Clinically guided core biopsy and cutaneous punch biopsy in the evaluation of breast lesions: a necessary test or an obsolete skill?

OBJECTIVE: The vast majority of breast cancers are diagnosed via image-guided procedures yet despite significant advances, imaging does not identify all breast malignancies. Clinically suspicious breast lesions with normal breast imaging remain a cause for concern. The aim of this study is to determine the diagnostic value of clinical core and cutaneous punch biopsies in the diagnosis of breast malignancy in clinically suspicious lesions with normal breast imaging. METHODS: All patients with suspicious clinical breast findings and normal imaging who underwent a clinical core and/or cutaneous punch biopsy from 2012 to 2019 were reviewed retrospectively. Patients with subsequent breast malignant diagnosis were analysed. RESULTS: A total of 283 biopsies (166 clinical core, 117 cutaneous punch) performed over the 7-year period were included in the analysis. A total of 263/283 (93%) yielded a benign outcome. A total of 2/283 (0.7%) yielded B3 lesions (probably benign). These lesions were benign on final surgical excision. A total of 18/283 (6.3%) yielded a malignant histopathology. Sixteen out of 18 were cutaneous punch biopsies, and 2/18 were clinical core biopsies. A total of 14/18 patients presented with nipple changes, while 4/18 had a palpable area of concern. Histopathological analysis demonstrated Paget's disease of the nipple in 8/18, invasive carcinoma in 9/18 out of which two represented a recurrence of breast malignancy. Cutaneous squamous cell carcinoma was diagnosed in 1/18. CONCLUSION: Clinical core and cutaneous punch biopsies remain a valuable tool in the diagnosis of breast cancer particularly in the management of clinically suspicious radiographically occult malignancies.

Value of Long-term Follow-up in Surgically Excised Lesions of Uncertain Malignant Potential in the Breast - Is 5 Years Necessary?

INTRODUCTION: B3 lesions are a heterogeneous group of breast lesions of uncertain malignant potential which usually require excision. The aim was to assess the efficacy of 5 years routine radiological or clinical follow-up of patients who had "high-risk" B3 lesions surgically excised, by analyzing recurrence and subsequent development of invasive/in-situ cancer. PATIENTS AND METHODS: A 10-year retrospective review from 2010 to 2019 was performed of B3 lesions diagnosed on core needle biopsy, including patients who proceeded to surgical excision with a high-risk lesion on final histology. The database recorded 6 specific B3 lesion categories: 1. Atypical ductal hyperplasia (ADH), 2. Radial scars/complex sclerosing lesions (CSLs) with epithelial atypia 3. Classical Lobular neoplasia (ALH/LCIS), 4. Papillary lesions with epithelial atypia, 5. Mixed, 6. Flat epithelial atypia (FEA), including radiological and clinical follow-up data. RESULTS: Six hundred sixteen patients had a B3 lesion after core biopsy. 110 patients had "high risk" lesions. This included 17 (15.5%) Atypical Ductal Hyperplasia (ADH), 22 (20%) radial scars/CSLs with epithelial atypia, 47 (42.7%) classical lobular neoplasia (LCIS/ALH), 7 (6.4%) papillary lesions with epithelial atypia, 13 (11.8%) mixed lesions & 4 (3.6%) Flat Epithelial Atypia (FEA) lesions. 4 of 110 (3.6%) developed invasive/in-situ disease and 4 of 110 (3.6%) developed recurrence during follow-up. 33 of 616 (5.4%) upgraded to invasive/preinvasive disease after surgical excision. CONCLUSION: Five years of routine radiological surveillance may not be necessary in patients who undergo surgical excision of "high-risk" B3 lesions. Clinical surveillance appears to be of little benefit, especially in patients with radial scars, papillary lesions, and FEA. Subsequent development of invasive/in-situ disease in patients who undergo surgical excision of atypical B3 lesions remains low.

Breast cancer patients with a negative axillary ultrasound may have clinically significant nodal metastasis.

INTRODUCTION: The non-invasive nature of the preoperative axillary ultrasound (AUS) fits the current trend of increasingly conservative axillary management. Recent publications suggest that early disease patients with clinically and radiologically negative axillae do not require sentinel lymph node biopsy (SLNB). This study aims to determine the true extent of axillary node disease in negative preoperative AUS patients. METHODS: A 10-year breast cancer registry was reviewed to identify women with pathologically confirmed T1-2 invasive breast cancer and a negative preoperative AUS. Patients who received neoadjuvant chemotherapy were excluded. Combined positive lymph node count of SLNB ± ALND was used to determine total nodal burden (TNB). Axillae were classified into low nodal burden (LNB) defined as 1-2 positive nodes and high nodal burden (HNB) defined as ≥ 3 positive nodes. RESULTS: 762 patients with negative AUS were included. There were 46.9% and 53.0% T1 and T2 tumours, respectively. 76.9% were node negative (0 LN +), 18.9% had LNB (1-2 LN +) and 4.2% had HNB (≥ 3LN +). Specifically, HNB disease was seen in 2% of T1 tumours and 6.2 % of T2 tumours with a negative AUS. In multivariate analysis, T2 strongly associated with ≥ 3 positive ALNs (OR 2.66 CI 1.09-6.51 p = 0.03) as did lymphovascular invasion (OR 3.56 CI 1.52-8.30 p = < 0.01). CONCLUSION: This study shows that AUS in its current form cannot exclude HNB axillary metastasis to the extent of eliminating the need for surgical staging of the axilla. This may impact axillary local-regional recurrence and disease-free survival. We caution that a negative AUS has a rate of 4.2% of HNB. Therefore, in cases of negative AUS with a T2 tumour, we advocate continued use of SLNB.

Phase Ib Trial of Copanlisib, A Phosphoinositide-3 Kinase (PI3K) Inhibitor, with Trastuzumab in Advanced Pre-Treated HER2-Positive Breast Cancer "PantHER".

BACKGROUND: Activation of the phosphoinositide-3 kinase (PI3K) pathway is a resistance mechanism to anti-human epidermal growth factor receptor 2 (HER2) therapy. This phase Ib trial was conducted to determine the maximum tolerated dose (MTD) of copanlisib, an intravenous (IV) pan-class I PI3K inhibitor, combined with trastuzumab. METHODS: Patients with advanced HER2-positive breast cancer and disease progression following at least one prior line of HER2 therapy in the metastatic setting were treated with copanlisib (45 or 60 mg) IV on days 1, 8 and 15 of a 28-day cycle with a fixed dose of trastuzumab 2 mg/kg weekly. RESULTS: Twelve patients were enrolled. The MTD was determined as copanlisib 60 mg plus trastuzumab 2 mg/kg weekly. The most common adverse events of any grade occurring in more than two patients were hyperglycaemia (58%), fatigue (58%), nausea (58%) and hypertension (50%). Stable disease was confirmed at 16 weeks in six participants (50%). PIK3CA mutations were detected in archival tumour of six participants (50%). PIK3CA hotspot mutations, were detectable in pre- and on-treatment plasma of all participants. Pre- and post-treatment tumour biopsies for two patients identified temporal genomic heterogeneity, somatic mutations in the TRRAP gene, which encodes a PI3K-like protein kinase, and emergent somatic mutations related to protein kinase signalling. CONCLUSION: Copanlisib and trastuzumab can be safely administered with fair overall tolerability. Preliminary evidence of tumour stability was observed in patients with heavily pre-treated, metastatic HER2 positive breast cancer. Several potential biomarkers were identified for further study in the current phase 2 clinical trial. NCT: 02705859.

Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies.

A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining region due to potential impact to the clinical efficacy. Here, we present and compare three analytical methods to monitor and/or quantify isoAsp formation in a monoclonal antibody. The methods include two peptide map based technologies with quantitation from either UV integration or total ion peak areas, as well as an alternative approach using IdeS digestion to generate Fc/2 and Fab'2 regions, followed by hydrophobic interaction chromatography (HIC) to separate the population of Fab'2 containing an isoAsp. The level of isoAsp detected by the peptide map and the digested-HIC methods presented here show similar trends although sample throughput varies by method.

Effects of anionic polyacrylamide products on gill histopathology in juvenile rainbow trout (Oncorhynchus mykiss).

Anionic polyacrylamide (PAM) products are commonly used to remove suspended materials from turbid waters and to help mitigate soil erosion. In the present study, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to 3 mg/L to 300 mg/L of 10 commercially available PAM products (Clearflow Water Lynx Polymer Log and Clearflow Soil Lynx Granular Polymer; Clearflow Enviro Systems Group), and gill histological parameters were measured following either 7 d or 30 d of polymer exposure. A cationic polymer product (≤0.38 mg/L MagnaFloc 368; Ciba Specialty Chemical) was also tested for comparison. Mild gill lesions were observed in fish exposed to polymer products. Lamellar fusion, interlamellar hyperplasia, epithelial lifting, mucous cell metaplasia, and cell counts of epithelial swelling and necrosis/apoptosis were minimal in fish exposed to environmentally relevant concentrations of anionic polymer (≤30 mg/L). Gill morphology was largely unaffected by exposure to concentrations up to 300 mg/L of many PAM products. Several anionic polymer products noticeably affected gill tissue by increasing epithelial hypertrophy, interlamellar hyperplasia, mucous cell metaplasia, and the frequency of necrotic cells. The severity of the lesions lessened with time, suggesting that fish may have experienced a short-term irritant effect. Similar levels of gill pathology were frequently observed in fish exposed to cationic polymer MagnaFloc 368 despite the concentration being 1000-fold lower than that of the PAM products. These observations highlight the increased toxicity of cationic polymers to aquatic life compared with anionic PAMs.

An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect.

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(-)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.

Trifluoromethylphenyl as P2 for ketoamide-based cathepsin S inhibitors.

The trifluoromethylphenyl P2 motif from previously reported heteroarylnitrile series has been successfully applied for the design and synthesis of highly potent novel ketoamide-based cathepsin S inhibitors. The key in this process is the change of the torsion angle between the P2 phenyl ring and the attached secondary amide by adding a small Cl, F, or Me group at the 2-position.

Optimisation of 2-cyano-pyrimidine inhibitors of cathepsin K: improving selectivity over hERG.

Several structure-guided optimisation strategies were explored in order to improve the hERG selectivity profile of cathepsin K inhibitor 1, whilst maintaining its otherwise excellent in vitro and in vivo profile. Ultimately, attenuation of clogP and pK(a) properties proved a successful approach and led to the discovery of a potent analogue 23, which, in addition to the desired selectivity over hERG (>1000-fold), displayed a highly attractive overall profile.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors.

Morphing structural features of HTS-derived chemotypes led to the discovery of novel 2-cyano-pyrimidine inhibitors of cathepsin K with good pharmacokinetic profiles, for example, compound 20 showed high catK potency (IC(50)=4nM), >580-fold selectivity over catL and catB, and oral bioavailability in the rat of 52%.

Analysis of the reaction coordinate of alpha-L-fucosidases: a combined structural and quantum mechanical approach.

The enzymatic hydrolysis of alpha-L-fucosides is of importance in cancer, bacterial infections, and fucosidosis, a neurodegenerative lysosomal storage disorder. Here we show a series of snapshots along the reaction coordinate of a glycoside hydrolase family GH29 alpha-L-fucosidase unveiling a Michaelis (ES) complex in a (1)C(4) (chair) conformation and a covalent glycosyl-enzyme intermediate in (3)S(1) (skew-boat). First principles metadynamics simulations on isolated alpha-L-fucose strongly support a (1)C(4)<-->(3)H(4)<-->(3)S(1) conformational itinerary for the glycosylation step of the reaction mechanism and indicate a strong "preactivation" of the (1)C(4) complex to nucleophilic attack as reflected by free energy, C1-O1/O5-C1 bond length elongation/reduction, C1-O1 bond orientation, and positive charge development around the anomeric carbon. Analysis of an imino sugar inhibitor is consistent with tight binding of a chair-conformed charged species.

Extension of hepatic necrosis secondary to current arcing to surgical clips: a potential complication of radiofrequency ablation.

The authors present a case report of a 67-year-old woman who underwent radiofrequency ablation of recurrent hepatic metastases. She was managed 2 years previously with a right hemi-hepatectomy. Subsequent to RF ablation she developed hepatic necrosis extending in a linear fashion to two of the metallic surgical clips at the free edge of the liver, consistent with current arcing.

Small bowel imaging: CT enteroclysis or barium enteroclysis? Critically appraised topic.

Barium enteroclysis has been traditionally used in the diagnosis of small bowel Crohn's disease. Recently CT enteroclysis has been developed as an alternative imaging technique for small bowel Crohn's disease. A search and critical appraisal of the literature was performed to determine which technique is better for diagnosis of Crohn's disease. The best current evidence indicates that CT enteroclysis is a good test for the diagnosis of Crohn's disease but barium enteroclysis may also be required in a small group of patients.

MRI as a problem-solving tool in unexplained failed total hip replacement following conventional assessment.

OBJECTIVE: To evaluate MRI as a problem-solving tool for patients with an unexplained failed total hip replacement following conventional radiological assessment. METHODS AND MATERIALS: Patients' informed consent was obtained in all cases. Institutional review board approval was obtained. Twenty-eight patients with unexplained failed total hip replacements following conventional radiological assessment underwent additional MR imaging with an optimised turbo-spin echo sequence. Images were reviewed by two musculoskeletal radiologists by consensus and compared with findings at surgery, or following response to image-guided intervention or clinical follow-up. RESULTS: Of the 28 patients, MRI revealed an unsuspected diagnosis explaining the cause of prosthesis failure in 15 patients. In eight of 15 patients in this group, subsequent minimally invasive image-guided intervention obviated the need for revision total hip replacement. No cause for prosthesis failure was identified in 13 patients. DISCUSSION: MRI may be successfully undertaken in patients following total hip replacement, and, when performed, it frequently leads to an unsuspected diagnosis, allowing informed patient treatment. In this study it allowed the identification of an unsuspected diagnosis in over 50% of cases.

MRI features after radiofrequency ablation of osteoid osteoma with cooled probes and impedance-control energy delivery.

OBJECTIVE: The purposes of our study were to determine the temporal changes in MR signal in bone after radiofrequency ablation of osteoid osteoma and the size of the zone of marrow signal change produced by the radiofrequency technique and to compare the size of the zone with published data for radiofrequency ablation with manual-control protocols. MATERIALS AND METHODS: Radiofrequency ablation was performed in 10 patients with a clinical and radiologic diagnosis of osteoid osteoma. A cooled radiofrequency probe was inserted in the nidus. Twelve minutes of radiofrequency energy was applied from a 200-W radiofrequency generator in an impedance-control setting. MRI with multiplanar turbo spin-echo T1-weighted and STIR sequences was performed at 1, 7, and 28 days after the procedure in seven patients. The three remaining patients had follow-up imaging at 28 days only. The images were reviewed by two radiologists who categorized the imaging features and measured the marrow zone of signal alteration when visible. The size of the zone of marrow signal change produced by the radiofrequency technique was compared with published data for radiofrequency ablation with manual-control protocols. RESULTS: A 1-mm band of homogeneous altered marrow signal distributed symmetrically parallel to the entire probe tract was seen earliest, at 1 day, in the femoral neck lesion treated with the 2-cm probe. The band was low signal on the T1 sequence and high signal on the STIR sequence, and the diameter of the zone was 27 mm. By 7 days, five of the seven treated bones showed a band of marrow signal alteration. By 28 days, all 10 treated bones had a band of marrow signal alteration. The interband distance at 90 degrees to the probe measured on STIR images at 28 days was a mean of 20.9 mm (confidence interval, 16.1-25.7 mm [p < 0.05]; range +/- measurement error, 10.5-35 +/- 1.64 mm) with a 1-cm probe and 30.5 mm (measurement error, +/- 0.78 mm) on T1 images without contrast material when a 2-cm exposed-tip probe was used. Higher-output generators with impedance-control software and internally cooled radiofrequency probes with longer exposed tips produce larger zones of marrow signal change than expected with manual-control protocols. CONCLUSION: MRI allows detection of temporal marrow signal change after radiofrequency ablation. The marrow signal change with a high-energy delivery protocol is larger than manual-control protocols.