trastuzumab and the dual tyrosine kinase inhibitor lapatinib. In the literature, the percentage of HER2-overexpressed/amplified breast carcinomas range from 3% to 30%. The routinely assigned low rate of 9% of HER2-overexpressed breast carcinomas alarmed one of our gynecologists who requested to confirm our HER2 test results.MethodsA small study of 83 patients with breast carcinoma was designed to reexamine the routinely assessed HER2 status using immunohistochemistry and fluorescence in situ hybridization.ResultsThe low rate of 9% of HER2-overexpressed/amplified breast tumors (DIN1C-3, invasive carcinoma) could be confirmed. However, FISH revealed two false positive cases and one false negative case. Moreover a case with an equivocal result in FISH was detected.ConclusionThe HER2 positivity rate may be as low as 9%. The novel ASCO/CAP criteria for assessing immunohistochemical results in HER 2 testing reduce the false positive rate of HER2. First-line testing with immunohistochemistry may obscure false positive and false negative test results. In heterogeneous carcinomas even fluorescence in situ hybridization may not succeed in a correct evaluation of HER2.

Abstract 3350: Cancer stem cells and lymph node metastasis in early stage breast cancer

Abstract Breast cancer is the leading cause of malignancy in women worldwide. Metastatic dissemination is the major cause of death. The mechanisms involved in cancer dissemination are unclear. The identification of tumor initiating cells in solid tumors has been reported. Initiating cells, also referred as cancer stem cells (CSCs), were defined as a CD44+/CD24— in breast carcinomas and have the ability to self-renewal, differentiate into any cell in the tumor and metastasize. The presence of CSCs is a suitable hypothesis for breast cancer metastasis and treatment failure. Nevertheless, there is no evidence of the relationship between CSCs in primary tumor and metastasis. Methods: we prospectively included 20 early-stage (I and II) breast cancer patients (17 invasive ductal carcinoma, 1 invasive lobular carcinoma, 1 metaplastic carcinoma and 1 tubular-lobular invasive carcinoma), and two patients with ductal carcinoma in situ (DCIS) subjected to primary surgery. The proportion of CSCs was analyzed in 22 primary tumors and in three lymph node metastasis by flow cytometry. Anti-human CD326 (EpCam; FITC), anti-human CD227 (MUC-1; FITC), anti-human CD44 (APC) and anti-human CD24 (PE) were used for phenotype analysis. The percentage of CD44+/CD24- cell population in primary invasive tumors was compared with standard prognostic factors. The mean age was 58.3 years (37 - 77), the mean pT was 1.8 cm (0.5 - 3.5), 13 patients (59%) had no axillary lymph node metastasis (NLN group) and 9 had positive axillary metastasis (PLN group) confirmed for standard histological evaluation. The ER, PR and HER2 positive expression rates were 84.2%, 78.9% and 15.7%, respectively. The Wilcoxon or Kruskal-Wallis tests were used for statistical analyses. Results: the percentage of CSCs in primary invasive ductal carcinoma (IDC) was 7.1% (0.2 - 29.4). In two DCIS we found 4.5% and 8.2% of CSCs. In lymph node metastases we observed a mean of 11.9% (1.5 - 31.1). In the other subtypes the mean percentage of CSCs was 12.7% (1.1 - 31.1). There was no association between CSC population in IDC and age (p= 0.8), pT (p= 0.7), tumor grade (p= 0.4), ER (p= 0.1), PR (p= 0.3) and HER2 (p= 0.4). We observed a significant increase in CSC population in PLN group [mean= 12.4% (4.1% - 29.4%)] when compared to patients without lymph node metastasis [mean= 2.4% (0.2% - 4.6%0] (p= 0.0008). Conclusion: this preliminary study indicates that the metastatic dissemination is associated with an increase in CSC population in early-stage breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3350.

Detect-Study: Comparison of Two Techniques for the Evaluation of the HER2 Expression on Circulating Tumor Cells in Metastatic Breast Cancer.

Abstract Introduction:There is a growing body of evidence that the HER2 status can change during disease progression (Gancberg et al. 2003). However, the re-assessment of the HER2 status to evaluate the eligibility for HER2 targeted therapy by a tissue biopsy of the metastatic lesion may not always be feasible. In this context, determination of the HER2 status using CTCs might be a strategy with potential clinical application. So far, no large prospective studies were performed comparing different methods for CTCs detection and characterization. The aim of this trial was to determine the HER2 status of CTCs in metastatic breast cancer patients using the antibody based CellSearch©assay and the RT-PCR approach AdnaTest Breast Cancer and to assess the concordance rate between these two techniques. Both techniques are frequently used in clinical trials.Method:A total of 252 metastatic breast cancer patients from 9 different certified breast cancer centers (Düsseldorf, Erlangen, Essen, Freiburg, Hamburg, Heidelberg, Muenchen, Regensburg, Tuebingen) were enrolled in this prospective open trial from 01/2008 until 04/2009. HER2 status of CTCs was assessed using both the FDA approved CellSearch©Assay (Veridex, Warren, NJ, USA) and the AdnaTest Breast Cancer (Adnagen, Langenhagen, Germany) according to the manufacturers' instructions. A web based databank was designed for data management and online-documentation (www. detect-study.de). The study design was approved by the ethics' comittee (2007/B01).Results:Using the CellSearch©Assay, 133 of 252 (55%) metastatic patients had CTCs at the time of first diagnosis or disease progression. HER2 positivity was observed in 39% of CTC positive patients (52 of 133) but only 22 of these had initially a HER2 positive primary tumor. Eighty-eight of 225 (39%) patients were CTC positive using the AdnaTest Breast Cancer. The HER2 positivity rate was 47% (41 of 88). Eight of these patients had a HER2 positive primary tumor whereas 33 patients were initially HER2 negative or unknown. Summarizing the data, the rate of breast cancer patients with HER2 negative primary tumors but HER2 positive circulating tumor cells was 38% (32 of 61) and 48% (28 of 41) using the CellSearch Assay and the AdnaTest Breast Cancer, respectively. Considering only those patients who had CTCs on both tests (n=61), the concordance rate regarding HER2 expression was 51% (32 of 61).Conclusion:Based on CTCs the HER2 status can change during disease progression. However, using different techniques, disconcordant results were obtained. To prove the clinical value of these CTC assays and to implement the HER2 status of CTCs as a stratification parameter in future clinical trials, it will be mandatory to correlate clinical response to HER2 targetd therapy on each method in patients with HER2 positive CTCs. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3004.

Poor Survival Outcomes in Elderly HER2 Positive Breast Cancer Patients.

Abstract IntroductionIncreasing age is a major risk factor for breast cancer. The majority of breast cancers in Europe and USA are in women over 65 and with an aging population this will become an increasing concern. Despite this, there are suggestions of bias in allocating treatment based on chronological rather than physiological age, with adverse effects on survival (1). In addition, women over 70 are usually excluded from randomised clinical trials. One particular area of controversy is the treatment of HER2 positive elderly women with trastuzumab +/- concurrent chemotherapy, particularly with the concerns over cardiac toxicity in these patients.MethodsWe analysed a cohort of 255 patients older than 70 years to assess the impact of HER2 positivity (IHC Herceptest 3+ or FISH positive) on survival in elderly women with breast cancer. These patients were selected from a database of 1400 breast cancers diagnosed between 1980-2002 which contains all clinicopathological details and full follow-up (median 5.3yrs). The group were 81% ER positive, 74% grade I or II, and 49% node positive. Only 2.7% received chemotherapy but 82% received endocrine therapy (tamoxifen).SPSS version 12 was used to calculate univariate Kaplan Meier Survival Curves and multivariate cox regression analysis using breast cancer specific death as an endpoint.ResultsThe HER2 positivity rate was 5.9% which is substantially lower than the 11.9% positive in the original unselected database of 1400 patients.HER2 positive elderly patients (n=15) had significantly poorer breast cancer specific survival rates of 65% compared to 79% at 5 years for the HER2 negative group (p=0.036). In cox regression analysis along side known prognostic variables of grade, size, nodal and ER status the hazard ratio for HER2 positivity was 2.58 (95% CI 1.079-6.147, p&amp;lt;0.033).ConclusionAs previously reported, our cohort of elderly women have low rates of HER2 positive disease, however HER2 status remains a significant predictor of breast cancer specific survival outcome. Whilst no adjuvant trastuzumab trials have directly targeted this age group (with minimal inclusion of patients &amp;gt;70 in any trial), we know that cardiac toxicity increases with age and with anthracycline use (2). This however is generally reversible and treatment certainly should be considered if there is adequate cardiac function. A clinical trial to assess the benefit of adjuvant trastuzumab alone (with endocrine therapy) in this group of patients is suggested in this largely ER positive cohort. The role of the dual inhibitor lapatinib should also be explored with suggestions of less cardiotoxicity with this agent (2).Reference List1. Eaker S, Dickman PW, Bergkvist L, Holmberg L 2006 Differences in management of older women influence breast cancer survival: results from a population-based database in Sweden. PLoS Med 3:e252. Perez EA 2008 Cardiac toxicity of ErbB2-targeted therapies: what do we know? Clin Breast Cancer 8 Suppl 3:S114-S120 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2010.

Assessment of Human Epidermal Growth Factor Receptor 2 (HER2) Amplification by In Situ Hybridization (ISH): Findings from a Nationwide Program in Australia.

Abstract Background: The HER2 gene is amplified in 18–20% of breast cancers. Trastuzumab (Herceptin®), an anti-HER2 monoclonal antibody, has survival benefits in HER2-positive breast cancer in both the metastatic and adjuvant settings. Evaluating HER2 status is, therefore, recommended in all primary breast cancers. The Australian In Situ Hybridization Program is the first nationwide program using ISH as the first-line testing platform. It was launched as a multicenter coordinated project to provide ISH testing for all primary breast cancers across Australia and to regulate HER2 testing.Methods: The following criteria were required for including a laboratory (lab) in the program: ≥50 ISH tests per pathologist, ≥150 per lab annually; training for pathologists and technicians; accreditation and certification of pathologists, technicians, and lab as per the Royal College of Pathologists of Australasia and National Association of Testing Authorities requirements; reporting time of 7 working days. A validated single-probe ISH test was used, with a chromosome 17 probe used for equivocal and possible polysomic cases. Cases that remained equivocal after dual-probe testing were tested by fluorescence ISH at a central lab. From March 2008, ISH scoring adhered to the 2007 ASCO/CAP recommendations. Only patients with ISH-positive primary tumors are eligible for adjuvant trastuzumab under the Australian government-funded Pharmaceutical Benefits Scheme.Results: 22 labs are approved for ISH testing: 10 use chromogenic ISH (CISH), 12 use silver ISH (SISH). Between October 2006 and September 2008, 20,406 ISH tests were reported: 8348 in the first 12 months (7835 primary, 513 metastatic) and 12,058 in the subsequent 12 months (11,594 primary, 464 metastatic). The HER2-positivity rate decreased in the second 12 months for primary (23.8% to 16.9%) but increased for metastatic (22.6% to 25.4%) cases. The number of core biopsies tested remained consistently low (3.7% and 4.5%), with testing performed primarily on excised tumor. Average ISH reporting time reduced slightly (4.9 to 4.7 days). Test repeat rates decreased for CISH labs (8.9% to 8.2%) but were higher for SISH labs (13.7%) because of a global issue of silver wash contamination that was subsequently addressed and resolved. Immunohistochemistry (IHC) was performed on 75% of samples and was used to allow evaluation of problematic or equivocal cases, or referrals from labs in the start-up phase of testing. Given this bias, the proportion of IHC 3+ samples amplified by ISH was relatively low but increased from 80.2% to 84.4% in the second 12 months.Discussion: The reduced HER2-positivity rate for primary cases, reporting times, and repeat rates (CISH labs only) reflects the increased number of ISH tests performed, increased lab experience, and improvements in testing accuracy. These data reflect the successful implementation of a regulated, nationwide testing program that continues to collect data on HER2 testing in patients with breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5100.