Development and Validation of a HER2-Low Focused IHC Scoring System with High Inter-Observer Concordance The Australian HER2-low Breast Cancer Concordance Study.

The DESTINY Breast-04 Trial (DB-04) revealed survival advantages of Trastuzumab Deruxtecan for women with metastatic HER2-low breast cancer (1+ or 2+ IHC, without amplification). While DB-04 applied the 2018 ASCO CAP HER2 IHC scoring criteria, the subjectivity and imprecision in IHC scoring have raised concerns that patients' treatment may be mis-aligned. Our group of 9 experienced breast pathologists collated a deidentified set of 60 breast cancer core biopsies from 3 laboratories, evaluated with the Ventana 4B5 HER2 assay and mostly scored locally as HER2 0 or 1+. Based on ASCO CAP 2018 criteria and our extensive experience of reporting HER2 IHC, we specified scoring conventions for cancers with low levels of HER2 protein expression, articulating specific scoring pitfalls. Each pathologist then reviewed digitised whole slide images of the IHC slides and scored the HER2 expression for each case. At a subsequent consensus workshop, we reviewed the cases jointly to i) establish consensus scores for each case and ii) determine the percentage of HER2 expressing tumour cells. Consensus was reached on all cases, 40 classified as 1+ and three as 2+ (not amplified), totalling 43 (71.7%) HER2-low cancers. The remaining cases were HER2 0. In 93.3% (56 of 60) cases the consensus score matched with the majority opinion of pathologists' independent scores. 7 of 17 (41.2%) cases reported locally as HER2 0 were classified as HER2-low. Conversely, among 32 cases with local scores of 1+, 7 (21.8%) were reclassified as ultralow or null. Individual pathologists' accuracy in matching the consensus scores ranged from 73.3-91.67% (mean 80.74%). Among HER2-low cancers those in which < 20% of the tumor cells expressed HER2 had the lowest concordance levels. Observers Cohen's Kappa coefficients for concordance was excellent for 4, good in 1 and moderate in the 4 observers. This reference set of cases with expert consensus HER2 scores will be invaluable for peer training and development of our national external quality assurance program for HER2-low cancers. For assessing breast cancers at the low end of HER2 protein expression, our targeted scoring criteria and explicit instruction on pitfalls, improved pathologists' accuracy and concordance.

Accuracy and precision of contrast enhanced mammography versus MRI for predicting breast cancer size: how "good" are they really?

OBJECTIVE: To evaluate and compare the accuracy and precision of contrast-enhanced mammography (CEM) vs MRI to predict the size of biopsy-proven invasive breast cancer. METHODS: Prospective study, 59 women with invasive breast cancer on needle biopsy underwent CEM and breast MRI. Two breast radiologists read each patient's study, with access limited to one modality. CEM lesion size was measured using low-energy and recombined images and on MRI, the first post-contrast series. Extent of abnormality per quadrant was measured for multifocal lesions. Reference standards were size of largest invasive malignant lesion, invasive (PathInvasive) and whole (PathTotal). Pre-defined clinical concordance ±10 mm. RESULTS: Mean patient age 56 years, 42 (71%) asymptomatic. Lesions were invasive ductal carcinoma 40 (68%) with ductal carcinoma in situ (31/40) in 78%, multifocal in 12 (20%). Median lesion size was 17 mm (invasive) and 27 mm (total), range (5-125 mm). Lin's concordance correlation coefficients for PathTotal 0.75 (95% CI 0.6, 0.84) and 0.71 (95% CI 0.56, 0.82) for MRI and contrast-enhanced spectral mammography (CESM) respectively. Mean difference for total size, 3% underestimated and 4% overestimated, and for invasive 41% and 50% overestimate on MRI and CESM respectively. LOAs for PathTotal varied from 60% under to a 2.4 or almost threefold over estimation. MRI was concordant with PathTotal in 36 (64%) cases compared with 32 (57%) for CESM. Both modalities concordant in 26 (46%) cases respectively. CONCLUSION: Neither CEM nor MRI have sufficient accuracy to direct changes in planned treatment without needle biopsy confirmation. ADVANCES IN KNOWLEDGE: Despite small mean differences in lesion size estimates using CEM or MRI, the 95% limits of agreement do not meet clinically acceptable levels.

Vacuum-assisted excision biopsy for definitive diagnosis of breast lesions of uncertain malignant potential (B3 lesions) on core biopsy - A single centre Western Australian experience.

INTRODUCTION: In Australia, the usual approach to breast lesions where core biopsy returns an uncertain result ("B3" breast lesion) is to perform surgical diagnostic open biopsy (DOB). This is associated with patient time off work, costs of hospital admission, risks of general anaesthesia and surgical complications. The majority of B3 lesions return benign results following surgery. Vacuum assisted excision biopsy (VAEB) is a less invasive, lower cost alternative, and is standard of care for selected B3 lesions in the United Kingdom. Similar use of VAEB in Australia, could save many women unnecessary surgery. The aim of this study was to document our experience during the introduction of VAEB as an alternative to DOB for diagnosis of selected B3 lesions. METHODS: The multidisciplinary team developed an agreed VAEB pathway for selected B3 lesions. Technically accessible papillary lesions, mucocele-like lesions and radial scars without atypia measuring ≤ 15mm were selected. RESULTS: Over a 7 month period, 18 women with 20 B3 lesions were offered VAEB. 16 women (18 lesions) chose VAEB over DOB. Papillomas were the commonest lesion type. All lesions were successfully sampled: 17/18 were benign. One lesion (6%) was upgraded to malignancy (ductal carcinoma in situ on VAEB, invasive ductal carcinoma at surgery). No major complications occurred. Patient satisfaction was high: 15/16 respondents would again choose VAEB over surgery. CONCLUSION: VAEB is a patient-preferred, safe, well-tolerated, lower-cost alternative to DOB for definitive diagnosis of selected B3 breast lesions.

Quantitative Micro-Elastography Enables In Vivo Detection of Residual Cancer in the Surgical Cavity during Breast-Conserving Surgery.

Breast-conserving surgery (BCS) is commonly used for the treatment of early-stage breast cancer. Following BCS, approximately 20% to 30% of patients require reexcision because postoperative histopathology identifies cancer in the surgical margins of the excised specimen. Quantitative micro-elastography (QME) is an imaging technique that maps microscale tissue stiffness and has demonstrated a high diagnostic accuracy (96%) in detecting cancer in specimens excised during surgery. However, current QME methods, in common with most proposed intraoperative solutions, cannot image cancer directly in the patient, making their translation to clinical use challenging. In this proof-of-concept study, we aimed to determine whether a handheld QME probe, designed to interrogate the surgical cavity, can detect residual cancer directly in the breast cavity in vivo during BCS. In a first-in-human study, 21 BCS patients were scanned in vivo with the QME probe by five surgeons. For validation, protocols were developed to coregister in vivo QME with postoperative histopathology of the resected tissue to assess the capability of QME to identify residual cancer. In four cavity aspects presenting cancer and 21 cavity aspects presenting benign tissue, QME detected elevated stiffness in all four cancer cases, in contrast to low stiffness observed in 19 of the 21 benign cases. The results indicate that in vivo QME can identify residual cancer by directly imaging the surgical cavity, potentially providing a reliable intraoperative solution that can enable more complete cancer excision during BCS. SIGNIFICANCE: Optical imaging of microscale tissue stiffness enables the detection of residual breast cancer directly in the surgical cavity during breast-conserving surgery, which could potentially contribute to more complete cancer excision.

Multi-class classification of breast tissue using optical coherence tomography and attenuation imaging combined via deep learning.

We demonstrate a convolutional neural network (CNN) for multi-class breast tissue classification as adipose tissue, benign dense tissue, or malignant tissue, using multi-channel optical coherence tomography (OCT) and attenuation images, and a novel Matthews correlation coefficient (MCC)-based loss function that correlates more strongly with performance metrics than the commonly used cross-entropy loss. We hypothesized that using multi-channel images would increase tumor detection performance compared to using OCT alone. 5,804 images from 29 patients were used to fine-tune a pre-trained ResNet-18 network. Adding attenuation images to OCT images yields statistically significant improvements in several performance metrics, including benign dense tissue sensitivity (68.0% versus 59.6%), malignant tissue positive predictive value (PPV) (79.4% versus 75.5%), and total accuracy (85.4% versus 83.3%), indicating that the additional contrast from attenuation imaging is most beneficial for distinguishing between benign dense tissue and malignant tissue.

Implant deformity on screening mammography, an unusual presentation of breast implant associated lymphoma.

Breast implant associated anaplastic large cell lymphoma (BIA-ALCL) is a rare condition related to textured breast implants. Recognition of characteristic imaging and pathological features are important, given the absence of symptoms can delay diagnosis, as illustrated by this case. Late-onset peri-implant effusion is commonly encountered whilst an associated mass or lymphadenopathy are rare. Clinical and radiological suspicion enables dedicated pathology work-up for diagnosis. Ultrasound is vital for initial work-up whilst MRI and PET-CT assist in staging. Surgical explantation is followed by adjuvant chemo-radiotherapy according to disease extent.

Bloody nipple discharge in Carney complex: A case report.

Carney complex (CNC) is an extremely rare, autosomal dominant genetic syndrome consisting of pigmented skin and mucosal changes with multiple endocrine and nonendocrine tumors, including the breast. Breast tumors are typically multiple and benign and are most commonly reported as myxoid fibroadenomas and/or intraductal papillomas. We present a young female patient with known CNC who presented with copious bloody nipple discharge with multiple breast lumps and discuss the breast imaging features regarding this complex and often underrecognized genetic condition.

Coherence function-encoded optical palpation.

Optical palpation maps stress at the surface of biological tissue into 2D images. It relies on measuring surface deformation of a compliant layer, which to date has been performed with optical coherence tomography (OCT). OCT-based optical palpation holds promise for improved clinical diagnostics; however, the complexity and cost hinder broad adoption. In this Letter, we introduce coherence function-encoded optical palpation (CFE-OP) using a novel optical profilometry technique that exploits the envelope of the coherence function rather than its peak position, which is typically used to retrieve depth information. CFE-OP utilizes a Fabry-Perot laser diode (bandwidth, 2.2 nm) and a single photodiode in a Michelson interferometer to detect the position along the coherence envelope as a function of path length. This technique greatly reduces complexity and cost in comparison to the OCT-based approach. We perform CFE-OP on phantom and excised human breast tissue, demonstrating comparable mechanical contrast to OCT-based optical palpation and the capability to distinguish stiff tumor from soft benign tissue.

Interobserver variability in the assessment of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative invasive breast carcinoma influences the association with pathological complete response: the IVITA study.

High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland-Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from -0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the 'ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

Diagnostic concordance among pathologists interpreting breast core biopsies on secondary review over a 1-year period at an Australian tertiary hospital.

This study provides data on the diagnostic concordance between initial and review diagnoses of all breast core biopsy cases at a single tertiary hospital in Western Australia over a 1-year period. A retrospective review of all breast core biopsy cases between January 1 and December 31, 2016, was carried out at PathWest, Fiona Stanley Hospital in Perth, Western Australia. Each biopsy is reported by a single pathologist and then reviewed within 1 week by a panel of intradepartmental subspecialist breast pathologists, who either agree with the original diagnosis, have a minor discordant diagnosis, or a major discordant diagnosis. Records for 2036 core biopsies were available between January 1 and December 31, 2016. Of these, 56.0% (n = 1141) were classified as benign, 34.3% (n = 699) as malignant, 7.2% (n = 147) as indeterminate, 2.3% (n = 46) as nondiagnostic, and 0.1% (n = 3) as suspicious for malignancy. In 99.1% (n = 2018) of cases, there was agreement between initial and review diagnoses. In total, 0.9% (n = 18) were disagreements: 0.49% (n = 10) were major discordant disagreements and 0.39% (n = 8) were minor discordant disagreements. All cases of major discordant disagreements would have resulted in significant changes to clinical management. This study demonstrates that an Australian institution is providing a high-quality pathology service with a low error rate between initial and review diagnoses of breast core biopsies. It reinforces the importance of secondary review of biopsies in a timely fashion for detecting potentially serious misdiagnoses that could lead to inappropriate management.

Optical palpation for tumor margin assessment in breast-conserving surgery.

Intraoperative margin assessment is needed to reduce the re-excision rate of breast-conserving surgery. One possibility is optical palpation, a tactile imaging technique that maps stress (force applied across the tissue surface) as an indicator of tissue stiffness. Images (optical palpograms) are generated by compressing a transparent silicone layer on the tissue and measuring the layer deformation using optical coherence tomography (OCT). This paper reports, for the first time, the diagnostic accuracy of optical palpation in identifying tumor within 1 mm of the excised specimen boundary using an automated classifier. Optical palpograms from 154 regions of interest (ROIs) from 71 excised tumor specimens were obtained. An automated classifier was constructed to predict the ROI margin status by first choosing a circle diameter, then searching for a location within the ROI where the circle was ≥ 75% filled with high stress (indicating a positive margin). A range of circle diameters and stress thresholds, as well as the impact of filtering out non-dense tissue regions, were tested. Sensitivity and specificity were calculated by comparing the automated classifier results with the true margin status, determined from co-registered histology. 83.3% sensitivity and 86.2% specificity were achieved, compared to 69.0% sensitivity and 79.0% specificity obtained with OCT alone on the same dataset using human readers. Representative optical palpograms show that positive margins containing a range of cancer types tend to exhibit higher stress compared to negative margins. These results demonstrate the potential of optical palpation for margin assessment.

Pre-operative localization of impalpable breast lesions using iodine 125 seeds: Placement accuracy and multidisciplinary challenges.

INTRODUCTION: The number of impalpable breast lesions requiring pre-operative lesion localization (PLL) continues to increase. The use of Radio-guided Occult Lesion Localization with Iodine 125 Seeds (ROLLIS) offers multiple benefits for the multidisciplinary team (MDT), but is not without challenges. AIMS: The aims of this audit were to review our multidisciplinary team's experience following introduction of ROLLIS as standard of care for PLL, identify challenges and evaluate seed placement accuracy (SPA). RESULTS/OUTCOMES: Over a nineteen month period, 327 seeds were inserted: 96% of single seed localizations were within 10 mm, 91% within 5 mm and 42% within or in contact with the lesion (or marker clip surrogate) on post-insertion two view mammography. Each component of the MDT reported on benefits of the ROLLIS program and challenges faced. Examples included: an undetectable seed in the operating room, a seed damaged in pathology during specimen processing, suboptimal seed position requiring hook-wire localization (HWL) and delayed seed removal in a patient who initially refused to return for surgery. CONCLUSION: ROLLIS results in high seed placement accuracy. Despite clear advantages, use of ROLLIS presents some multidisciplinary challenges. Robust patient information, training of new staff and adherence to strict policies and protocols are required to ensure safe delivery of a ROLLIS program.

Camera-based optical palpation.

Optical elastography is undergoing extensive development as an imaging tool to map mechanical contrast in tissue. Here, we present a new platform for optical elastography by generating sub-millimetre-scale mechanical contrast from a simple digital camera. This cost-effective, compact and easy-to-implement approach opens the possibility to greatly expand applications of optical elastography both within and beyond the field of medical imaging. Camera-based optical palpation (CBOP) utilises a digital camera to acquire photographs that quantify the light intensity transmitted through a silicone layer comprising a dense distribution of micro-pores (diameter, 30-100 µm). As the transmission of light through the micro-pores increases with compression, we deduce strain in the layer directly from intensity in the digital photograph. By pre-characterising the relationship between stress and strain of the layer, the measured strain map can be converted to an optical palpogram, a map of stress that visualises mechanical contrast in the sample. We demonstrate a spatial resolution as high as 290 µm in CBOP, comparable to that achieved using an optical coherence tomography-based implementation of optical palpation. In this paper, we describe the fabrication of the micro-porous layer and present experimental results from structured phantoms containing stiff inclusions as small as 0.5 × 0.5 × 1 mm. In each case, we demonstrate high contrast between the inclusion and the base material and validate both the contrast and spatial resolution achieved using finite element modelling. By performing CBOP on freshly excised human breast tissue, we demonstrate the capability to delineate tumour from surrounding benign tissue.

Diagnostic Accuracy of Quantitative Micro-Elastography for Margin Assessment in Breast-Conserving Surgery.

Inadequate margins in breast-conserving surgery (BCS) are associated with an increased likelihood of local recurrence of breast cancer. Currently, approximately 20% of BCS patients require repeat surgery due to inadequate margins at the initial operation. Implementation of an accurate, intraoperative margin assessment tool may reduce this re-excision rate. This study determined, for the first time, the diagnostic accuracy of quantitative micro-elastography (QME), an optical coherence tomography (OCT)-based elastography technique that produces images of tissue microscale elasticity, for detecting tumor within 1 mm of the margins of BCS specimens. Simultaneous OCT and QME were performed on the margins of intact, freshly excised specimens from 83 patients undergoing BCS and on dissected specimens from 7 patients undergoing mastectomy. The resulting three-dimensional images (45 × 45 × 1 mm) were coregistered with postoperative histology to determine tissue types present in each scan. Data from 12 BCS patients and the 7 mastectomy patients served to build a set of images for reader training. One hundred and fifty-four subimages (10 × 10 × 1 mm) from the remaining 71 BCS patients were included in a blinded reader study, which resulted in 69.0% sensitivity and 79.0% specificity using OCT images, versus 92.9% sensitivity and 96.4% specificity using elasticity images. The quantitative nature of QME also facilitated development of an automated reader, which resulted in 100.0% sensitivity and 97.7% specificity. These results demonstrate high accuracy of QME for detecting tumor within 1 mm of the margin and the potential for this technique to improve outcomes in BCS. SIGNIFICANCE: An optical imaging technology probes breast tissue elasticity to provide accurate assessment of tumor margin involvement in breast-conserving surgery.

Three-dimensional mapping of the attenuation coefficient in optical coherence tomography to enhance breast tissue microarchitecture contrast.

Effective intraoperative tumor margin assessment is needed to reduce re-excision rates in breast-conserving surgery (BCS). Mapping the attenuation coefficient in optical coherence tomography (OCT) throughout a sample to create an image (attenuation imaging) is one promising approach. For the first time, three-dimensional OCT attenuation imaging of human breast tissue microarchitecture using a wide-field (up to ~45 × 45 × 3.5 mm) imaging system is demonstrated. Representative results from three mastectomy and one BCS specimen (from 31 specimens) are presented with co-registered postoperative histology. Attenuation imaging is shown to provide substantially improved contrast over OCT, delineating nuanced features within tumors (including necrosis and variations in tumor cell density and growth patterns) and benign features (such as sclerosing adenosis). Additionally, quantitative micro-elastography (QME) images presented alongside OCT and attenuation images show that these techniques provide complementary contrast, suggesting that multimodal imaging could increase tissue identification accuracy and potentially improve tumor margin assessment.

Handheld volumetric manual compression-based quantitative microelastography.

Compression optical coherence elastography (OCE) typically requires a mechanical actuator to impart a controlled uniform strain to the sample. However, for handheld scanning, this adds complexity to the design of the probe and the actuator stroke limits the amount of strain that can be applied. In this work, we present a new volumetric imaging approach that utilizes bidirectional manual compression via the natural motion of the user's hand to induce strain to the sample, realizing compact, actuator-free, handheld compression OCE. In this way, we are able to demonstrate rapid acquisition of three-dimensional quantitative microelastography (QME) datasets of a tissue volume (6 × 6 × 1 mm3 ) in 3.4 seconds. We characterize the elasticity sensitivity of this freehand manual compression approach using a homogeneous silicone phantom and demonstrate comparable performance to a benchtop mounted, actuator-based approach. In addition, we demonstrate handheld volumetric manual compression-based QME on a tissue-mimicking phantom with an embedded stiff inclusion and on freshly excised human breast specimens from both mastectomy and wide local excision (WLE) surgeries. Tissue results are coregistered with postoperative histology, verifying the capability of our approach to measure the elasticity of tissue and to distinguish stiff tumor from surrounding soft benign tissue.

Interobserver variability in upfront dichotomous histopathological assessment of ductal carcinoma in situ of the breast: the DCISion study.

Histopathological assessment of ductal carcinoma in situ, a nonobligate precursor of invasive breast cancer, is characterized by considerable interobserver variability. Previously, post hoc dichotomization of multicategorical variables was used to determine the "ideal" cutoffs for dichotomous assessment. The present international multicenter study evaluated interobserver variability among 39 pathologists who performed upfront dichotomous evaluation of 149 consecutive ductal carcinomas in situ. All pathologists independently assessed nuclear atypia, necrosis, solid ductal carcinoma in situ architecture, calcifications, stromal architecture, and lobular cancerization in one digital slide per lesion. Stromal inflammation was assessed semiquantitatively. Tumor-infiltrating lymphocytes were quantified as percentages and dichotomously assessed with a cutoff at 50%. Krippendorff's alpha (KA), Cohen's kappa and intraclass correlation coefficient were calculated for the appropriate variables. Lobular cancerization (KA = 0.396), nuclear atypia (KA = 0.422), and stromal architecture (KA = 0.450) showed the highest interobserver variability. Stromal inflammation (KA = 0.564), dichotomously assessed tumor-infiltrating lymphocytes (KA = 0.520), and comedonecrosis (KA = 0.539) showed slightly lower interobserver disagreement. Solid ductal carcinoma in situ architecture (KA = 0.602) and calcifications (KA = 0.676) presented with the lowest interobserver variability. Semiquantitative assessment of stromal inflammation resulted in a slightly higher interobserver concordance than upfront dichotomous tumor-infiltrating lymphocytes assessment (KA = 0.564 versus KA = 0.520). High stromal inflammation corresponded best with dichotomously assessed tumor-infiltrating lymphocytes when the cutoff was set at 10% (kappa = 0.881). Nevertheless, a post hoc tumor-infiltrating lymphocytes cutoff set at 20% resulted in the highest interobserver agreement (KA = 0.669). Despite upfront dichotomous evaluation, the interobserver variability remains considerable and is at most acceptable, although it varies among the different histopathological features. Future studies should investigate its impact on ductal carcinoma in situ prognostication. Forthcoming machine learning algorithms may be useful to tackle this substantial diagnostic challenge.

Invasive lobular carcinoma of the breast: assessment of proliferative activity using automated Ki-67 immunostaining.

Invasive lobular carcinoma (ILC) is almost always classified as Nottingham histological grade 2. Despite this, prognosis is markedly varied, with some ILCs behaving more akin to grade 3 invasive ductal carcinoma (IDC). Methods to separate these aggressive ILCs are needed. Digital image analysis (DIA) of the Ki-67 biomarker has potential in this regard; thus, we sought to determine the feasibility of its use for automated evaluation of ILC. An initial pilot study demonstrated no ILC specific changes were required to our Ki-67 DIA algorithm for reproducible results. Subsequently, 42 consecutive cases of ILC were evaluated by visual mitosis counting in H&E stained sections and by DIA on Ki-67 stained sections. Ki-67 proliferative index (PI) DIA showed significant correlation with visual mitosis counting on H&E stained sections (rs=0.63; p<0.05), significant strong correlation (rs=0.78; p<0.05) and substantial agreement (κ=0.62) with manual/visual Ki-67 assessment and significant positive associations with grade, nodal status and 'pleomorphic' ILC subtype, and a wide stratification of values in classical/grade 2 ILC. In conclusion, DIA of Ki-67 PI in ILC is feasible, correlates with mitotic index, manual/visual Ki-67 PI and clinico-pathological variables. The broad stratification of Ki-67 PI in classical/grade 2 ILC supports its practicability as a biomarker with prognostic and predictive potential, although large studies with outcome data are required for validation.

Tumour suppression by targeted intravenous non-viral CRISPRa using dendritic polymers.

Aberrant gene expression is a hallmark of cancer. Although transcription is traditionally considered 'undruggable', the development of CRISPR-associated protein 9 (Cas9) systems offers enormous potential to rectify cancer-associated transcriptional abnormalities in malignant cells. However delivery of this technology presents a critical challenge to overcome in order to realize clinical translation for cancer therapy. In this article we demonstrate for the first time, a fully synthetic strategy to enable CRISPR-mediated activation (CRISPRa) of tumour suppressor genes in vivo using a targeted intravenous approach. We show this via highly efficient transcriptional activation of two model tumour suppressor genes, Mammary Serine Protease Inhibitor (MASPIN, SERPINB5) and cysteine-rich 61/connective tissue growth factor/nephroblastoma-overexpressed 6 (CCN6, WISP3), in a mouse model of breast cancer. In particular, we demonstrate that targeted intravenous delivery of can be achieved using a novel nanoscale dendritic macromolecular delivery agent, with negligible toxicity and long lasting therapeutic effects, outlining a targeted effective formulation with potential to treat aggressive malignancies.