Eric L. Rosen

Gene Expression Profiles of Multiple Breast Cancer Phenotypes and Response to Neoadjuvant Chemotherapy

Purpose: Breast cancer is a heterogeneous disease, and markers for disease subtypes and therapy response remain poorly defined. For that reason, we employed a prospective neoadjuvant study in locally advanced breast cancer to identify molecular signatures of gene expression correlating with known prognostic clinical phenotypes, such as inflammatory breast cancer or the presence of hypoxia. In addition, we defined molecular signatures that correlate with response to neoadjuvant chemotherapy. Experimental Design: Tissue was collected under ultrasound guidance from patients with stage IIB/III breast cancer before four cycles of neoadjuvant liposomal doxorubicin paclitaxel chemotherapy combined with local whole breast hyperthermia. Gene expression analysis was done using Affymetrix U133 Plus 2.0 GeneChip arrays. Results: Gene expression patterns were identified that defined the phenotypes of inflammatory breast cancer as well as tumor hypoxia. In addition, molecular signatures were identified that predicted the persistence of malignancy in the axillary lymph nodes after neoadjuvant chemotherapy. This persistent lymph node signature significantly correlated with disease-free survival in two separate large populations of breast cancer patients. Conclusions: Gene expression signatures have the capacity to identify clinically significant features of breast cancer and can predict which individual patients are likely to be resistant to neoadjuvant therapy, thus providing the opportunity to guide treatment decisions.

Tissue harmonic imaging sonography of breast lesions: improved margin analysis, conspicuity, and image quality compared to conventional ultrasound.

The purpose of this preference study is to determine if tissue harmonic imaging (THI) is preferred over conventional sonography for imaging breast masses. A prospective evaluation of 73 identical image pairs (one obtained with conventional sonography, one with THI sonography) was performed, examining 25 cysts, 36 solid masses, and 12 indeterminate lesions. Each image was evaluated for lesion contrast, margins, and overall image quality using a graduated score. Statistical analysis was performed using a modified t test. For cystic and solid lesions, THI was preferred for lesion conspicuity, margin, and overall quality (P<.001). For indeterminate lesions, THI was significantly preferred for lesion conspicuity and overall quality (P<.05), but the preference for margins was not significant. Overall, THI of breast lesions was significantly preferred for lesion contrast and margin evaluation compared to conventional sonography. This modality deserves further evaluation and may improve detection and evaluation of breast lesions.

Neoadjuvant comparisons of aromatase inhibitors and tamoxifen: pretreatment determinants of response and on-treatment effect.

Adjuvant endocrine therapy reduces the risk of relapse and death from early stage hormone receptor positive breast cancer. However, tamoxifen is only partially effective because of the development of tumor resistance. Aromatase inhibitors (letrozole, anastrozole and exemestane) are also prone to the development of resistance but the pharmacologic action (estrogen deprivation) is distinct and so different mechanisms may be responsible. The problem of endocrine resistance can be directly studied in patients by examining the relationship between predictive tumor biomarkers and clinical outcome. In an example of a prospectively planned biomarker study, tumor samples were examined from a randomized trial of neoadjuvant endocrine treatment in which letrozole proved more effective than tamoxifen in terms of the rate of breast conservation and tumor regression. Interestingly letrozole was more effective at all levels of ER expression and was particularly more efficacious than tamoxifen for tumors that expressed HER1 and/or HER2 (with ER). This suggests that HER1/2 predicts primary tamoxifen resistance and relative sensitivity to potent estrogen deprivation, perhaps because HER1/2 signaling promotes the partial agonist effects of tamoxifen. A Phase 2 study of neoadjuvant letrozole is now underway to focus on gene expression profiling as a mechanism to further investigate the transcriptional programs that underlie resistance and sensitivity to estrogen deprivation. Expression profiles taken at baseline and after 1 month of therapy reveal dramatic reductions in the expression from genes responsible for DNA replication and synthesis, cell cycle progression, suppression of apoptosis and tissue invasion. When enough profiles have been generated it should be possible to detect complex interaction patterns that correctly reclassify ER+ disease into treatment responsive and resistant categories with high probability.

DCE-MRI Parameters Have Potential to Predict Response of Locally Advanced Breast Cancer Patients to Neoadjuvant Chemotherapy and Hyperthermia: A Pilot Study

Combined therapies represent a staple of modern medicine. For women treated with neoadjuvant chemotherapy (NA ChT) for locally advanced breast cancer (LABC), early determination of whether the patient will fail to respond can enable the use of alternative, more beneficial therapies. This is even more desirable when the combined therapy includes hyperthermia (HT), an efficient way to improve drug delivery, however, more costly and time consuming. There is data showing that this goal can be achieved using magnetic resonance imaging (MRI) with contrast agent (CA) enhancement. This work for the first time proposes combining the information extracted from pre-treatment MR imaging into a morpho-physiological tumour score (MPTS) with the hypothesis that this score will increase the prognostic efficacy, compared to each of its MR-derived components: morphological (derived from the shape of the tumour enhancement) and physiological (derived from the CA enhancement variance dynamics parameters). The MPTS was correlated with response as determined by both pathologic residual tumour and MRI imaging, and was shown to have potential to predict response. The MPTS was extracted from pre-treatment MRI parameters, so independent of the combined therapy used. Purpose: To use a novel morpho-physiological tumour score (MPTS) generated from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to predict response to treatment. Materials and methods: A protocol was designed to acquire DCE-MRI images of 20 locally advanced breast cancer (LABC) patients treated with neoadjuvant chemotherapy (NA ChT) and hyperthermia (HT). Imaging was done over 30 min following bolus injection of gadopentetate-based contrast agent. Parametric maps were generated by fitting the signal intensity to a double exponential curve and were used to derive a morphological characterisation of the lesions. Enhancement-variance dynamics parameters, wash-in and wash-out parameters (WiP, WoP), were extracted. The morphological characterisation and the WiP and WoP were combined into a MPTS with the intent of achieving better prognostic efficacy. The MPTS was correlated with response to NA therapy as determined by pathological residual tumour and MRI imaging. Results: The contrast agent in all tumours typically peaked in the first 1–4 min. The tumours’ WiP and WoP varied considerably. The MPTS was highly correlated with whether the patients had a pathological response. This scoring system has a specificity of 78% and a sensitivity of 91% for predicting response to NA chemotherapy. The kappa was 0.69 with a 95% confidence interval of [0.38, 1] and a p-value of 0.002. Conclusions: This pilot study shows that the MPTS derived using pre-treatment MRI images has the potential to predict response to NA ChT and HT in LABC patients. Further prospective studies are needed to confirm the validity of these results.

MRI-guided vacuum-assisted breast biopsy with a handheld portable biopsy system.

OBJECTIVE The purpose of this study was to evaluate a compact portable 10-gauge handheld battery-operated vacuum-assisted biopsy system for MRI-guided breast biopsy. CONCLUSION The compact portable battery-operated biopsy system can be used successfully for MRI-guided core breast biopsy and is an alternative to current systems.

Ultrasound guided pO2 measurement of breast cancer reoxygenation after neoadjuvant chemotherapy and hyperthermia treatment.

The objective of this study was to determine whether neoadjuvant chemotherapy in combination with hyperthermia (HT) would improve oxygenation in locally advanced breast tumours. The study describes a new optimized ultrasound guided technique of pO 2 measurement using Eppendorf polarographic oxygen probes in 18 stage IIB-III breast cancer patients. Prior to treatment, tumour hypoxia (median pO 2 < 10 mmHg) was present in 11/18 patients (average median pO 2 = 3.2 mmHg). Seven patients had well oxygenated tumours (median pO 2 of 48.3 mmHg). Eight patients with hypoxic tumours prior to treatment had a significant improvement ( p = 0.0008) in tumour pO 2 after treatment (pO 2 increased to 19.2 mmHg). In three patients, tumours remained hypoxic (average median pO 2 = 4.5 mmHg). The advantages of the ultrasound guided pO 2 probe are in the accuracy of the Eppendorf electrode placement in tumour tissue, the ability to monitor electrode movement through the tumour tissue during the measurement and the ability to avoid electrode placement near or in large blood vessels by using colour Doppler imaging. The results of this preliminary study suggest that the combination of neoadjuvant chemotherapy and hyperthermia improves oxygenation in locally advanced breast tumours that are initially hypoxic.

Streaming detection for evaluation of indeterminate sonographic breast masses : A pilot study

OBJECTIVE Streaming detection is a novel sonography technique that uses ultrasonic energy to induce movement in cyst fluid that is detected on Doppler sonography. This pilot study evaluates the utility of streaming detection for differentiating cysts from solid masses in breast lesions that are indeterminate on sonography. SUBJECTS AND METHODS Thirty-nine lesions-11 simple cysts and seven solid masses (control group) and 21 masses with indeterminate findings for the diagnosis of a cyst versus a solid lesion (study group)-in 34 patients were evaluated using streaming detection. All lesions underwent cyst aspiration or biopsy (n = 35) or were diagnosed simple cysts (n = 4) on sonography. Lesion size and depth were recorded. Streaming detection software was placed on conventional sonography units. Acoustic pulses were focused on the lesion, and if fluid movement was generated, it was seen on the spectral Doppler display as velocity away from the transducer. Lesions were then aspirated or underwent biopsy, and the viscosity of the aspirated fluid was recorded. The sensitivity and specificity of the technique and the effect of cyst size, cyst depth, and fluid viscosity in diagnosing fluid-filled cysts were assessed. RESULTS Overall, 31 cysts and eight solid masses (seven benign, one carcinoma) were diagnosed in the study and control groups. Aspiration of indeterminate lesions resulted in 20 cysts and one solid mass. Lesions ranged in size from 4 to 47 mm and in depth from 4 to 29 mm. In the control group, streaming detection correctly showed nine of the 11 simple cysts (sensitivity, 82%; positive predictive value, 100%), and acoustic streaming was absent in all seven solid masses (specificity, 100%; negative predictive value, 78%). Of the indeterminate lesions, streaming detection allowed correct identification of 10 of 20 cysts (sensitivity, 50%; positive predictive value, 100%). Acoustic streaming was not detected in the one solid study group lesion. Neither cyst size or depth nor fluid viscosity had a significant effect on the ability to detect fluid. CONCLUSION The streaming detection technique improved differentiation of cysts from solid masses in indeterminate lesions and has potential for reducing the number of recommended cyst aspirations for the diagnosis of indeterminate breast masses.

Characterizing tumor changes during neoadjuvant treatment of locally advanced breast cancer patients (LABC) using dynamic-enhanced magnetic resonance imaging (DE-MRI)

At Duke University Medical Center, selective LABC patients were treated on a protocol using neoadjuvant Myocet/Paclitaxel (ChT) and HT. With the purpose of generating perfusion/permeability parametric maps and to use gadolinium (Gd) enhancement curves to score and predict response to neoadjuvant treatment, a study was designed to acquire 3 sets of DE-MRI images along the 4 cycles of combined ChT and HT. A T1-weighted three-dimensional fast gradient echo technique was used over 30 minutes following bolus injection of Gd-based contrast agent. Perfusion/permeability maps were generated by fitting the signal intensity to a double exponential curve that generates washin (WiP) and washout (WoP), parameters that are associated with the tumors vascularity/permeability and cellularity. Based on the values of the WiP, the tumors were divided in lowWI (WiP < 100), mediumWI (100 200). During the HT treatments temperatures in the breast were measured invasively via a catheter inserted under CT guidance. Although minimum sampled temperatures give a crude indication of the temperature distribution, several thermal dose metrics were calculated for each of the HT fractions (e.g. T90, T50, T10). As expected, tumors that were more vascularized (i.e. higher WiP) heated less than tumors with low WiP, a degree on average. The adjuvant treatment also changed the shape and inhomogeneity of the perfusion/permeability maps, with dramatic changes after the first fraction in responders. The correlation between the thermal metrics and pathological response will be discussed, as well as possible correlation with other tumor physiology parameters. In conclusion, the Gd-enhancement analysis of DE-MRI images is able to generate information related to the tumor vascularity, permeability and cellularity that can correlate with the tumors response to the neoadjuvant treatment in general, and to HT in particular. Work supported by a grant from the NCI CA42745.

Neoadjuvant chemotherapy and hyperthermia improve tumor reoxygenation in patients with locally advanced breast carcinoma

organizing alveolitis and sacrificed just prior to death due to development of organizing alveolitis. When 80% of control, irradiated mice had been sacrificed with organizing alveolitis, the remaining mice in all groups were sacrificed. The lungs were expanded with Optimum Cutting Temperature (OCT), removed, frozen in OCT, sectioned, and hematoxylin and eosin (H&E)-stained. These sections were examined microscopically and percent of alveolitis was determined using an Optimus Image Analysis System.

Ultrasound-guided pO2 measurement in breast cancer patients before and after hyperthermia treatment

The significance of tumor hypoxia extends beyond conventional radiation resistance. It has been found that tumor hypoxia affects drug resistance, angiognesis, cytokine production, cell cycle control, apoptosis and development of distant metastases. Recently, it has been reported that hyperthermia improves tumor oxygenation in both canine as well as human soft tissue sarcoma. This study describes a new optimized technique for pO2 measurement in breast cancer patients using ultrasound-guided placement of Eppendorf polarographic oxygen probes. Locally advanced breast cancer patients, participating in a phaseI/II study of neoadjuvant liposomal doxorubicin/paclitaxel/hyperthermia treatment, were the subjects of this study. Tumor oxygenation was measured before and 24 h after hyperthermia treatment.Advantages of the ultrasound-guided pO2 probe placement are the following: accuracy with visualization and verification of the Eppendorf electrode placement in tumor tissue; monitoring of the electrode movement through the tumor tissue during the measurement; ability to avoid electrode placement near or in large blood vessels by using color Doppler imaging; and spatial reproducibility of the second measurement. Despite progress in the technology that can be used to measure tumor hypoxia, accurate and verifiable placement of the oxygen probes in tumor tissue is of tremendous importance. Ultrasound-guided pO2 probe placement should become standard technique to improve accuracy and reliability in the assessment of tumor oxygenation for disease sites in which it is appropriate. Supported by a grant from the NIH CA42745.