The Mutational Spectrum of Pre- and Post-Neoadjuvant Chemotherapy Triple-Negative Breast Cancers.

The response of triple-negative breast cancer (TNBC) patients to pre-operative (neoadjuvant chemotherapy) is a critical factor of their outcome. To determine the effects of chemotherapy on the tumor genome and to identify mutations associated with chemoresistance and sensitivity, we performed whole exome sequencing on pre/post-chemotherapy tumors and matched lymphocytes from 26 patients. We observed great inter-tumoral heterogeneity with no gene mutated recurrently in more than four tumors besides TP53. Although the degree of response to chemotherapy in residual tumors was associated with more subclonal changes during chemotherapy, there was minimal evolution between pre/post-tumors. Indeed, gene sets enriched for mutations in pre- and post-chemotherapy tumors were very similar and reflected genes involved in the biological process of neurogenesis. Somatically mutated genes present in chemosensitive tumors included COL1A2, PRMD15, APOBEC3B, PALB2 and histone protein encoding genes, while BRCA1, ATR, ARID1A, XRCC3 and genes encoding for tubulin-associated proteins were present in the chemoresistant tumors. We also found that the mutational spectrum of post-chemotherapy tumors was more reflective of matching metastatic tumor biopsies than pre-chemotherapy samples. These findings support a portrait of modest ongoing genomic instability with respect to single-nucleotide variants induced by or selected for by chemotherapy in TNBCs.

Autologous Hematopoietic Stem Cell Transplantation for Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) refractory to conventional therapy can lead to marked disability and represents a therapeutic challenge. OBJECTIVE: To report five cases of treatment-refractory disabling CIDP treated with autologous hematopoietic stem cell transplantation (AHSCT). METHODS: This was a retrospective cohort study from a tertiary care referral center for both neuromuscular disease and AHSCT. Patients with CIDP treated with AHSCT between 2008 and 2020 were included. All patients had major persistent and disabling neuropathic deficits despite combinations of intensive immunosuppressive therapy. The primary outcome measures were: Medical Research Council sum score, Overall Neuropathy Limitations Scale and requirement for ongoing CIDP immunotherapy after transplantation. We also analyzed safety outcomes by documenting all severe AHSCT-related complications. RESULTS: Five patients with refractory CIDP underwent AHSCT. Three were classified as manifesting a typical syndrome, two were classified as the multifocal Lewis Sumner variant. The mean age at time of CIDP diagnosis was 33.4 years (range 24-46 years), with a median delay of 46 months (range 21-135 months) between diagnosis and AHSCT. The median follow-up period was 41 months. All five patients were able to wean off CIDP-related immunotherapy. Marked improvements in Medical Research Council scale and overall Neuropathy Limitations Scale were noted in 4/5 patients. One patient with longstanding neurogenic atrophy showed no improvement in disability scales. There were no treatment-related deaths or critical illnesses. CONCLUSIONS: AHSCT can achieve marked sustained clinical improvement of refractory CIDP and may allow for weaning off long-term complex immunotherapies.

Performance characteristics of Fungitell STAT™, a rapid (1→3)-ß-D-glucan single patient sample in vitro diagnostic assay.

Serum (1→3)-ß-D-glucan (BDG), is an adjunct test in the diagnosis of invasive fungal disease (IFD). Fungitell STAT™, a facile, rapid, single patient option, executable for one or more patient specimens in approximately an hour, has been developed to address a need for rapid in-house testing. This method presents qualitative information concerning serum BDG levels, using an index value that allows the rapid categorization of patients as positive, negative, or indeterminate relative to serum BDG titer. The categorical and analytical performance of Fungitell STAT was evaluated. The categorical agreement between methods was established by testing patient samples which had been previously categorized with Fungitell. Receiver Operating Characteristic curves were used to identify cut-offs using 93 de-identified patient specimens. Subsequently, using these cutoffs, an independent group of 488 patient specimens was analyzed. Positive percent agreement (PPA) with, and without, indeterminate results was 74% and 99%, respectively. Negative percent agreement (NPA) was 91% and 98% with, and without, indeterminate results, respectively. Additionally, commercially available normal off-the-clot sera were spiked with Saccharomyces cerevisiae-derived (1→3)-ß-D-glucan to produce analytical samples. Analytical reproducibility using spiked samples was excellent with 94% of the CV (coefficient of variation) values ≤10% among three independent laboratories. Good correlation with the predicate method was demonstrated with correlation coefficients of 0.90 or better with patient samples and 0.99 with spiked samples. The Fungitell STAT index assay provides a rapid and suitable method for serum BDG testing.

Temporal evolution of nerve conduction study abnormalities in anti-myelin-associated glycoprotein neuropathy.

BACKGROUND: A distal-predominant demyelinating symmetric pattern is most frequent in patients with neuropathy associated with anti-myelin-associated glycoprotein (MAG) antibodies. The literature however lacks longitudinal data to describe whether this is consistent over time. METHODS: From the Ottawa Neuromuscular Center database, we identified 23 patients with both immunoglobulin M gammopathy and anti-MAG antibodies. For median, ulnar and fibular motor conduction studies, we analyzed distal latency and amplitude, negative peak duration, terminal latency index (TLI), and conduction velocity. For median, ulnar, sural, and superficial fibular sensory conduction studies, we analyzed distal latency and amplitude. Results were compared for the earliest and the latest data sets. RESULTS: The mean time interval between the two assessment points was 6.5 years. Median and ulnar motor nerve conduction studies did not show a significant change for any of the parameters tested. There was disproportionate prolongation of median distal motor latency and reduction in TLI, compared to the ulnar nerve. Deep fibular motor conduction studies showed a marked reduction in amplitudes over time. Sensory potentials were recordable in the upper limb in less than 50% at the first study and less than 25% on the most recent study. There was an even larger attrition of recordable sural and superficial fibular sensory potentials. CONCLUSIONS: Our results highlight the stability of median and ulnar motor conduction study results over a mean observation period of 6.5 years. In contrast, lower limb motor and all sensory potentials show a marked trend toward becoming unrecordable.

Prognostic and predictive value of circulating tumor DNA during neoadjuvant chemotherapy for triple negative breast cancer.

Response to neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) is highly prognostic and determines whether adjuvant chemotherapy is needed if residual tumor is found at surgery. To evaluate the predictive and prognostic values of circulating tumor DNA (ctDNA) in this setting, we analyzed tumor and serial bloods from 26 TNBC patients collected prior, during, and after NAC. Individual digital droplet PCR assays were developed for 121 variants (average 5/patient) identified from tumor sequencing, enabling ctDNA detection in 96% of patients at baseline. Mutant allele frequency at baseline was associated with clinical characteristics. Levels drastically fell after one cycle of NAC, especially in patients whose tumors would go on to have a pathological complete response (pCR), but then rose significantly before surgery in patients with significant residual tumor at surgery (p = 0.0001). The detection of ctDNA early during treatment and also late at the end of NAC before surgery was strongly predictive of residual tumor at surgery, but its absence was less predictive of pCR, especially when only TP53 variants are considered. ctDNA detection at the end of neoadjuvant chemotherapy indicated significantly worse relapse-free survival (HR = 0.29 (95% CI 0.08-0.98), p = 0.046), and overall survival (HR = 0.27 95% CI 0.075-0.96), p = 0.043). Hence, individualized multi-variant ctDNA testing during and after NAC prior to surgery has prognostic and predictive value in early TNBC patients.

A Unique Morphological Phenotype in Chemoresistant Triple-Negative Breast Cancer Reveals Metabolic Reprogramming and PLIN4 Expression as a Molecular Vulnerability.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

The identification of challenges in tissue collection for biomarker studies: the Q-CROC-03 neoadjuvant breast cancer translational trial experience.

One of the major challenges in biomarker development is the collection of tumor tissue of adequate quality for analysis. A prospective clinical trial was initiated to collect tissues from triple negative breast cancers prior to and after neoadjuvant chemotherapy in order to study the mechanisms of chemoresistance. Sixty patients had pre-chemotherapy biopsies performed by either a surgeon or a radiologist, while those with residual tumor after chemotherapy had research-only biopsies and/or surgical samples collected in liquid nitrogen, RNA-later and formalin. We examined each core for tumor cellularity, stromal content, and necrosis after which, RNA and DNA extraction was performed. We found that biopsies collected with ultrasound guidance were more likely to contain tumor than those collected by the surgeon. Patient reluctance to undergo research-only biopsies after chemotherapy was not a problem. Pre-chemotherapy tumor biopsies frequently did not contain any tumor cells (15%) or did not have ≥50% tumor content (63%). Indeed, 50% of patients had at least 2 pre-chemotherapy core biopsies with <50% tumor content. After chemotherapy, 30% of biopsy or surgical samples in patients with incomplete response did not contain any tumor. Finally, RNA-later not only made histopathological assessment of tumor content difficult, but yielded less DNA than fresh snap frozen samples. We recommend that high-quality tissue procurement can be best accomplished if at least three image-guided core biopsies be obtained per sample, each of these cores be examined for tumor cellularity and that at least some of them be freshly snap frozen in liquid nitrogen.

Comparative short-term efficacy of Oridermyl(®) auricular ointment and Revolution(®) selamectin spot-on against feline Otodectes cynotis and its associated secondary otitis externa.

The efficacy of Oridermyl(®) (acaricidal/antibiotic/antifungal/anti-inflammatory ointment) and Revolution(®) (selamectin spot-on) was compared in a blinded randomized study on 24 adult cats with otoacariasis. Twelve cats were treated once daily for 10 d with Oridermyl(®) and 12 cats were treated on Day 0 with Revolution(®). Otitis was assessed with cytological counts of mean number of bacteria and yeast on Days 0 and 10, and scored clinically every other day. All auricular secretions were removed for mite count on Day 10. On Day 0, cytological examination confirmed the presence of secondary bacterial (24/24) and fungal (21/24) infections. No live mites were observed otoscopically after Day 4 and in auricular secretions at Day 10 in both groups. On Day 10, secondary infections were resolved for all cats treated with Oridermyl(®) but were present in all cats treated with Revolution(®). Improvement in clinical signs of otitis over time was superior in the Oridermyl(®) group (P < 0.001).

Treatment of feline otitis externa due to Otodectes cynotis and complicated by secondary bacterial and fungal infections with Oridermyl auricular ointment.

A blinded randomized study was conducted on 24 cats to confirm the presence of bacterial and/or fungal secondary infections associated with otoacariasis and to verify the efficacy of Oridermyl, an acaricidal/antibiotic/antifungal/anti-inflammatory ointment, for treatment of the primary infestation and secondary infections. Sixteen cats were treated once daily for 10 d; 4 cats were not treated and 4 were treated with a placebo ointment. On Days 0 and 10, ears were swabbed for counts of bacteria and yeasts, for bacterial culture and sensitivity, and examined for determination of the degree of clinical otitis. Auricular secretions were removed for mite counts on Day 10, except for 8 treated cats that were done on Day 30. There was a high number of bacteria and yeasts in most cats and Oridermyl treatment significantly decreased those numbers. Staphylococci were the most frequently isolated bacteria. No live ear mites were found in cats treated with Oridermyl or the placebo ointment.

Magnesium in a polyethylene glycol formulation provides neuroprotection after unilateral cervical spinal cord injury.

STUDY DESIGN: Experimental animal study. OBJECTIVE: To investigate the neuroprotective efficacy of this magnesium in polyethylene glycol (PEG) formulation in a contusive model of cervical spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: Intravenously administered magnesium has been extensively investigated as a neuroprotective agent in animal models of SCI, stroke, and traumatic brain injuries, and has been evaluated in large scale clinical trials for the latter 2 indications. We have developed a novel formulation of magnesium chloride (MgCl2) within PEG, and have previously demonstrated the neuroprotective benefit of this formulation in animal models of thoracic SCI. METHODS: Twenty-two Sprague Dawley rats underwent a unilateral cervical hemicontusion at C4-C5 and were randomized 2 hours later to either the MgCl2 in PEG formulation, or normal saline. Each treatment was administered in 5 intravenous infusions spaced 6 hours apart. Behavioral recovery was assessed over 6 weeks, after which the cord was analyzed to measure the extent of gray matter and white matter sparing through the injury site. RESULTS: In the horizontal ladder test, the percentage of forelimb errors made by the animals treated with MgCl2 in PEG formulation was significantly lower than the saline-treated controls. Histologic analysis also revealed a significantly higher cumulative white matter sparing through the injury site in the MgCl2 in PEG group. CONCLUSION: MgCl2 in a PEG formulation reduced secondary damage and improved behavioral recovery when administered 2 hours after a unilateral cervical hemicontusion injury. These findings are consistent with the neurologic benefit observed when administering this magnesium formulation in contusive and compressive models of thoracic SCI. Demonstrating the robustness of this neuroprotective effect in multiple injury models (and in the cervical injury model in particular) is important when considering the applicability of such a therapy for human SCIs.

Magnesium chloride in a polyethylene glycol formulation as a neuroprotective therapy for acute spinal cord injury: preclinical refinement and optimization.

Intravenously administered magnesium has been extensively investigated as a neuroprotective agent traumatic brain injuries and stroke. Numerous investigators have reported the neuroprotective benefits of magnesium in animal models of spinal cord injury (SCI) as well, but typically with doses that far exceed human tolerability. To develop magnesium into a clinically relevant therapy for SCI, further refinement and improvement of the magnesium formulation is necessary. In this series of experiments, we evaluated the neuroprotective efficacy of magnesium in a polyethylene glycol (PEG) formulation using an acute model of thoracic SCI. Following thoracic contusion (Infinite Horizon) rat SCI model, we independently confirmed the neuroprotective efficacy of the magnesium and PEG combination which had been previously reported in a thoracic clip compression model of SCI (Ditor et al., 2007). We established that the 254 micromol/kg dose of MgCl(2) was superior to 127 micromol/kg MgCl(2) with respect to tissue sparing and locomotor recovery. Additionally, the number of infusions (2, 4, or 6), time between infusions (6 vs 8 hours), and different magnesium salts (MgCl(2) vs MgSO(4)) were evaluated to determine an "optimal" treatment regimen. We observed that an "optimized" regimen of MgCl(2) within PEG conferred greater tissue neuroprotection and improved locomotor recovery compared to methylprednisolone. Further a 4 hour time window of histologic and behavioral efficacy was established. The goal of these experiments was to help guide the treatment parameters for a clinical trial of magnesium within a polyethylene glycol formulation in acute human spinal cord injury.

Clinical and in vitro efficacy of amoxicillin against bacteria associated with feline skin wounds and abscesses.

A clinical trial involving 122 cats with infected skin wounds or abscesses presented to 10 veterinary clinics was conducted to evaluate the efficacy of 2 oral amoxicillin drug products (a paste and a suspension). A 2nd objective of the study was to identify bacteria involved in such infections and verify their in vitro sensitivity to amoxicillin. Samples of wound exudate were harvested at the time of presentation and submitted for aerobic and anaerobic culture. The sensitivity to amoxicillin of isolates thought to be infecting agents was tested, using a standard minimum inhibitory concentration method. Pasteuralla multocida and obligate anaerobes of the genera Prevotella, Fusobacterium, and Porphyromonas were the most frequently isolated pathogens. Overall, their in vitro susceptibility to amoxicillin was very good. Both drug products were clinically efficacious with a global success rate of 95.1% for cats administered oral amoxicillin at 11-22 mg/kg bodyweight (mean 13.8 mg/kg bodyweight) twice daily for 7 to 10 days.

Effects of polyethylene glycol and magnesium sulfate administration on clinically relevant neurological outcomes after spinal cord injury in the rat.

The purpose of this study was to determine the long-term effects of polyethylene glycol (PEG) and magnesium sulfate (MgSO(4)) on clinically relevant motor, sensory, and autonomic outcomes after spinal cord injury (SCI). Rats were injured by clip compression (50 g; T4) and treated 15 min and 6 hr postinjury intravenously (tail vein) with PEG (1 g/kg, 30% w/w in saline; n = 11), MgSO(4) (300 mg/kg; n = 5), PEG + MgSO(4) (n = 6), or saline (n = 10). Behavioral testing lasted for 6 weeks, followed by histological analysis of the spinal cord. Both PEG and MgSO(4) resulted in enhanced locomotor recovery and lower susceptibility to neuropathic pain (mechanical allodynia) compared with saline. At 6 weeks, BBB scores were 7.3 +/- 0.2, 7.7 +/- 0.4, and 6.4 +/- 0.6 in PEG-treated, MgSO(4)-treated, and saline-treated control groups, respectively. Likewise, at 6 weeks PEG-, MgSO(4)-, and saline-treated control animals showed 3.5 +/- 0.4, 2.8 +/- 0.9, and 5.0 +/- 0.5 avoidance responses to at-level touch, respectively. PEG + MgSO(4) improved locomotor recovery and reduced pain but did not provide additional benefit compared with either treatment alone. Neither treatment, nor their combination, attenuated mean arterial pressure (MAP) increases during autonomic dysreflexia. However, saline-treated controls had significantly lower resting MAP than PEG-treated rats and tended to have lower resting MAP than MgSO(4)-treated rats 6 weeks postinjury. MgSO(4) treatment and PEG + MgSO(4) treatment resulted in significant increases in dorsal myelin sparing, and the latter resulted in significant reductions in lesion volume, compared with saline-treated controls. Furthermore, mean lesion volumes correlated negatively with the corresponding mean BBB scores and positively with the corresponding mean pain scores. In conclusion, both PEG and MgSO(4) enhanced long-term clinical outcomes after SCI.

Quality of life in ovarian cancer patients: comparison of paclitaxel plus cisplatin, with cyclophosphamide plus cisplatin in a randomized study.

PURPOSE: Formal quality-of-life (QOL) assessments may contribute important information on patient symptoms. Despite many trials of systemic chemotherapy in ovarian cancer, reports of its effect on QOL are few. PATIENTS AND METHODS: QOL was assessed in an Intergroup randomized trial comparing paclitaxel plus cisplatin to cyclophosphamide plus cisplatin in women with advanced ovarian cancer. One hundred fifty-two eligible patients accrued in Canada completed the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 and a trial-specific checklist at baseline (after surgical debulking) and at regular intervals during and after chemotherapy. Mean change scores over time in the two arms were calculated. RESULTS: Compliance with QOL questionnaire completion was excellent (81% to 93%). In general, deterioration was seen in the QOL domains immediately after chemotherapy (day 8 of cycle 1), followed by clinically meaningful improvements compared with baseline (change scores > or = 10) in both arms during the treatment period in a number of domains and items, including global QOL, emotional function, social function, fatigue, pain, sleep, constipation, appetite, abdominal swelling, and abdominal cramps. Improvements in global QOL persisted for the duration of follow-up. More neurosensory effects and myalgia were documented in the paclitaxel arm; however, this did not adversely affect global or other domains of QOL and improved once chemotherapy was completed. CONCLUSION: Improvement from baseline in QOL measures was seen in both treatment arms. The greater neurologic and muscle toxicity of paclitaxel did not adversely influence QOL. QOL data can contribute useful information on the experience of symptoms and their time course, which may assist patients and physicians in their discussion about the anticipated effects of therapy.

Particulate matter inflammation and receptor sensitivity are target cell specific.

The complexity of primary source particulate matter (PM) and the various cell types encountered by its inhalation raise the possibility that target cells are differentially activated. Since epithelial cells, which line the nasal-tracheal-bronchial airways, and sensory C fibers, which terminate throughout this epithelial layer, are initially targeted by inhaled PM, we compared their relative biological response in vitro to PM originating from volcanic (MSH), anthropogenic (diesel), residential (woodstove), urban ambient (St. Louis, Ottawa), and industrial emission (coal fly ash, CFA; residual oil fly ash, ROFA; oil fly ash, OFA) sources. Increases in intracellular calcium (i.e., [Ca(2+)](i)) are a second-messenger event that indicates cellular activation and signal transduction, in both nerve and epithelial cells. Single-cell calcium imaging recordings were taken of human bronchial epithelial cells (BEAS-2B) exposed to selected PM (50 microg/ml or 30 microg/cm(2)). These cells responded with variable increases in [Ca(2+)](i) ranging from abrupt increases, which returned to baseline upon washing of the cells, to oscillations of the [Ca(2+)](i) that did not wash out. Increases in [Ca(2+)](i) and inflammatory cytokine (i.e., interleukin 6, IL-6) release were measured in populations of BEAS-2B cells exposed to PM (50 microg/ml) and were shown to significantly correlate (r(2) =.80). BEAS-2B cells, stained histochemically with cobalt, displayed a concentration-dependent precipitation in response to acid pH and capsaicin, indicating the presence of acid-sensitive pathways (e.g., VR1 and acid-sensitive receptors). To demonstrate the relevance of these pathways to inflammatory cytokine (i.e., IL-6) release, BEAS-2B cells were pretreated (15 min) with antagonists to the vanilloid (VR1) receptor (i.e., capsazepine, CPZ) or acid-sensitive pathways (i.e., amiloride) before their exposure to the selected PM. A significant reduction of IL-6 release occurred in response to all PM, except for MSH and diesel exhaust. Dorsal root ganglia (DRG), which innervate the tracheal airways, were dissociated from fetal mice and pretreated with CPZ or amiloride before exposure (4 h) to the selected PM (50 microg/ml). Overall, significantly higher release occurred in PM-exposed sensory neurons relative to that of BEAS-2B epithelial cells. Although both CPZ and amiloride significantly reduced IL-6 release for all PM, the degree of inhibition was less for the PM-exposed DRG relative to BEAS-2B cells. These data show that differential increases in [Ca(2+)](i) and IL-6 release occur in BEAS-2B epithelial cells and DRG sensory neurons, when exposed to PM derived from different sources. The degree of this activation, however, depends not only on the source of the PM, but also on its cellular target. This differential sensitivity of target cells may contribute to the organism's overall inflammatory response to PM exposure.