Fast label-free multilayered histology-like imaging of human breast cancer by photoacoustic microscopy

The goal of breast-conserving surgery is to completely remove all of the cancer. Currently, no intraoperative tools can microscopically analyze the entire lumpectomy specimen, which results in 20 to 60% of patients undergoing second surgeries to achieve clear margins. To address this critical need, we have laid the foundation for the development of a device that could allow accurate intraoperative margin assessment. We demonstrate that by taking advantage of the intrinsic optical contrast of breast tissue, photoacoustic microscopy (PAM) can achieve multilayered histology-like imaging of the tissue surface. The high correlation of the PAM images to the conventional histologic images allows rapid computations of diagnostic features such as nuclear size and packing density, potentially identifying small clusters of cancer cells. Because PAM does not require tissue processing or staining, it can be performed promptly and intraoperatively, enabling immediate directed re-excision and reducing the number of second surgeries

High-throughput, label-free, single-cell photoacoustic microscopy of intratumoral metabolic heterogeneity

Intratumoral heterogeneity, which is manifested in almost all of the hallmarks of cancer, including the significantly altered metabolic profiles of cancer cells, represents a challenge to effective cancer therapy. High-throughput measurements of the metabolism of individual cancer cells would allow direct visualization and quantification of intratumoral metabolic heterogeneity, yet the throughputs of current measurement techniques are limited to about 120 cells per hour. Here, we show that single-cell photoacoustic microscopy can reach throughputs of approximately 12,000 cells per hour by trapping single cells with blood in an oxygen-diffusion-limited high-density microwell array and by using photoacoustic imaging to measure the haemoglobin oxygen change (that is, the oxygen consumption rate) in the microwells. We demonstrate the capability of this label-free technique by performing high-throughput single-cell oxygen-consumption-rate measurements of cultured cells and by imaging intratumoral metabolic heterogeneity in specimens from patients with breast cancer. High-throughput single-cell photoacoustic microscopy of oxygen consumption rates should enable the faster characterization of intratumoral metabolic heterogeneity

Photoacoustic microscopy enables multilayered histological imaging of human breast cancer without staining

In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming for large specimens. Thus, 20-60% of patients undergoing BCS still require second surgeries due to positive surgical margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional histology and measuring features related to the histological landscape without the need for labels. We demonstrate that our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast tissue

Impact of consensus guidelines for breast-conserving surgery in patients with ductal carcinoma in situ

BACKGROUND: Consensus guidelines published in 2016 recommended a 2 mm free margin as the standard for negative margins in patients undergoing breast-conserving surgery (BCS) for ductal carcinoma in situ (DCIS). The goal of the guideline recommendation was standardization of re-excision practices. AIMS: To evaluate the impact of this consensus guideline on our institutional practices. METHODS: We identified all patients at our institution with pure DCIS who were initially treated with BCS from September 2014 to August 2018 using a prospectively-maintained institutional database. A retrospective chart review was performed to determine margin status and re-excision rates during the 2 years before and the 2 years after the guideline was published in order to determine the effect on our re-excision rates. Close margins were defined as \u3c2 mm. RESULTS: In the 2 years before the consensus guideline was published, 184 patients with DCIS underwent BCS. Twenty-six patients had positive margins and 24 underwent re-excision, including three who had completion mastectomy. Of the remaining 159 patients, 76 had ≥2 mm (negative) margins. The remaining 82 patients had close margins and 48 of these patients (58.5%) underwent re-excision, including one who had a completion mastectomy. Excluding the patients with positive margins, our re-excision rate was 30.4% prior to the guideline. In the 2 years after the consensus guideline was published, 192 patients with DCIS underwent initial BCS. Twenty-four patients had positive margins and 22 underwent re-excision, including three who had completion mastectomy. Of the remaining 168 patients, 95 patients had ≥2 mm (negative) margins. The remaining 73 patients had close margins and 45 of those patients (61.6%) underwent re-excision, including six who had completion mastectomy. Excluding the patients with positive margins, our re-excision rate was 26.8% after the guideline. CONCLUSIONS: Our institution\u27s re-excision rate did not change significantly during the 2 years before and after the publication of the consensus guideline on adequate margins for patients undergoing BCT for DCIS. Our overall re-excision rate decreased slightly. However, of the patients who had close margins, a larger proportion underwent re-excision after the guideline was published. The guideline publication appears to have affected our institutional practices slightly, but not dramatically as many of our surgeons\u27 practices were comparable to the guideline recommendations prior to 2016. We continue to use clinical judgment based on patient and tumor characteristics in deciding which patients will benefit from margin re-excision

Metastatic disease to the breast: the Washington University experience

<p>Abstract</p> <p>Background</p> <p>Metastases to the breast occur rarely, but may be increasing in incidence as patients live longer with malignant diseases. The aim of this study is to characterize metastatic disease to the breast and to describe the management and prognosis of patients who present with this diagnosis.</p> <p>Methods</p> <p>A retrospective review of our institution's pathology and breast cancer databases was performed in order to identify patients with breast malignancies that were not of primary breast origin. Chart review provided additional information about the patients' primary malignancies and course of illness.</p> <p>Results</p> <p>Between 1991 and 2006, eighteen patients with metastatic disease to the breast of non-hematologic origin were identified and all had charts available for review. Among the 18 patients with disease metastatic to the breast, tissues of origin included 3 ovarian, 6 melanoma, 3 medullary thyroid, 3 pulmonary neuroendocrine, 1 pulmonary small cell, 1 oral squamous cell, and 1 renal cell. Overall mean survival after diagnosis of metastatic disease to the breast was 22.4 months. Treatment of metastases varied and included combinations of observation, surgery, radiation, and chemotherapy. Five patients (27.8%) required a change in management of their breast disease for local control.</p> <p>Conclusion</p> <p>Due to the variable course of patients with metastatic disease, a multi-disciplinary approach is necessary for each patient with disease metastatic to the breast to determine optimal treatment. Based on our review, many patients survive for long periods of time and local treatment of metastases to the breast may be beneficial in these patients to prevent local complications.</p

High-throughput, label-free, single-cell photoacoustic microscopy of intratumoral metabolic heterogeneity

Intratumoral heterogeneity, which is manifested in almost all of the hallmarks of cancer, including the significantly altered metabolic profiles of cancer cells, represents a challenge to effective cancer therapy. High-throughput measurements of the metabolism of individual cancer cells would allow direct visualization and quantification of intratumoral metabolic heterogeneity, yet the throughputs of current measurement techniques are limited to about 120 cells per hour. Here, we show that single-cell photoacoustic microscopy can reach throughputs of approximately 12,000 cells per hour by trapping single cells with blood in an oxygen-diffusion-limited high-density microwell array and by using photoacoustic imaging to measure the haemoglobin oxygen change (that is, the oxygen consumption rate) in the microwells. We demonstrate the capability of this label-free technique by performing high-throughput single-cell oxygen-consumption-rate measurements of cultured cells and by imaging intratumoral metabolic heterogeneity in specimens from patients with breast cancer. High-throughput single-cell photoacoustic microscopy of oxygen consumption rates should enable the faster characterization of intratumoral metabolic heterogeneity

Photoacoustic microscopy enables multilayered histological imaging of human breast cancer without staining

In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming for large specimens. Thus, 20-60% of patients undergoing BCS still require second surgeries due to positive surgical margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional histology and measuring features related to the histological landscape without the need for labels. We demonstrate that our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast tissue

Lymphovascular space invasion and lack of downstaging after neoadjuvant chemotherapy are strong predictors of adverse outcome in young women with locally advanced breast cancer

Younger age diagnosis of breast cancer is a predictor of adverse outcome. Here, we evaluate prognostic factors in young women with locally advanced breast cancer (LABC). We present a retrospective review of 104 patients younger than 40 years with LABC treated with surgery, radiotherapy (RT), and chemotherapy from 2003 to 2014. Patient‐, tumor‐, and treatment‐related factors important for overall survival (OS), local/regional recurrence (LRR), distant metastasis (DM), and recurrence‐free survival (RFS) were evaluated. Mean age at diagnosis was 34 years (23–39 years) with a median follow‐up of 47 months (8–138 months). Breast‐conserving surgery was performed in 27%. Axillary lymph node dissection was performed in 85%. Sixty percent of patients received neoadjuvant chemotherapy with 19% achieving pathologic complete response (pCR), and 61% downstaged. Lymph node positivity was present in 91% and lymphovascular space invasion (LVSI) in 35%. Thirty‐two percent of patients had triple negative tumors (TN, ER‐/PR‐/HER2 nonamplified). Four‐year OS and RFS was 84% and 71%, respectively. Factors associated with worse OS on multivariate analysis include TN status, LVSI, and number of positive lymph nodes. LVSI was also associated with DM and LRR, as well as worse RFS. Downstaging was associated with improved 4 year RFS in patients receiving neoadjuvant chemotherapy (74% vs. 38%, P = 0.002). With high risks of recurrence and inferior OS compared to older women, breast cancer in young women can be difficult to treat. Among additional factors, presence of LVSI and lack of downstaging portends a particularly worse prognosis

IL-12p70–producing patient DC vaccine elicits Tc1-polarized immunity

Background. Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients, but dose-limiting toxicities have hindered its incorporation in vaccine formulations. Here, we report on the immunological and clinical outcomes upon vaccination with CD40L/IFN-γ–matured, IL-12p70–producing DCs. Methods. 7 HLA-A*0201(+) newly diagnosed stage IV melanoma patients were immunized against the gp100 melanoma antigen using autologous peptide-pulsed, CD40L/IFN-γ–matured DCs. PBMCs were taken weekly for immune monitoring by tetramer analysis and functional assays. CT imaging was performed at baseline, week 9, and week 18 for clinical assessment using RECIST. Results. 6 of 7 treated patients developed sustained T cell immunity to all 3 melanoma gp100 antigen–derived peptides. 3 of the 6 immunological responders developed confirmed clinical responses (1 complete remission >4 years, 2 partial response). Importantly, DC vaccine–derived IL-12p70 levels positively correlated with time to progression (P = 0.019, log-rank), as did T-cytotoxic 1 (Tc1) immunity, as assessed by IFN-γ/IL-13 and IFN-γ/IL-5 ratios (P = 0.035 and P = 0.030, respectively, log-rank). In contrast, a pathway-specific defect in IL-12p35 transcription was identified upon CD40L/IFN-γ activation in clinical nonresponder patient DCs, and gp100-specific T cells from these patients displayed a Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ activation protocol corrected the IL-12p70 production defect in DCs derived from clinical nonresponder patients. Conclusion. These findings underscore the essential role of IL-12p70 in the development of therapeutic type 1 antigen–specific CD8(+) T cell immunity in humans with cancer. Trial registration. Clinicaltrials.gov NCT00683670. Funding. Barnes-Jewish Hospital Foundation, Siteman Cancer Frontier Fund, Washington University/JNJ Translational Medicine Award, and NCI (P30 CA91842)