Metastatic Breast Cancer Lesions Research Articles

The Impact That Number of Analyzed Metastatic Breast Cancer Lesions Has on Response Assessment by 18F-FDG PET/CT Using PERCIST.

The PET Response Criteria in Solid Tumors (PERCIST) are not specific regarding the number of lesions that should be analyzed per patient. This study evaluated how the number of analyzed lesions affects response assessment in metastatic breast cancer. In 60 patients, response was assessed by the change in SUVpeak, normalized to lean body mass, of the most (18)F-FDG-avid lesion (PERCIST 1) and by the change in the sum of normalized SUVpeak for up to 5 lesions (PERCIST 5). The correlation between response by PERCIST and progression-free and disease-specific survival was evaluated. In responders and nonresponders, the respective progression-free survival at 2 y was 37.26% and 6.43% for PERCIST 1 (P < 0.0001) and 33.65% and 7.14% for PERCIST 5 (P < 0.0001) and the respective disease-specific survival at 4 y was 58.96% and 25.44% for PERCIST 1 (P < 0.012) and 59.12% vs 20.01% for PERCIST 5 (P < 0.002). The number of analyzed lesions does not appear to have a major impact on the prognostic value of response assessment with (18)F-FDG PET/CT in metastatic breast cancer.

Highlights of This Issue

Biopsy of metastatic breast cancer (MBC) lesions are recommended to confirm the diagnosis and re-evaluate hormone receptors (HR) and HER2 status. Circulating tumor cells (CTC) might serve as “liquid biopsy,” which could give insight into cancer heterogeneity. Paoletti and colleagues explored the feasibility of performing different markers related to endocrine therapy sensitivity/resistance using the CellSearch assay. They developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which is based on CTC enumeration and a highly validated semiquantitative analysis of CTC-ER, BCL-2, HER2, and Ki67 expression. CTC-ETI may serve as a real-time predictive factor for resistance to ET in patients with HR-positive MBC.In HER2 (+) gastric cancer (GC), although trastuzumab-based chemotherapy is the standard of care, no biomarker for patient selection has been identified. To define the optimal patients who benefit from trastuzumab, Ock and colleagues analyzed the association of HER2/CEP17 ratio and HER2 gene copy number and overall survival (OS) using training set and validation set. In all HER2 (+) GC, patients with HER2/CEP17 ratio ≥ 4.48 showed the better OS. Especially, in patients with HER2 IHC ≤ 2+, HER2/CEP17 ratio ≥ 3.69 conferred the better OS. These findings are helpful for optimal patient selection for trastuzumab in HER2(+) GC.Preconditioning melanoma patients with total body irradiation (TBI) before adoptive transfer of T cells increases objective and curative responses. Bowers and colleagues report the novel mechanism of TBI-induced IL12 secretion by activated dendritic cells, which significantly enhanced the antitumor response of infused Tc17 cells. Further, adding IL12 to Tc17 cultures ex vivo before transfer mimicked effects of TBI by enhancing their function and engraftment in the tumor. Administering low dose IL12 to the mice along with IL12-primed Tc17 cells abrogated the need for TBI — highlighting an alternative to TBI that effectively enhances adoptive immunotherapy for metastatic disease.To unearth the comprehensive genomic landscape of lung adenocarcinoma (LA), Choi and colleagues performed whole exome sequencing for 247 LA patients and discovered 16 novel driver candidates, including COL11A1, CENPF, and SLIT2. They also found that genomic alternations to the RB pathway are strongly linked to a shorter survival outcome for early-stage LA patients. Although the current treatment guideline for early-stage LA patients is composed mainly of follow-up without adjuvant therapy after resection, the current study raises an interesting possibility that additional clinical interventions might provide benefits for early-stage LA patients who have genomic alterations to the RB pathway.

Abstract P5-01-06: Characterization of metastatic breast cancer lesions with ferumoxytol MRI and treatment response to MM-398, nanoliposomal irinotecan (nal-IRI)

Abstract Introduction Irinotecan has known activity in metastatic breast cancer (MBC). MM-398, nanoliposomal irinotecan (nal-IRI), is designed to exploit leaky tumor vasculature for enhanced drug delivery to tumors. Tumor deposition of nal-IRI and subsequent conversion to SN-38 in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with activity. Predictive biomarkers to measure tumor deposition could identify patients likely to benefit from nal-IRI. Ferumoxytol (FMX), an iron-oxide superparamagnetic nanoparticle with MRI contrast properties, is taken up by TAMs with similar distribution patterns to nal-IRI in preclinical models. Our previous work has shown the feasibility of quantitative FMX MRI (Fe-MRI) of tumor lesions, and we developed a quantitative mechanistic PK model of FMX deposition (AACR 2014, abstract #CT224). Here we report nal-IRI activity and FMX levels in MBC patients on the study. Patients and methods Patients (n=15) with refractory solid tumors and at least two metastatic lesions &amp;gt;2 cm accessible for percutaneous biopsy were enrolled in a Phase 1 study. Fe-MRI scans were performed using T2* iron sensitive sequences prior to and following FMX infusion (1 h, 24 h, 72 h). T2* signal was used to calculate FMX levels in total lesions by comparison to a standard curve. Comparison of quantified FMX lesion uptake with a mechanistic PK model previously indicated that tissue permeability to FMX contributed to early Fe-MRI signals at 1 h and 24 h, while FMX binding contributed at 72 h. Patients then received nal-IRI (80 mg/m2 q2w) until progression. Core biopsies were obtained 72 h after both FMX and nal-IRI infusions. RECIST evaluation was done by CT every 8 weeks. Results FMX was well tolerated, and adverse events to nal-IRI were consistent with previous studies. Three of the 13 patients receiving nal-IRI had ER/PR+ MBC (median # of prior Rx: 8 compared to 4 for all study patients). Thirteen liver lesions (4-5/pt) were evaluated by FMX-MRI and CT for these 3 patients. Average lesion size: 26.9±11.2 mm diameter and 8.1±11.3 cm3 (median 4.7 cm3). Time on treatment for the 3 patients was 57, 126 and 256 days (study median 57 days). Best overall response was 1 stable disease (SD) and 1 partial response (PR) in these 3 patients. The patient with a PR had an average lesion size reduction of 44.5%, while the patient with SD had an average lesion size increase of 12.5% at final evaluation. Lesions that shrank after nal-IRI showed higher early levels of FMX compared to the study median (median 39.6 vs. 32.6 mcg/mL at 1 h; median 37.7 vs. 34.5 mcg/mL at 24 h). This relationship between lesion response and FMX levels was consistent with the lesion behavior in the full data set (n=31 lesions/9 patients across 7 indications) of the study. Conclusions Clinical activity of nal-IRI was observed in a subset of heavily treated ER/PR+ MBC patients. The relationship between FMX levels in tumor lesions and nal-IRI activity suggests that lesion permeability to FMX may be a useful biomarker for nal-IRI deposition and tumor response in MBC and potentially other indications. A multi-institution expansion of this study in HER2-negative MBC is planned to confirm these findings. Citation Format: Jasgit C Sachdev, Ramesh K Ramanathan, Natarajan Raghunand, Jaeyeon Kim, Stephan G Klinz, Eliel Bayever, Jonathan B Fitzgerald, Ronald L Korn. Characterization of metastatic breast cancer lesions with ferumoxytol MRI and treatment response to MM-398, nanoliposomal irinotecan (nal-IRI) [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 P5-01-06.

Receptor Expression Discrepancy between Primary and Metastatic Breast Cancer Lesions

SummaryBackground: Recent studies have shown that the expression status of hormone receptors and human epidermal growth factor receptor 2 (HER2) in breast cancer may change during disease progression. The aim of this study was to determine and compare the estrogen receptor (ER), progesterone receptor (PR), and HER2 expression status in primary breast cancer and metastatic lesions. Methods: 58 patients with registered biopsy reports or available samples of the primary tumor and distant metastases were included in the final analysis. Biopsy samples were re-stained using immunohistochemical methods to determine receptor status (if not already recorded in previous reports) and re-examined by 2 independent pathologists. Results: Discordance rates for receptor expression status of the primary tumor and distant metastases for ER, PR, and HER2 were 17.4, 45.4, and 13.3%, respectively. No statistically significant difference in overall survival due to receptor expression discordance between the primary tumor and metastatic sites (p > 0.05) was found, although a tendency toward worse survival time was observed in patients with HER2 expression discrepancies. Conclusion: This study showed receptor discordance rates between primary and metastatic breast cancer sites for ER, PR, and HER2 of 17.8, 45.4, and 13.3%, respectively. Re-biopsy and IHC evaluation of metastatic sites for receptor status may change treatment decisions in patients with relapsed/progressed BC.

The discordance between primary breast cancer lesions and pulmonary metastatic lesions in expression of aldehyde dehydrogenase 1-positive cancer cells

We evaluated the expression of aldehyde dehydrogenase 1 (ALDH1) between primary breast lesions and pulmonary metastatic (PM) lesions in breast cancer patients. We retrospectively analyzed the clinicopathological features and the expression statuses of ER, PR, HER2, Ki-67 and ALDH-1 in both primary and metastatic breast cancer lesions and evaluated the discordance rates in the expressions of these markers between the primary and metastatic lesions, and also the prognostic value of these factors. None of the PM patients had metastases at any other sites, and all had undergone curative breast cancer surgery. The pulmonary operation was partial resection in 15 (88 %) patients and lobectomy in 2 (12 %) patients. The median overall survival (OS) after resection of the PNs (OS) was 48 months. The discordance rates in the expressions of ER, PR, HER2, Ki67 and ALDH-1 between the primary and metastatic lesions were 0, 29, 21, 43 and 50 %, respectively. There was significant discordance in the biomarkers between the primary tumors and the metastatic lesions.

Assessing the Relation Between Bone Marrow Signal Intensity and Apparent Diffusion Coefficient in Diffusion-Weighted MRI

The purposes of this study were to observe the relation between signal intensity (SI) on MR images with a high b value and the apparent diffusion coefficient (ADC) of bone marrow on body diffusion-weighted MR images, to determine cutoff values that enable separation of malignant and normal bone marrow, and to identify the upper ADC values of untreated multiple myeloma lesions and bone metastatic lesions of breast cancer. Retrospective evaluations of 16 patients without bone disease, 21 patients with untreated metastases of breast cancer, and 12 patients with myeloma undergoing body diffusion-weighted MRI were performed (b values, 50 s/mm(2) and 800 or 900 s/mm(2)). Normal yellow and red bone marrow regions were compared with metastatic breast and myeloma bone marrow lesions (one to five regions of interest per patient). SI values were normalized to kidney, muscle, and spinal cord SI. Signal-to-noise ratio and ADC for each lesion were recorded. Nonparametric, receiver operating characteristic, and nonlinear regression analyses were performed. Yellow bone marrow and red bone marrow ADC values were lower than the tumor values (p < 0.001; area under the curve, 0.94; cutoff, 774 μm(2)/s). Tissue-normalized SI and the signal-to-noise ratio of normal bone marrow were also lower than those in tumor regions (p < 0.001; area under the curve, 0.86-0.88). Second-order polynomial curve fitting between SI and ADC was observed (muscle normalized SI, R(2) = 0.4). The 95th percentile and maximum values for mean tumor ADC distribution were 1209 μm(2)/s and 1433 μm(2)/s. Both tissue-normalized SI and ADC measurements allow differentiation between normal bone marrow and tumors of myeloma and breast cancer. The presence of a nonlinear relation between bone marrow SI and ADC values enables definition of an upper limit of ADC value for untreated myeloma lesions and metastatic lesions of breast cancer.

Comparison of the oestrogen and progesterone receptor status in primary breast carcinomas as evaluated by immunohistochemistry and immunocytochemistry: a consecutive series of 267 patients

The use of cytological specimens to evaluate tumour biomarkers in metastatic breast cancer lesions has attracted increased interest because of the considerable number of reports that have shown discordance between the primary tumour and metastatic lesion. Oestrogen receptor (ER) and progesterone receptor (PgR) assays are crucial for the management of patients with breast cancer, in both adjuvant and palliative settings. The aim of this study was to compare the ER and PgR immunocytochemical analysis of fine needle aspiration (FNA) samples with the immunohistochemistry (IHC) of surgical specimens and core biopsies from primary breast cancers. The FNA specimens were prepared as cell blocks (n = 25) or ThinPreps (n = 258) for the immunocytochemistry (IC) ER and PgR analyses. Sixteen patients were excluded because of lack of follow-up (n = 1), neoadjuvant therapy (n = 3) or cell counts in their fine needle aspirates that were too low (n = 12). The results of IC on 25 cell blocks and 242 ThinPreps were compared with IHC on the corresponding core needle biopsies (n = 16) or excised tumours (n = 251). The ER and PgR status was defined as negative (when less than 10% of the nuclei were stained) or positive (when equal or more than 10% of the nuclei were stained). Kappa statistics were used to evaluate the concordance. The ER concordance was 98% with ThinPrep (κ = 0.93) and 92% with cell block (κ = 0.82). The corresponding values for PgR were 96% (κ = 0.91) and 96% (κ = 0.92). Our results confirm that, in cases in which biopsies or surgical specimens are not available, IC (with either cell block or ThinPrep techniques) is a reliable method for the determination of the ER and PgR status performed under strict conditions using primary breast carcinomas, and is therefore potentially useful in metastatic settings.

Site-specific CpG methylation in the CCAAT/enhancer binding protein delta (CEBPδ) CpG island in breast cancer is associated with metastatic relapse

Background:The CCAAT/enhancer binding protein delta (CEBPδ) is a member of a highly conserved family of basic region leucine zipper transcription factors. It has properties consistent with a tumour suppressor; however, other data suggest that CEBPδ may be involved in the metastatic process.Methods:We analysed the expression of CEBPδ and the methylation status of the CpG island in human breast cancer cell lines, in 107 archival cases of primary breast cancer and in two series of metastatic breast cancers using qPCR and pyrosequencing.Results:Expression of CEBPδ is downregulated in primary breast cancer by site-specific methylation in the CEBPδ CpG island. Expression is also downregulated in 50% of cases during progression from primary carcinoma to metastatic lesions. The CEBPδ CpG island is methylated in 81% metastatic breast cancer lesions, while methylation in the CEBPδ CpG island in primary cancers is associated with increased risk of relapse and metastasis.Conclusion:CCAAT/enhancer binding protein delta CpG island methylation is associated with metastasis in breast cancer. Detection of methylated CEBPδ genomic DNA may have utility as an epigenetic biomarker of primary breast carcinomas at increased risk of relapse and metastasis.

Quantitative HER2 measurement and PI3K mutation profile in matched primary and metastatic breast cancer tissues.

614 Background: HER2 status of primary breast cancer (PBC) is routinely used to determine systemic treatment for metastatic breast cancer (MBC) patients. Discordance rates of HER2 status between PBC and MBC range from 5.5% to 29% based on published meta-analyses. The clinical benefit of re-assessing HER2 in MBC tissues remains controversial. In this study, we measured quantitative HER2 expression in matched PBC and MBC tissues and correlated changes of HER2 with mutations in the catalytic domain of PI3 kinase(PIK3CA). Methods: Total HER2 protein expression (H2T) was quantified by the HERmark assay in 41 matched PBC and MBC formalin-fixed, paraffin-embedded specimens. PIK3CA mutation status in exons 9 (E545K and E542K) and 20 (H1047R) was determined using a validated pyrosequencing assay. Results: MBC samples included 5 lymph node, 13 viscera, 6 brain, and 17 soft tissue lesions (N=41). 27 (66%) cases showed higher H2T in MBC than in matched PBC; and 14 (34%) cases had higher H2T in PBC than in matched MBC, indicating an overall increase of H2T in matched MBC lesions (fold change 0.25-17.57; p=0.005, paired Wilcoxon rank sum test). HER2 positive conversion (HERmark negative/equivocal in PBC, but positive in matched MBC) was found in 6 (15%) cases, while HER2 negative conversion (HERmark positive in PBC, but negative/equivocal in matched MBC) was seen in 2 (5%) cases. HER2 status was unchanged in 33 (80%) cases. PIK3CA mutations were detected in 13 (32%) of PBC and 19 (46%) of MBC samples. Among the HER2 positive conversion cases, PIK3CA mutation was identified in 50% (3/6) PBC and 67% (4/6) MBC, compared to 0% (0/2, PBC or MBC) in the HER2 negative conversion cases. Among cases with unchanged HER2 status, PIK3CA mutation was observed in 30% (10/33) PBC and 42% (14/33) MBC. Conclusions: Quantitative HER2 assessment revealed a 20% discordance in HER2 status between matched PBC and MBC tissues, with more frequent conversion from low HER2 in PBC to high HER2 in MBC. PIK3CA mutation was observed more frequently in patients who converted from HER2 negative PBC to HER2 positive MBC. These results suggest that re-assessment of biomarkers in MBC tissues may better inform the selection of therapeutic options for patients with MBC.

Abstract 683: Evaluation of PTEN and PIK3CA status in breast cancer for patient selection: Cross-validation between institutions

Abstract PTEN and PIK3CA status are potential predictors of response to PI3K-pathway inhibitors. Therefore, we searched the most reliable platform to assess both PTEN expression and PIK3CA mutations in primary and metastatic breast cancer (BC). Cross-validation across platforms at SU2C sites was performed using three different sample sets: 18 formalin-fixed paraffin-embedded (FFPE) primary triple negative breast cancer (pTNBC) samples from VHUH/VHIO, 12 FFPE pTNBC samples from Columbia University and 21 FFPE all-comers BC from VHUH/VHIO. Moreover, we evaluated the transitions occurring between early and disseminated disease in 14 paired FFPE samples from VHUH/VHIO. PTEN alterations were genotyped in 18 triple pTNBCs by OncoscanTM platform (Affymetrix) and were correlated to immunohistochemistry (IHC, 138G6 CST antibody). PTEN loss of heterozygosity (LOH, n=4) or frameshift mutation (n=1) was concordant to PTEN protein loss (H-Score&amp;lt;50) by IHC in 4 samples; with the discordant LOH having an H-Score of 60. In the other two non-concordant samples (total of 3/18, 17% discordance), presence of non-tumor DNA, as assessed by genomic heterogeneity, impeded an accurate DNA copy number variation determination by OncoscanTM. PTEN protein loss by IHC was also cross-validated between two institutions (Columbia and VHIO). In a cohort of 12 pTNBCs containing eight PTEN low samples, only two samples were discordant (2/12, 17% discordance). Paired primary versus metastatic tumors were identified to transit either way, in the PTEN assessment by IHC (2/8 paired samples, 25% transition). PTEN IHC failed in most (3/5) metastatic bone samples due to Bouin's fixative required for handling of this tissue. PIK3CA mutations by OncoscanTM were concordant to MassARRAY (Sequenom) in 4 out of 5 mutated samples within the panel of 18 pTNBCs. The discrepancy (1/18, 6%) was most likely due to differences in the sensitivity of the two assays. In another cohort of 21 BC samples, PIK3CA mutational status was cross-validated by MassARRAY at two institutions (MDACC and VHIO). Using identical, customized panels we found that only two samples were discordant because of mutant allele frequency close to the sensitivity of the assay (10%). Manual review of the individual assays increased the concordance to 100%. Among 14 paired primary vs metastatic breast cancers we detected two transitions from wild type (WT) to H1047R mutation and one transition from E545K to WT (3/14, 21% transition), underscoring the need to determine PIK3CA status in metastatic lesions. The divergence evaluating both PTEN and PIK3CA status between institutions was due to cut-off and sensitivity of the assays respectively. For patient pre-screening purposes, MassARRAY and IHC can be performed at each SU2C site both in primary and metastatic breast cancer lesions. OncoscanTM is a valid, centralized platform for evaluation of PTEN and PIK3CA genomic alterations in BC. 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 683. doi:1538-7445.AM2012-683

To Biopsy or Not to Biopsy: Is That the Only Question?

The clinical effects of discordance in receptor status between primary and metastatic breast cancer lesions are discussed in relation to the study of Macfarlane et al. published in this issue of The Oncologist.

Abstract A20: The Six1-regulated miR-106b-25 cluster is a mediator of the tumor promotional effects of TGF-β signaling in human breast cancer

Abstract Recent studies have highlighted the developmental transcription factor Six1 as an important mediator of breast cancer progression and metastasis. Six1 is overexpressed in a striking 90% of metastatic breast cancer lesions, and patients whose tumors overexpress Six1 have a decreased time to metastasis and relapse, as well as an overall decrease in survival. Six1 mediates metastasis via multiple mechanisms, including its ability to induce an epithelial-to-mesenchymal transition (EMT) and tumor initiating cell (TIC) characteristics, both of which are dependent on an upregulation of TGF-β signaling. Indeed, the Six1-induced increase in TGF-β signaling is critical for the ability of Six1 to induce late stage metastasis. Interestingly, Six1 not only activates TGF-β signaling, but it also enables the switch of TGF-β signaling from tumor suppressive to tumor promotional, a phenomenon of considerable importance in cancer pathogenesis. This event, coined the “TGF-β paradox,” has been an area of extensive research, but remains largely elusive. To further investigate the mechanism by which Six1 mediates the switch in TGF-β signaling, we performed a miRNA microarray screen and identified a cluster of miRNAs, the miR-106b-25 cluster, that is upregulated in response to Six1 overexpression. The miR-106b-25 cluster consists of three miRNAs, miR-106b, miR-93, and miR-25, which reside together in the intron of the MCM7 gene. Importantly, overexpression and knockdown experiments demonstrate that Six1 regulates all three miRNAs within the cluster. Interestingly, these miRNA have previously been implicated in the impairment of TGF-β-mediated growth suppression through repression of the cell cycle inhibitor, p21, and pro-apoptotic factor, Bim. These data suggest that Six1-induced upregulation of these miRNA may mediate the switch in TGF-β signaling from tumor suppressive to tumor promotional. Surprinsingly, bioinformatic analysis revealed that the miR-106b-25 cluster may also contribute to the activation of TGFβ signaling through repression of the TGF-β signaling inhibitor, Smad7, which mediates the degradation of TβRI. Indeed, overexpression of the miR-106b-25 cluster results in repression of the Smad7 protein, with concominant upregulation of TβRI. Furthermore, activation of TGF-β signaling is observed with miR-106b-25 overexpression, as demonstrated by an increase in phosphorylation of the downstream effector of the TGF-β pathway, Smad3, and by an upregulation of downstream TGF-β transcriptional targets. miRNA inhibition also demonstrates that miR-106b and miR-93 are necessary for Six1 induced TGF-β activation. Additionally, like Six1, the miR-106b-25 cluster is sufficient to induce features of EMT, including a redistribution of E-cadherin, and increased β-catenin transcriptional activation. Furthermore, the miR-106b-25 cluster is also sufficient to increase the TIC population as measured by flow cytometry, mammosphere formation, and in vivo serial dilution assays. Finally, we demonstrate a significant correlation between miR-106b, Six1, and activated TGF-β signaling in human breast cancers, and further show that high levels of miR-106b and miR-93 in breast tumors significantly predicts shortened time to relapse. These findings expand the spectrum of oncogenic functions of miR-106b-25, and may provide a novel molecular explanation, through the Six1 regulated miR-106b-25 cluster, by which TGF-β signaling shifts from tumor suppressive to tumor promotional. Citation Format: Anna L. Smith, Ritsuko Iwanaga, David J. Drasin, Douglas S. Micalizzi, Rebecca L. Vartuli, Heide L. Ford. The Six1-regulated miR-106b-25 cluster is a mediator of the tumor promotional effects of TGF-β signaling in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A20.

P5-13-10: Discordance of Estrogen Receptor and HER-2/neu Status Can Be Seen between First and Subsequently Biopsied Metastatic Lesions in Breast Cancer.

Abstract Background We have previously reported discordance between primary and first metastasis for both estrogen receptor (ER) and HER-2/neu (HER 2) status (Breast Cancer Res Treat 2005;90:65–70 and Breast Cancer Res Treat 2009;113:301–6). Because biomarker status is the primary determinant for targeted therapy, changes in biomarkers can impact recommendations for systemic treatment. Materials and Methods: In order to evaluate the possibility of further discordance with the development of additional metastatic sites, we examined the ER and HER 2 status of primary and first and subsequently biopsied breast cancer metastasis. In most cases, the subsequent site of metastasis was the second detected metastasis. Pathology reports were reviewed from 65 patients with biopsy proven first and additional metastasis; however, immunostain results were not available for all three timepoints for all patients. Sites of biopsied metastasis included lung, liver, bone, chest wall, and central nervous system. Results: The time between the initial diagnosis and first metastasis was 102 (3-676) months [Median (Range)], and the time between the first and subsequent metastasis was 60 (6-273) months. For ER, there was discordance between the primary lesion and either the first or subsequent metastasis in 18 of 65 cases (27.7%). Discordance of the ER occurred between the first and subsequent metastasis in 12 (18%) of cases. Three patterns of ER discordance occurred in these 12 cases: 4 cases of both primary and first metastasis negative with subsequent metastasis positive; 3 cases of primary positive, first metastasis negative, and subsequent metastasis positive, and 5 cases of primary and first metastasis positive and subsequent metastasis negative. For HER 2, discordance was detected between the primary and either first or subsequent metastasis in 11 of 36 cases (30.6%). Further discordance between the first and subsequent metastatic lesions occurred in 8 of 51 (16%) of cases. Conclusions: A significant level of discordance for either ER or HER 2 was detected between first and subsequent metastatic breast cancer lesions. For breast cancer patients with new metastasis, consideration of reevaluating ER and HER-2/neu status may be worthwhile. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-10.

Expression of antigen processing and presenting molecules in brain metastasis of breast cancer

Defects in human leukocyte antigen class I antigen processing machinery (APM) component expression can have a negative impact on the clinical course of tumors and the response to T cell-based immunotherapy. Since brain metastases of breast cancer are of increasing clinical significance, the APM component expression levels and CD8(+) T cell infiltration patterns were analyzed in primary breast and metastatic brain lesions of breast cancer by immunohistochemistry. Comparison of unpaired 50 primary and 33 brain metastases showed lower expression of β2-microglobulin, transporter associated with antigen processing (TAP) 1, TAP2 and calnexin in the brain lesions. Although no significant differences were found in APM component scores between primary breast and brain lesions in 15 paired cases, primary breast lesions of which patients eventually developed brain metastases showed lower levels of β2-microglobulin, TAP1 and calnexin compared with breast lesions without known brain metastases. The extent of CD8(+) T cell infiltration was significantly higher in the lesions without metastasis compared with the ones with brain metastases, and was positively associated with the expression of TAP1 and calnexin. Furthermore, mouse tumor cells stably transfected with silencing hairpin (sh)RNA for TAP1 demonstrated a decreased susceptibility to cytotoxic T lymphocytes in vitro and enhanced spontaneous brain metastasis in vivo. These data support the functional significance of TAP1 expression in tumor cells. Taken together, our data suggest that patients with low or defective TAP1 or calnexin in primary breast cancers may be at higher risks for developing brain metastasis due to the defects in T cell-based immunosurveillance.

Role of serum HER2 (sHER2) levels to predict acquisition of HER2 overexpression in patients with metastatic lesions of breast cancer with HER2-negative/unknown at the primary site.

e11114 Background: HER2 overexpression occur in 20%-30% of breast cancer (BC) patients being associated with poor prognosis and having a predictive value regarding response to anti-HER2 agents. sHER2 is a circulating oncoprotein cleaved from full-length, membrane-bound HER2 indicator of poor prognosis and of poor response to chemotherapeutic and hormone treatment in metastatic breast cancer when it is elevated. HER2 overexpression in BC metastatic tissue (BCMT) occur in 5,1% of patients that were HER2 negative in the primary tumor (Locatelli et al. 2010). We investigated whether sHER2 correlates with an immunophenothype change in BCMT of patients that were HER2 negative or unknown (primary samples not available) in primary BC. Methods: We determined sHER2 in 27 BC patients that were selected to perform BCMT biopsy because HER2 status was negative or not possible to determine. sHER2 was measured by immunoassay (H2n Advia Centaur Siemens), N< 15μg/L. Localization of BCMT was: 19 liver, 7 bone and 1 skin. Me...

The frequency of methylation of the NT5E gene in metastatic breast cancer.

e11553 Background: Ecto-5-prime-nucleotidase (NT5E; CD73) catalyzes the conversion of purine 5-prime mononucleotides to nucleosides, the preferred substrate being AMP. Deficiency of NT5 occurs in a variety of immunodeficiency diseases and some studies have associated over-expression of NT5E with clinically aggressive neoplastic disease. Here we describe transcriptional down-regulation of the NT5E gene in breast cancer and show that this is associated with aberrant methylation in the CpG island located in its 5’ regulatory sequences. Methods: Methylation in the NT5E CpG island was analysed by methylation specific PCR (MSP) and quantitative pyrosequencing in a panel of 10 breast carcinoma cell lines and two independent clinical series of breast carcinomas, comprising 80 and 140 cases respectively and 17 brain metastatic breast cancer lesions. The breast carcinomas were predominantly invasive ductal carcinomas, untreated at the time of surgery and randomly selected from our clinical practice. Expression was assessed by qRT-PCR. Results: NT5E mRNA was down-regulated in 4/10 breast carcinoma cell lines analysed. Down-regulation was associated with aberrant methylation in the CpG island located in the 5’ regulatory sequences of the NT5E gene: methylation was present only in cell lines with down-regulation and the CpG island was unmethylated in cell lines which express the gene. Moreover, expression of NT5E was up-regulated by azacytidine. In clinical cases, methylation (assessed by pyrosequencing and/or MSP) was present in 28% and 41% of cases in independent clinical series of primary carcinomas. Methylation was more common in primary cases which ultimately relapsed at distant metastatic sites. Strikingly, methylation was particularly common in 9/16 brain metastatic lesions. Conclusions: In contrast to previous reports, we show that methylation of NT5E is common in metastatic breast cancer, particularly in intra-cranial metastases. Our data implicate transcriptional silencing of NT5E as a common epigenetic event in metastatic breast cancer and imply that detection of methylated DNA may have utility as a prognostic and/or predictive biomarker in breast cancer.

Abstract 2439: Association of antigen processing machinery components and CD8+ T-cell infiltration in breast cancer brain metastasis

Abstract Purpose: Defects in human leukocyte antigen (HLA) class I antigen processing machinery (APM) component expression can have a negative impact on the clinical course of tumors and the response to T cell-based immunotherapy. Since brain metastases of breast cancer are of increasing clinical significance and only scant information is available about the expression of APM component abnormalities in these lesions, the APM component expression and CD8+ T cell infiltration pattern was analyzed in primary breast and metastatic brain lesions of breast cancer. Experimental design: Immunohistochemistry was performed to evaluate the expression of APM components HLA class I heavy chain, β2-microglobulin, tapasin, antigen peptide transporter (TAP)1, TAP2, LMP2 and calnexin. Results: Comparison of unpaired 50 primary and 33 brain metastases showed lower expression of β2-microglobulin, TAP1, TAP2 and calnexin in the brain lesions. Although no significant differences were found in APM component scores between primary breast and brain lesions in 15 paired cases, primary breast lesions of which patients eventually developed brain metastases showed lower levels of β2-microglobulin, TAP1 and calnexin compared with newly diagnosed breast lesions without brain metastases. Furthermore, the extent of CD8+ T cell infiltration was significantly higher in the newly diagnosed breast lesions compared with the ones of which the patients developed brain metastases, and was positively associated with the expression of TAP1 and calnexin. Conclusions: Our data suggest that patients with low or defective TAP1 or calnexin in the primary breast cancers may be at higher risks for developing brain metastases due to the reduced immunosurveillance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2439. doi:10.1158/1538-7445.AM2011-2439

Repeated Chemoembolization Followed by Laser-Induced Thermotherapy for Liver Metastasis of Breast Cancer

The purpose of this study was to evaluate local tumor control and survival after use of a downstaging protocol of repeated transarterial chemoembolization (TACE) with two chemotherapeutic combinations followed by laser-induced thermotherapy in the care of patients with liver metastasis of breast cancer. This prospective study included 161 patients with liver metastasis of breast cancer origin. TACE (mean, 3.5 [SD, 1.3] sessions per patient; range, 1-9 sessions) was performed as downstaging treatment to achieve the size and number of metastatic lesions that met the requirements for laser-induced thermotherapy (diameter < 5 cm, number ≤ 5). The TACE protocol was performed with either mitomycin C alone (n = 53) or mitomycin C in combination with gemcitabine (n = 108). In response to TACE overall, the mean reduction in diameter based on the longest diameter of the target lesions was 27%. The difference between diameter reduction in the mitomycin C group and that in the mitomycin C-gemcitabine group was not statistically significant (p = 0.65). The mean survival time of all patients was 32.5 months, calculation starting from the first TACE treatment. The mean local tumor control period calculated as of completion of therapy was 13 months, and the mean time to progression was 8 months. In the mitomycin-gemcitabine group, mean time to progression was 10.7 months, and in the mitomycin group it was 6.9 months (p = 0.5). TACE can be used for sufficient downstaging of liver metastatic lesions of breast cancer to allow laser-induced thermotherapy. A combination of mitomycin C and gemcitabine seems to improve the reduction achieved with TACE.

Abstract P5-14-20: Clinical Features of Surgical Resection for Solitary Pulmonary Metastasis and the Discrepancy in Immunopathological Features between Primary and Metastatic Breast Cancer Lesions

Abstract Background: The treatment for metastatic breast cancer (MBC) is usually systemic therapy decided by the hormone and HER2 status of primary breast cancer lesion. There are some reports about the discrepancy in hormone and HER2 status between primary and metastatic lesion in these days. We have reported previously that the expression of ALDH-1 which considered the pathological markers of breast cancer stem cell in axillary lymph node matastases is a significant predictor of poor outcome in primary breast cancer patients (Figure 1). We evaluated the role of surgery in the diagnosis and treatment of a solitary pulmonary nodule (SPN) in breast cancer patients and demonstrated the significance of analyzing the immunopathological features in metastatic breast cancer lesion. Methods: Between 1990 and 2006, 17 patients (PM) with SPN which were diagnosed breast cancer metastases underwent complete pulmonary resection and 36 MBC patients (AP) underwent autopsy after breast cancer related death in our hospitals. We retrospectively analyzed clinicopathological features and the expression of ER, HER2, Ki-67 and ALDH-1 in both primary and metastatic breast cancer lesions. Results: The median age of PM patients was 53 (range 30-80). All PM patients had not other metastases and had undergone curative breast cancer operation. The pulmonary operation were partial pulmonary resection in 15(88%) and lobectomy in 2 (12%) patients. The median disease free survival (DFS) was 63 months (range 9-175). Median overall survival (OS) was 181 months and the median OS after pulmonary operation was 48 months. The median DFS and OS of AP patients were 21 and 43 months. All AP patients had multiple visceral metastases. We compared the immunohistlogical status between primary and metastatic lesion and evaluated the colletation with these status in metastatic lesion and prognosis in all patients. Conclusion: Pulmonary resection was effective to diagnose and the OS after resection was relatively long in PM patients. We report the value of the expression of ER, HER2, Ki67 and ALDH-1 in distant metastatic lesions. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-14-20.

Determination of the Her-2/neu Gene Amplification Status in Cytologic Breast Cancer Specimens Using Automated Silver-enhanced In-situ Hybridization (SISH)

Silver-enhanced in-situ hybridization (SISH) is an emerging tool for the determination of the Her-2/neu amplification status in breast cancer. SISH is technically comparable to fluorescence in-situ hybridization (FISH) but does not require a fluorescence microscope for its interpretation. Although recent studies on histologic evaluations of SISH are promising, we aimed to evaluate its performance on 71 cytologic breast cancer specimens with the new combined Her-2/Chr17 probe. Her-2/neu status as routinely determined by FISH was available for all patients. We found SISH signals in cytologic cell blocks and smear specimens easy to evaluate in most cases. Small numbers of tumor cells and difficulties in identifying tumor cells in lymphocyte-rich backgrounds were limiting factors. Her-2/neu status, as determined by Her-2/Chr17 SISH, was basically identical to the results of the corresponding FISH. The discrepancies were mainly owing to the heterogeneity of Her-2/neu amplification in the tumor tissue. Interobserver agreement for the SISH evaluation was high (kappa value: 0.972). We conclude that Her-2/Chr17 SISH is a useful and accurate method for the evaluation of the Her-2/neu gene amplification status in cytologic breast cancer specimens, particularly in metastatic breast cancer lesions. The advantages of signal permanency and bright-field microscopic result interpretation make this technique an attractive alternative to the current FISH-based gold standard.