Susan J. Done

Associations between the uptake of 111In-DTPA-trastuzumab, HER2 density and response to trastuzumab (Herceptin) in athymic mice bearing subcutaneous human tumour xenografts

PurposeThe purpose of the study was to investigate the associations between uptake of 111In-DTPA-trastuzumab, tumour HER2 density and response to trastuzumab (Herceptin) of human breast cancer (BC) xenografts in athymic mice.Materials and methodsThe tumour uptake of 111In-DTPA-trastuzumab in athymic mice bearing BC xenografts with increasing HER2 density (0 to 3+) was evaluated. Specific uptake ratios were established in biodistribution (SUR) and imaging studies (ROI-SUR) using 111In-labeled mouse IgG (111In-DTPA-mIgG). Further corrections were made for circulating radioactivity using tumour-to-blood ratios defined as a localization index (LI) and region-of-interest localization index (ROI-LI), respectively. Mice were treated with trastuzumab (Herceptin). A tumour growth inhibition index (TGI) was calculated and relative TGIs calculated by dividing the TGI of control by that of trastuzumab-treated mice.ResultsStrong, nonlinear associations with HER2 density were obtained if the uptake of 111In-DTPA-trastuzumab was corrected for nonspecific IgG localization (i.e., SUR; r2 = 0.99) and circulating radioactivity (i.e., LI; r2 = 0.87), but without these corrections, the association between HER2 density and tumour uptake was poor (r2 = 0.22). There was a strong association between ROI-SUR and ROI-LI values and HER2 expression (r2 = 0.90 and r2 = 0.95, respectively. All tumours were imaged. Relative TGI values were associated with increasing uncorrected tumour uptake of 111In-DTPA-trastuzumab but not always with HER2 density (i.e., MCF-HER2-18 cells with trastuzumab-resistance).ConclusionHER2 expression (0 to 3+) can be differentiated using 111In-DTPA-trastuzumab, but requires correction of tumour uptake for nonspecific IgG localization and circulating radioactivity. The uncorrected uptake of 111In-DTPA-trastuzumab was associated with tumour response to trastuzumab.

Somatic mutations in the BRCA1 gene in Chinese sporadic breast and ovarian cancer

Inherited mutations in the BRCA1 gene confer increased susceptibility to breast and ovarian cancer. Its role in sporadic carcinogenesis is not well defined. Somatic mutations in breast cancers have not been reported and to date there are only three reports of somatic mutations in sporadic ovarian cancers. To investigate the contribution of BRCA1 mutations to sporadic breast and ovarian cancer in the Chinese population, we analysed 62 samples from Chinese women using the protein truncation test. There were 40 cases of breast cancer under age 50 and 22 cases of ovarian cancer, all unselected for family history. There was no age selection for the ovarian cancers. We found two somatic BRCA1 mutations in exon 11, one in a breast cancer and the other in an ovarian cancer, both of which result in truncated proteins. Our results indicate that somatic BRCA1 mutations, like somatic mutations in the BRCA2 gene, though very rare, can be found in both breast and ovarian cancers and support a tumor suppressor function for BRCA1 in sporadic tumors.

Identification of a Low-Risk Luminal A Breast Cancer Cohort That May Not Benefit From Breast Radiotherapy

PURPOSE To determine the prognostic and predictive value of intrinsic subtyping by using immunohistochemical (IHC) biomarkers for ipsilateral breast relapse (IBR) in participants in an early breast cancer randomized trial of tamoxifen with or without breast radiotherapy (RT). PATIENTS AND METHODS IHC analysis of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6, epidermal growth factor receptor, and Ki-67 was conducted on 501 of 769 available blocks. Patients were classified as luminal A (n = 265), luminal B (n = 165), or high-risk subtype (luminal HER2, n = 22; HER2 enriched, n = 13; basal like, n = 30; or triple-negative nonbasal, n = 6). Median follow-up was 10 years. RESULTS Classification by subtype was prognostic for IBR (10-year estimates: luminal A, 5.2%; luminal B, 10.5%; high-risk subtypes, 21.3%; P < .001). Luminal subtypes seemed to derive less benefit from RT (luminal A hazard ratio [HR], 0.40; luminal B HR, 0.51) than high-risk subtypes (HR, 0.13); however, the overall subtype-treatment interaction term was not significant (P = .26). In an exploratory analysis of women with clinical low-risk (age older than 60 years, T1, grade 1 or 2) luminal A tumors (n = 151), 10-year IBR was 3.1% versus 11.8% for the high-risk cohort (n = 341; P = .0063). Clinical low-risk luminal A patients had a 10-year IBR of 1.3% with tamoxifen versus 5.0% with tamoxifen plus RT (P = .42). Multivariable analysis showed that RT (HR, 0.31; P < .001), clinical risk group (HR, 2.2; P = .025), and luminal A subtype (HR, 0.25; P < .001) were significantly associated with IBR. CONCLUSION IHC subtyping was prognostic for IBR but was not predictive of benefit from RT. Further studies may validate the exploratory finding of a low-risk luminal A group who may be spared breast RT.

Role of Antibody-Mediated Tumor Targeting and Route of Administration in Nanoparticle Tumor Accumulation in Vivo

In this study, we have looked at enhancing tumor uptake and intracellular delivery of gold nanoparticles (AuNPs) while reducing the systemic exposure by systematic evaluation of the impact of targeting and route of administration on organ distribution. High-resolution microSPECT/CT imaging was used to track the in vivo fate of (111)In-labeled nontargeted and human epidermal growth factor receptor-2 (HER-2) targeted AuNPs following intravenous (i.v.) or intratumoral (i.t.) injection. For i.v. injection, the effects of GdCl3 (for deactivation of macrophages) and nonspecific (anti-CD20) antibody rituximab (for blocking of Fc mediated liver and spleen uptake) were studied. It was found that HER-2 targeting via attachment of trastuzumab paradoxically decreased tumor uptake as a result of faster elimination of the targeted AuNPs from the blood while improving internalization in HER-2-positive tumor cells as compared to nontargeted AuNPs. I.T. injections with HER-2 targeted AuNPs resulted in high tumor retention with low systemic exposure and represents an attractive delivery strategy. Our results provide a strategy for optimizing tumor delivery and quantifying organ distribution of this widely studied class of nanomaterial.

Micro-SPECT/CT with 111In-DTPA-Pertuzumab Sensitively Detects Trastuzumab-Mediated HER2 Downregulation and Tumor Response in Athymic Mice Bearing MDA-MB-361 Human Breast Cancer Xenografts

Pertuzumab is a HER2 dimerization inhibitor that binds to an epitope unique from that of trastuzumab. Our objective was to determine whether SPECT with 111In-diethylenetriaminepentaacetic acid–pertuzumab (111In-DTPA-pertuzumab) could sensitively detect an early molecular response to trastuzumab manifested by HER2 downregulation and a later tumor response revealed by a decreased number of HER2-positive viable tumor cells. Methods: Changes in HER2 density in SKBr-3 and MDA-MB-361 BC cells exposed to trastuzumab (14 μg/mL) in vitro were measured by saturation binding assays using 111In-DTPA-pertuzumab and by confocal immunofluorescence microscopy and flow cytometry with fluorescein isothiocyanate–labeled HER2/neu antibodies. Imaging of HER2 downregulation was studied in vivo in athymic mice with subcutaneous MDA-MB-361 tumors treated for 3 d with trastuzumab (4 mg/kg) or nonspecific human IgG (hIgG) or phosphate-buffered saline (PBS). Imaging of tumor response to trastuzumab was studied in mice bearing subcutaneous MDA-MB-361 xenografts treated with trastuzumab (4 mg/kg), followed by weekly doses of nonspecific hIgG or rituximab or PBS (2 mg/kg). Mice were imaged on a micro-SPECT/CT system at 72 h after injection of 111In-DTPA-pertuzumab. Tumor and normal-tissue biodistribution was determined. Results: 111In-DTPA-pertuzumab saturation binding to SKBr-3 and MDA-MB-361 cells was significantly decreased at 72 h after exposure in vitro to trastuzumab (14 μg/mL), compared with untreated controls (62% ± 2%, P < 0.0001; 32% ± 9%, P < 0.0002, respectively). After 3 d of trastuzumab, in vivo tumor uptake of 111In-DTPA-pertuzumab decreased 2-fold in trastuzumab- versus PBS-treated mice (13.5 ± 2.6 percentage injected dose per gram [%ID/g] vs. 28.5 ± 9.1 %ID/g, respectively; P < 0.05). There was also a 2-fold decreased tumor uptake in trastuzumab- versus PBS-treated mice by image volume-of-interest analysis (P = 0.05), suggesting trastuzumab-mediated HER2 downregulation. After 3 wk of trastuzumab, tumor uptake of 111In-DTPA-pertuzumab decreased 4.5-fold, compared with PBS-treated mice (7.6 ± 0.4 vs. 34.6 ± 9.9 %ID/g, respectively; P < 0.001); this decrease was associated with an almost-completed eradication of HER2-positive tumor cells determined immunohistochemically. Conclusion: 111In-DTPA-pertuzumab sensitively imaged HER2 downregulation after 3 d of treatment with trastuzumab and detected a reduction in viable HER2-positive tumor cells after 3 wk of therapy in MDA-MB-361 human breast cancer xenografts.

Local control with conventional and hypofractionated adjuvant radiotherapy after breast-conserving surgery for ductal carcinoma in-situ

PURPOSE Adjuvant whole breast radiotherapy (WBRT) for ductal carcinoma in situ (DCIS) improves local control, however an optimal dose fractionation remains undefined. WBRT following breast-conserving surgery for invasive breast cancer demonstrates equivalent efficacy and morbidity for conventional and hypofractionated treatment. Our group policy allowed for the use of both schedules, therefore we compared local control in women with DCIS following breast-conserving surgery. PATIENTS AND METHODS Two hundred and sixty-six patients treated between January 1999 and December 2004 with conventional (50Gy in 25 fractions) or hypofractionated (42.4Gy in 16 fractions or 40Gy/16+12.5Gy boost) WBRT after breast-conserving surgery for DCIS were retrospectively reviewed. Median follow-up was 3.76years (range 0.1-8.9 years). RESULTS One hundred and four patients (39%) were treated with conventional and 162 (61%) with hypofractionated WBRT. The median age was 56.7 years (range 32.2-83.8 years), and prognostic features were well matched in both groups, apart from a small increase in tumour size in the conventional arm (1.75 vs. 2.12 cm, p=0.05). Actuarial risk of recurrence at 4 years was 7% with hypofractionated WBRT and 6% with the conventional schedule (p=0.9). Univariate analysis showed an increased risk of recurrence with high nuclear grade tumours (11% at 4 years for grade 3 vs. 4% for grade 1/2, p=0.029). CONCLUSIONS Hypofractionated adjuvant WBRT following breast-conserving surgery for DCIS has comparable local control to a conventional radiation schedule. Hypofractionated WBRT is more convenient for patients, has equivalent morbidity and should be considered in this patient group.

Proteomic Analyses Reveal High Expression of Decorin and Endoplasmin (HSP90B1) Are Associated with Breast Cancer Metastasis and Decreased Survival

Background Breast cancer is the most common malignancy among women worldwide in terms of incidence and mortality. About 10% of North American women will be diagnosed with breast cancer during their lifetime and 20% of those will die of the disease. Breast cancer is a heterogeneous disease and biomarkers able to correctly classify patients into prognostic groups are needed to better tailor treatment options and improve outcomes. One powerful method used for biomarker discovery is sample screening with mass spectrometry, as it allows direct comparison of protein expression between normal and pathological states. The purpose of this study was to use a systematic and objective method to identify biomarkers with possible prognostic value in breast cancer patients, particularly in identifying cases most likely to have lymph node metastasis and to validate their prognostic ability using breast cancer tissue microarrays. Methods and Findings Differential proteomic analyses were employed to identify candidate biomarkers in primary breast cancer patients. These analyses identified decorin (DCN) and endoplasmin (HSP90B1) which play important roles regulating the tumour microenvironment and in pathways related to tumorigenesis. This study indicates that high expression of Decorin is associated with lymph node metastasis (p<0.001), higher number of positive lymph nodes (p<0.0001) and worse overall survival (p = 0.01). High expression of HSP90B1 is associated with distant metastasis (p<0.0001) and decreased overall survival (p<0.0001) these patients also appear to benefit significantly from hormonal treatment. Conclusions Using quantitative proteomic profiling of primary breast cancers, two new promising prognostic and predictive markers were found to identify patients with worse survival. In addition HSP90B1 appears to identify a group of patients with distant metastasis with otherwise good prognostic features.

Genomic Differences Between Pure Ductal Carcinoma In Situ of the Breast and that Associated with Invasive Disease: a Calibrated aCGH Study

Purpose: In the quest for new targets, genomes of ductal carcinoma in situ (DCIS) and infiltrating duct carcinoma (IDC) have been compared previously; however, genomic alterations associated with cancer progression were difficult to identify. We hypothesized that significant events can be detected by comparing lesions with a broader range of behavior: from pure DCIS to IDC associated with lymph node metastasis. Experimental Design: Array comparative genomic hybridization, calibrated by self-self hybridization tests, was used to study 6 cases of pure DCIS and 17 cases of DCIS paired with IDC where 8 tumors had spread to the local lymph nodes. Results: Pure DCIS exhibited a marginally higher degree of genomic complexity than DCIS and IDC components of invasive tumors. The latter two showed similarity between tumors and between components of the same tumor with several regions detected preferentially compared with pure DCIS. IDC associated with lymph node metastases showed similarity of genomic profiles as a group. Gain on 17q22-24.2 was associated with higher histologic grade, large IDC size, lymphatic/vascular invasion, and lymph node metastasis (P < 0.05). Conclusions: Our findings suggest that DCIS and IDC are associated with specific genomic events. DCIS associated with IDC is genomically similar to the invasive component and therefore may represent either a clone with high invasive potential or invasive cancer spreading through the ducts. Specifically, gain on 17q22-24.2 is a candidate region for further testing as a predictor of invasion when detected in DCIS and predictor of nodal metastasis when detected in DCIS or IDC.

Digital compared with screen-film mammography: performance measures in concurrent cohorts within an organized breast screening program.

PURPOSE To evaluate the performance of digital direct radiography (DR) and computed radiography (CR) compared with that of screen-film mammography (SFM) in large concurrent cohorts. MATERIALS AND METHODS This study was approved by the University of Toronto Research Ethics Board and did not require informed consent. Concurrent cohorts of women aged 50-74 years screened with DR (n = 220 520), CR (n = 64 210), or SFM (n = 403 688) between 2008 and 2009 were identified and followed for 12 months. Performance was compared between cohorts, with SFM as the referent cohort. Associations were examined by using mixed-effect logistic regression. RESULTS The cancer detection rate was similar for DR (4.9 per 1000; 95% confidence interval [CI]: 4.7, 5.2) and SFM (4.8 per 1000; 95% CI: 4.7, 5.0); however, the rate was significantly lower for CR (3.4 per 1000; 95% CI: 3.0, 3.9) (odds ratio, 0.79; 95% CI: 0.68, 0.93). Recall rates were higher for DR (7.7%; 95% CI: 7.6%, 7.8%) and lower for CR (6.6%; 95% CI: 6.5%, 6.7%) than for SFM (7.4%; 95% CI: 7.3%, 7.5%). Positive predictive value was lower for CR (5.2%; 95% CI: 4.7%, 5.8%) than for SFM (6.6%; 95% CI: 6.4%, 6.8%); however, the adjusted odds were not significant. CONCLUSION Although DR is equivalent to SFM for breast screening among women aged 50-74 years, the cancer detection rate was lower for CR. Screening programs should monitor the performance of CR separately and may consider informing women of the potentially lower cancer detection rates.

Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells

Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.