The Impact of Temporal Variation in Indocyanine Green Administration on Tumor Identification During Fluorescence Guided Breast Surgery.

BACKGROUND: On average, 21% of women in the USA treated with Breast Conserving Surgery (BCS) undergo a second operation because of close positive margins. Tumor identification with fluorescence imaging could improve positive margin rates through demarcating location, size, and invasiveness of tumors. We investigated the technique's diagnostic accuracy in detecting tumors during BCS using intravenous indocyanine green (ICG) and a custom-built fluorescence camera system. METHODS: In this single-center prospective clinical study, 40 recruited BCS patients were sub-categorized into two cohorts. In the first 'enhanced permeability and retention' (EPR) cohort, 0.25 mg/kg ICG was injected ~ 25 min prior to tumor excision, and in the second 'angiography' cohort, ~ 5 min prior to tumor excision. Subsequently, an in-house imaging system was used to image the tumor in situ prior to resection, ex vivo following resection, the resection bed, and during grossing in the histopathology laboratory to compare the technique's diagnostic accuracy between the cohorts. RESULTS: The two cohorts were matched in patient and tumor characteristics. The majority of patients had invasive ductal carcinoma with concomitant ductal carcinoma in situ. Tumor-to-background ratio (TBR) in the angiography cohort was superior to the EPR cohort (TBR = 3.18 ± 1.74 vs 2.10 ± 0.92 respectively, p = 0.023). Tumor detection reached sensitivity and specificity scores of 0.82 and 0.93 for the angiography cohort and 0.66 and 0.90 for the EPR cohort, respectively (p = 0.1051 and p = 0.9099). DISCUSSION: ICG administration timing during the angiography phase compared with the EPR phase improved TBR and diagnostic accuracy. Future work will focus on image pattern analysis and adaptation of the camera system to targeting fluorophores specific to breast cancer.

Can we see what is invisible? The role of MRI in the evaluation and management of patients with pathological nipple discharge.

INTRODUCTION: The aim of this study was to determine the ability of MRI to identify and assess the extent of disease in patients with pathological nipple discharge (PND) with an occult malignancy not evident on standard pre-operative evaluation with mammography and ultrasound. METHODS: Patients presenting to the breast unit of Imperial College Healthcare NHS Trust between December 2009 and December 2018 with PND and normal imaging were enrolled in the study. Pre-operative bilateral breast MRI was performed in all patients as part of our protocol and all patients were offered diagnostic microdochectomy. RESULTS: A total of 82 patients fulfilled our selection criteria and were enrolled in our study. The presence of an intraductal papilloma (IDP) was identified as the cause of PND in 38 patients (46.3%), 14 patients had duct ectasia (DE-17%) and 5 patients had both an IDP and DE. Other benign causes were identified in 11 patients (13.4%). Despite normal mammography and ultrasound a malignancy was identified in 14 patients (17%). Eleven patients had DCIS (13.4%), two had invasive lobular carcinoma and one patient had an invasive ductal carcinoma. The sensitivity of MRI in detecting an occult malignancy was 85.71% and the specificity was 98.53%. The positive predictive value was 92.31% and the negative predictive value was 97.1%. CONCLUSIONS: Although a negative MRI does not exclude the presence of an occult malignancy the high sensitivity and specificity of this diagnostic modality can guide the surgeon and alter the management of patients with PND.

Rapid evaporative ionisation mass spectrometry of electrosurgical vapours for the identification of breast pathology: towards an intelligent knife for breast cancer surgery.

BACKGROUND: Re-operation for positive resection margins following breast-conserving surgery occurs frequently (average = 20-25%), is cost-inefficient, and leads to physical and psychological morbidity. Current margin assessment techniques are slow and labour intensive. Rapid evaporative ionisation mass spectrometry (REIMS) rapidly identifies dissected tissues by determination of tissue structural lipid profiles through on-line chemical analysis of electrosurgical aerosol toward real-time margin assessment. METHODS: Electrosurgical aerosol produced from ex-vivo and in-vivo breast samples was aspirated into a mass spectrometer (MS) using a monopolar hand-piece. Tissue identification results obtained by multivariate statistical analysis of MS data were validated by histopathology. Ex-vivo classification models were constructed from a mass spectral database of normal and tumour breast samples. Univariate and tandem MS analysis of significant peaks was conducted to identify biochemical differences between normal and cancerous tissues. An ex-vivo classification model was used in combination with bespoke recognition software, as an intelligent knife (iKnife), to predict the diagnosis for an ex-vivo validation set. Intraoperative REIMS data were acquired during breast surgery and time-synchronized to operative videos. RESULTS: A classification model using histologically validated spectral data acquired from 932 sampling points in normal tissue and 226 in tumour tissue provided 93.4% sensitivity and 94.9% specificity. Tandem MS identified 63 phospholipids and 6 triglyceride species responsible for 24 spectral differences between tissue types. iKnife recognition accuracy with 260 newly acquired fresh and frozen breast tissue specimens (normal n = 161, tumour n = 99) provided sensitivity of 90.9% and specificity of 98.8%. The ex-vivo and intra-operative method produced visually comparable high intensity spectra. iKnife interpretation of intra-operative electrosurgical vapours, including data acquisition and analysis was possible within a mean of 1.80 seconds (SD ±0.40). CONCLUSIONS: The REIMS method has been optimised for real-time iKnife analysis of heterogeneous breast tissues based on subtle changes in lipid metabolism, and the results suggest spectral analysis is both accurate and rapid. Proof-of-concept data demonstrate the iKnife method is capable of online intraoperative data collection and analysis. Further validation studies are required to determine the accuracy of intra-operative REIMS for oncological margin assessment.

Cutaneous epithelioid malignant peripheral nerve sheath tumour: a clinicopathological analysis of 11 cases.

AIMS: Epithelioid malignant peripheral nerve sheath tumour (E-MPNST) is a distinctive variant of malignant peripheral nerve sheath tumour characterized by the predominance of epithelioid cells, diffuse S100 positivity and infrequent association with neurofibromatosis type 1. The aim of this study was to further delineate clinicopathological features of cutaneous E-MPNST, correlate them with disease outcome and discuss differential diagnosis. METHODS AND RESULTS: We analysed 11 cutaneous E-MPNSTs (six males, five females, median age 49 years, median size 1.6 cm). Tumours showed a predilection for lower extremities (45%) and trunk (45%), followed by upper extremity (9%). Follow-up was available for nine of 11 patients (range 24-100 months, median 52 months). Four patients had an uneventful clinical course (44%), two developed local recurrence(s) (22%) and three died due to disseminated disease (33%). No histological parameters were found to predict local recurrence(s), development of distant metastases or disease outcome, including size, percentage of epithelioid component, number of mitoses per 10 high-power fields, degree of nuclear atypia or site of occurrence (dermis, dermis/subcutis, subcutis) (P > 0.05). Immunohistochemically, all tumours were diffusely S100-positive, with a subset displaying loss of integrase interactor 1 (INI1) expression (50%). CONCLUSIONS: Cutaneous E-MPNST has the potential to pursue an aggressive clinical course, associated with wide dissemination and unfavourable disease outcome.

Imaging breast cancer morphology using probe-based confocal laser endomicroscopy: towards a real-time intraoperative imaging tool for cavity scanning.

Current techniques for assessing the adequacy of tumour excision during breast conserving surgery do not provide real-time direct cytopathological assessment of the internal cavity walls within the breast. This study investigates the ability of probe-based confocal laser endomicroscopy (pCLE), an emerging imaging tool, to image the morphology of neoplastic and non-neoplastic breast tissues, and determines the ability of histopathologists and surgeons to differentiate these images. Freshly excised tumour samples and adjacent non-diseased sections from 50 consenting patients were stained with 0.01 % acriflavine hydrochloride and imaged using pCLE. All discernible pCLE features were cross-examined with conventional histopathology. Following pattern recognition training, 17 histopathologists and surgeons with no pCLE experience interpreted 50 pCLE images independently whilst blinded to histopathology results. Three-hundred and fifty pCLE image mosaics were analysed. Consistent with histopathology findings, the glandular structures, adipocytes and collagen fibres of normal breast were readily visible on pCLE images. These were distinguishable from the morphological architecture exhibited by invasive and non-invasive carcinoma. The mean accuracy of pCLE image interpretation for histopathologists and surgeons was 94 and 92 %, respectively. Overall, inter-observer agreement for histopathologists was 'almost perfect', κ = 0.82; and 'substantial' for surgeons, κ = 0.74. pCLE morphological features of neoplastic and non-neoplastic breast tissues are readily visualized and distinguishable with high accuracy by both histopathologists and surgeons. Further research is required to investigate a potential role for the use of pCLE intraoperatively for in situ detection of residual cancerous foci, thereby guiding operating decision-making based on real-time breast cavity scanning.

Quantitative Imaging In Vivo of Functioning Lymphatic Vessels Around Human Melanoma and Benign Nevi.

OBJECTIVES: The density of functioning human lymphatics in vivo and of immunohistochemically defined lymphatics was quantified around melanomas, benign nevi, and matched normal skin, to assess the current lymphangiogenesis paradigm. We investigated whether histological and functioning density increased around melanomas compared with benign nevi or matched skin; whether functioning and histological density increased similarly; and whether larger increases occurred around metastatic melanomas. METHODS: Functioning density was quantified in vivo as the total amount of human dermal microlymphatics taking up fluorescent marker injected at the lesion margin. After tissue excision, perilesion histological density was quantified using podoplanin marker D2-40. RESULTS: Histological density was raised similarly around metastasising and non-metastasising melanomas compared with normal skin (+71%, p < 0.0001, n = 32); but was also raised significantly around benign nevi (+17%, p = 0.03, n = 20). In contrast, functioning lymphatic density was substantially reduced around the margins of melanomas (both metastasising and non-metastasising) compared with benign nevi (by 65%, p = 0.02) or normal skin (by 53%, p = 0.0014). CONCLUSIONS: Raised perilesion histological lymphatic density is not unique to melanoma but occurs also around benign nevi. The findings indicated that the number of functioning lateral lymphatics around human melanomas in vivo but not benign nevi is reduced, despite histologically increased numbers of lymphatics.

Indocyanine green fluorescence image processing techniques for breast cancer macroscopic demarcation.

Re-operation due to disease being inadvertently close to the resection margin is a major challenge in breast conserving surgery (BCS). Indocyanine green (ICG) fluorescence imaging could be used to visualize the tumor boundaries and help surgeons resect disease more efficiently. In this work, ICG fluorescence and color images were acquired with a custom-built camera system from 40 patients treated with BCS. Images were acquired from the tumor in-situ, surgical cavity post-excision, freshly excised tumor and histopathology tumour grossing. Fluorescence image intensity and texture were used as individual or combined predictors in both logistic regression (LR) and support vector machine models to predict the tumor extent. ICG fluorescence spectra in formalin-fixed histopathology grossing tumor were acquired and analyzed. Our results showed that ICG remains in the tissue after formalin fixation. Therefore, tissue imaging could be validated in freshly excised and in formalin-fixed grossing tumor. The trained LR model with combined fluorescence intensity (pixel values) and texture (slope of power spectral density curve) identified the tumor's extent in the grossing images with pixel-level resolution and sensitivity, specificity of 0.75 ± 0.3, 0.89 ± 0.2.This model was applied on tumor in-situ and surgical cavity (post-excision) images to predict tumor presence.

Malignancies confined to disused arteriovenous fistulae in renal transplant patients: an important differential diagnosis.

BACKGROUND: Swelling in an arteriovenous fistula (AVF) is commonly caused by thrombosis, aneurysm and infection. However, due to the increased risk of malignancy after transplantation, this should also be considered. PATIENTS: We discuss 4 patients with malignancy confined to an AVF after renal transplantation presenting in a 2-year period. Angiosarcoma was diagnosed in 3 patients and the other had post-transplant lymphoproliferative disorder (PTLD). Angiosarcoma behaves aggressively and 2 of our patients died within 6 months of diagnosis. There are 6 previous cases and 5 died within 16 months of diagnosis. PTLD at AVFs has not been documented previously. CONCLUSION: Malignancy at an AVF is a rare but important differential that can impact significantly on patient morbidity and mortality. Predilection for malignancy at an AVF is not understood. We review the literature and discuss possible aetiologies.

Spatially resolved metabolic phenotyping of breast cancer by desorption electrospray ionization mass spectrometry.

Breast cancer is a heterogeneous disease characterized by varying responses to therapeutic agents and significant differences in long-term survival. Thus, there remains an unmet need for early diagnostic and prognostic tools and improved histologic characterization for more accurate disease stratification and personalized therapeutic intervention. This study evaluated a comprehensive metabolic phenotyping method in breast cancer tissue that uses desorption electrospray ionization mass spectrometry imaging (DESI MSI), both as a novel diagnostic tool and as a method to further characterize metabolic changes in breast cancer tissue and the tumor microenvironment. In this prospective single-center study, 126 intraoperative tissue biopsies from tumor and tumor bed from 50 patients undergoing surgical resections were subject to DESI MSI. Global DESI MSI models were able to distinguish adipose, stromal, and glandular tissue based on their metabolomic fingerprint. Tumor tissue and tumor-associated stroma showed evident changes in their fatty acid and phospholipid composition compared with normal glandular and stromal tissue. Diagnosis of breast cancer was achieved with an accuracy of 98.2% based on DESI MSI data (PPV 0.96, NVP 1, specificity 0.96, sensitivity 1). In the tumor group, correlation between metabolomic profile and tumor grade/hormone receptor status was found. Overall classification accuracy was 87.7% (PPV 0.92, NPV 0.9, specificity 0.9, sensitivity 0.92). These results demonstrate that DESI MSI may be a valuable tool in the improved diagnosis of breast cancer in the future. The identified tumor-associated metabolic changes support theories of de novo lipogenesis in tumor tissue and the role of stroma tissue in tumor growth and development and overall disease prognosis.

Features of sentinel lymph nodes for melanoma may lead to re-diagnosis of the cutaneous primary: an unusual case and review of literature.

Although sentinel lymph-node biopsy is accepted as a reliable method of staging of melanoma, it is not without problems to the pathologist. It has been re-emphasised that aggregates of benign naevus cells are not uncommon. Usually these are easily identified by a combination of their benign cytology and location in the fibrous skeleton of lymph nodes. This case represents a combination of an unusual pseudo-malignant pattern in the primary lesion with unusual morphology of the sentinel lymph node. The latter prompted reassessment of the cutaneous lesion as a benign naevus. Confirmation of the diagnosis as cutaneous melanoma by a positive sentinel-node biopsy was averted only by a careful comparison of unusual features of the putative primary and the sentinel lymph node. This case illustrates the need for a rigorous protocol for pathological assessment of sentinel lymph nodes for melanomas to assure detection of all metastases but also to avoid misdiagnosis and over-treatment. It also supports "benign metastases" as the mechanism underlying at least some melanocytes in regional lymph nodes.

Primary cutaneous adenoid cystic carcinoma: a clinicopathologic and immunohistochemical study of 27 cases.

AIMS: This study examines clinical and pathologic features of primary cutaneous adenoid cystic carcinoma (ACC), with emphasis on biological behavior of these tumors. A total of 27 cases of primary cutaneous ACC with detailed follow-up information were evaluated. Clinically, these were solitary, slow-growing lesions, half of which were in the head and neck area. The median age was 62 years with a male predilection. Surgical excision was the treatment of choice. Histologically, the lesions were similar to those seen in the salivary glands. Tumors were classified as grade 1 (17), grade 2 (3), and grade 3 (7). The mitotic count was generally low (mean=1.9/mm), except in 2 high-grade tumors (>10 mitotic figures/mm). Sixteen cases showed perineural invasion. Immunohistochemically, cytokeratin positivity was noted in 13/13 cases, and CD117 was observed in 10/10 cases, with luminal/cytoplasmic staining for epithelial membrane antigen (14/16) and at least focal luminal expression for carcinoembryonic antigen (11/16), smooth muscle actin (10/13), and S100 staining (9/13). Eighteen cases had follow-up data (median 54 mo), 9 of which had local recurrences (50%). Three cases showed metastatic disease. No statistical difference was noted between tumor grade and local recurrence (P=0.77). Primary cutaneous ACC is a distinct entity with a more indolent behavior compared with its salivary counterpart. The cutaneous lesions tend to recur locally but have a low metastatic potential.

Inflammation-based prognostic indices in malignant pleural mesothelioma.

INTRODUCTION: Chronic inflammation plays a key role in the pathogenesis of malignant pleural mesothelioma (MPM) as a result of asbestos exposure. Biomarkers of systemic inflammation have been shown to predict the natural history of MPM; however, this observation lacks independent validation. Our aim was to compare the prognostic performance of three inflammation-based biomarkers in predicting overall survival (OS) in MPM. METHODS: In patients with histologically proven MPM, the inflammation-based prognostic scores modified Glasgow Prognostic Score (mGPS), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio were studied and compared with the European Organization for the Research and Treatment of Cancer Prognostic Score (EPS) and other known potential prognostic factors such as gender, histologic subtype, Eastern Cooperative Oncology Group performance status, and baseline blood parameters. RESULTS: A total of 171 MPM patients presenting to Imperial College NHS Trust were studied. In univariate analyses, the following parameters were predictors of OS: female gender (p = 0.03), epithelioid histology (p = 0.03), normal C-reactive protein (p = 0.03), baseline white blood cell count <8.3 × 10/liter (p = 0.04), EPS (p = 0.003), mGPS (p < 0.001), NLR (p = 0.006), and platelet-to-lymphocyte ratio (p = 0.03). Multivariate survival analysis confirmed the mGPS (hazard ratio = 2.6; p < 0.001) and NLR (hazard ratio = 2.0; p = 0.008), but not the EPS, as independent predictors of OS. Tissue expression of Ki-67 (p < 0.001) and vascular endothelial growth factor (p < 0.001) was higher in a subgroup of patients with high-risk inflammatory scores. CONCLUSIONS: The mGPS and NLR are externally validated prognostic indices in patients with MPM and correlate with sustained neoangiogenesis and increased proliferative index.

Morphological changes in breast tissue with menstrual cycle.

Whether the breast tissue undergoes morphologic changes in relation to the menstrual cycle had been a subject of debate. Elegant studies performed in the early 1980s provided conclusive evidence of cyclical changes in the normal breast lobules. These studies were almost entirely based on autopsy material and have not been validated in the clinical setting. In the present study, we examine breast tissues from surgical specimens from 73 premenopausal women and use morphological criteria to characterize the stage of the menstrual cycle. Patients taking oral contraceptives or hormonal therapy were excluded from this study. The following histological parameters were used to assess the menstrual stage: number of cell layers in the acini and presence and degree of vacuolation of the myoepithelial cells, stromal edema, infiltrate, mitosis, and apoptosis. The morphological stage was then correlated with the stage of the cycle, as determined by last menstrual period and the usual menstrual cycle length and in some patients with serum estrogen and progesterone levels. The morphologic stage was concordant with dates in 54 of the 73 patients (74%, P =.001). In 31 of these patients, serum levels of estradiol and progesterone at the time of surgery were available for correlation. Twenty-five (80%) of these were phase concordant by morphology and progesterone levels (P =.01), and 25 (80%), by dates and progesterone levels (P =.007). Women with a high morphologic score were seven times as likely to be in luteal phase as were women with a low score (odds ratio, 7.1; 95% confidence interval). Menstrual phase can be determined by the morphology of the normal lobules present within the surgically excised breast specimens. This will permit retrospective analysis of large archival databases to analyze the effect of timing of surgery in relation to menstrual cycle phase. It will also aid the design of epidemiological studies for breast cancer risk assessment.

Expression of interleukin-6 and tumor necrosis factor alpha and histopathologic findings in painful and nonpainful breast tissue.

Mastalgia is a common condition that is thought to be hormonally related, but the mechanisms of pain causation are unknown. Inflammatory cytokines are implicated in pain modulation, but have not been studied with regard to mastalgia. We compared the relationship of mastalgia to the expression of the cytokines interleukin (IL)-6, IL-1beta, and tumor necrosis factor (TNF)-alpha and the degree of tissue infiltration with inflammatory cells in breast tissue from 29 premenopausal women with breast pain and 29 age-matched pain-free controls. Paraffin sections from breast biopsy samples were scored for the presence of inflammatory infiltrate and were evaluated for the expression of IL-6, IL-1beta, and TNF-alpha using standard immunohistochemical procedures. TNF-alpha and IL-6 expression displayed a trend toward slightly lower values in patients with pain (median TNF-alpha score, 3 versus 5; median IL-6 score, 3 versus 4). In the luteal phase, patients with mastalgia showed a trend toward lower expression of IL-6 (p = 0.4) in comparison to those without pain. A similar trend was also seen with TNF-alpha expression (p = 0.4). IL-1beta expression was extremely scant in the first 30 samples and was not investigated further. The degree of inflammatory infiltrate in the tissue was unrelated to the presence of breast pain. These data suggest that the three cytokines tested in this study do not play a significant role in the causation of mastalgia and lend weight to the previous finding that there are no identifiable histologic correlates of this troubling condition. Further investigation of the role of cytokines in breast pain is warranted, especially in view of the possible association between mastalgia and breast cancer risk.

Normal breast lobular architecture in breast biopsy samples from breast cancer cases and benign disease controls.

Rodent studies suggest a relationship between lobular maturation and breast cancer risk. Human data are sparse, and were developed using whole mounts of mastectomy or mammoplasty samples, without consideration of menstrual phase in premenopausal women. We studied normal breast lobules in relation to cancer risk in 284 women, using surgical biopsy material (mean two sections and 43.2 lobular structures per subject): 167 were premenopausal; 89 with breast cancer (cases) and 78 undergoing benign breast biopsy (controls). Of 117 postmenopausal women, 67 were cases and 50 were controls. Normal lobular type was classified based on size, and was designated predominant if it constituted 60% or more of the total lobules classified. The control group showed 66% type I, 34% type II and 1% type III lobules while cases showed 69% type I, 31% type II and 7% type III structures. Predominant lobule type showed no association with cancer (p = 0.9). Postmenopausal women had a substantially higher proportion of type I lobules compared to premenopausal women, irrespective of the parity or cancer status (p < 0.001). Lobule type was not associated with menstrual phase classified by dates; however, when menstrual phase was classified using breast morphological characteristics, type I lobules were more abundant in follicular phase and type II in the luteal phase (p < 0.001). In conclusion, we did not observe a relationship between lobular architecture and breast cancer susceptibility when using smaller breast samples usually available in epidemiological studies, but these data highlight the need for menstrual phase stratification in future investigations.

Ductal lavage findings in women with known breast cancer undergoing mastectomy.

BACKGROUND: Ductal lavage has the potential to detect cancer by sampling breast epithelium in asymptomatic high-risk women. To assess the utility of ductal lavage as a cancer diagnostic test, we investigated the association between ductal lavage cytologic findings and histologic findings in women with known breast cancer undergoing mastectomy. METHODS: Ductal lavage was performed in the operating room before mastectomy on 44 breasts from 32 women with known cancer and on eight breasts from seven women undergoing prophylactic mastectomy, two with occult malignancy. If the ductal lavage sample from one or more ducts contained enough epithelial cells for a cytologic diagnosis, lavaged ducts were injected with a mixture of colored dye, gelatin, and a radiographic contrast compound after mastectomy, and breast tissue was radiographed and sectioned. Histologic findings in ducts with and without dye were recorded. Associations between cytologic results and histologic results were examined by univariate and multivariable analyses. RESULTS: At least one duct was lavaged in 36 breasts (mean = 1.4 ducts per breast); all histologic and cytologic procedures were completed in 28 breasts and in 39 ducts. Markedly atypical or malignant cytology was found in five cancer-containing breasts. In 39 ducts with complete cytologic and histologic data and when marked atypia or malignant cells defined a positive cytologic test, sensitivity was 43% (95% confidence interval [CI] = 23% to 72%), specificity was 96% (95% CI = 86% to 100%), and accuracy was 77% (95% CI = 63% to 89%). When mild or marked atypia or malignant cells defined a positive cytologic test, sensitivity was 79% (95% CI = 57% to 96%), specificity was 64% (95% CI = 46% to 83%), and accuracy was 69% (95% CI = 55% to 83%). When all 31 cytologically evaluable breasts were analyzed, sensitivity was 17% (95% CI = 7% to 35%), specificity was 100% (95% CI = 5% to 100%), and accuracy was 19% (95% CI = 9% to 38%). CONCLUSION: In breasts with cancer, ductal lavage appears to have low sensitivity and high specificity for cancer detection, possibly because cancer-containing ducts fail to yield fluid or have benign or mildly atypical cytology.