Association of artificial intelligence-powered and manual quantification of programmed death-ligand 1 (PD-L1) expression with outcomes in patients treated with nivolumab ± ipilimumab.

Assessment of programmed death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) has emerged as an important predictive biomarker across multiple tumor types. However, manual quantitation of PD-L1 positivity can be difficult and leads to substantial inter-observer variability. Although the development of artificial intelligence (AI) algorithms may mitigate some of the challenges associated with manual assessment and improve the accuracy of PD-L1 expression scoring, use of AI-based approaches to oncology biomarker scoring and drug development has been sparse, primarily due to the lack of large-scale clinical validation studies across multiple cohorts and tumor types. We developed AI-powered algorithms to evaluate PD-L1 expression on tumor cells by IHC and compared it with manual IHC scoring in urothelial carcinoma, non-small cell lung cancer, melanoma, and squamous cell carcinoma of the head and neck (prospectively determined during the phase II and III CheckMate clinical trials). 1,746 slides were retrospectively analyzed, the largest investigation of digital pathology algorithms on clinical trial datasets performed to date. AI-powered quantification of PD-L1 expression on tumor cells identified more PD-L1-positive samples compared with manual scoring at cutoffs of ≥1% and ≥5% in most tumor types. Additionally, similar improvements in response and survival were observed in patients identified as PD-L1-positive compared with PD-L1-negative using both AI-powered and manual methods, while improved associations with survival were observed in patients with certain tumor types identified as PD-L1-positive using AI-powered scoring only. Our study demonstrates the potential for implementation of digital pathology-based methods in future clinical practice to identify more patients who would benefit from treatment with immuno-oncology therapy compared with current guidelines using manual assessment.

Local Tissue Response to Subcutaneous Administration of Ceftriaxone in an Animal Model.

Subcutaneous administration is a novel way to deliver antibiotics for an infection, but intolerability has been reported. Evaluating the local tolerability of subcutaneously administered antibiotics is not standardized. The goal of this study was to develop an animal model to assess the subcutaneous administration of ceftriaxone. Sprague-Dawley rats were given daily subcutaneous injections for 12 days. The back of each animal was divided into 4 quadrants, with injections rotating each day among the quadrants. Ceftriaxone (1,000 mg/kg of body weight daily) was given in different concentrations and durations. Normal saline and potassium chloride solutions (2 meq/2 ml) were used as negative and positive controls, respectively. After the treatment course, skin samples were biopsied, and the local inflammatory response was assessed histologically using a semiquantitative scoring system. The histopathology scores were compared using a Kruskal-Wallis test. Injections with potassium chloride resulted in full-thickness skin necrosis with subcutaneous atrophy that was not seen in the saline-injected animals; inflammation of the muscular panniculus was observed, with various degrees of myocyte injury. Serosanguinous cavity formation in the subcutaneous compartment was observed when ceftriaxone (125 mg/ml) was given as a bolus injection, but the extent of the local tissue response was remarkably reduced when the same ceftriaxone dose was given at a lower concentration (25 mg/ml) over 120 min (P = 0.63, compared to saline controls). At a low concentration, ceftriaxone infusion was found to be well tolerated in this animal tissue necrosis model. If validated, the model could be an instrumental platform to evaluate different pharmaceutical formulations for subcutaneous delivery.

A Primary Cutaneous CD30-Positive T-Cell Lymphoproliferative Disorder Arising in a Patient With Multiple Myeloma and Cutaneous Amyloidosis.

CD30-positive cutaneous lymphoproliferative disorders, a group of T-cell neoplasms, including lymphomatoid papulosis (LyP) and cutaneous anaplastic large cell lymphoma, require careful clinicopathologic correlation for diagnosis. An association between LyP and the development of a second hematolymphoid malignancy has been established in the literature. LyP has also been reported with systemic amyloidosis, but no such reports have documented coexisting cutaneous amyloid deposition with LyP to our knowledge. A 66-year-old woman with cutaneous amyloidosis, secondary to multiple myeloma, in remission, presented with erythematous and dark-brown papules involving the right arm, scalp, and torso. Punch biopsy of the arm showed a dermal infiltrate of intermediate-sized lymphocytes, some of which displayed a plasmacytoid morphology and prominent nodular subepidermal amyloid deposition. Punch biopsy of the scalp similarly showed a nonepidermotropic dense dermal infiltrate of intermediate-sized plasmacytoid lymphocytes and multifocal amyloid deposition. Both infiltrates were immunophenotypically CD30-positive, anaplastic lymphoma kinase-negative T-cell lymphoproliferative processes. Subsequent studies showed no systemic involvement, and clinical correlation suggested a final diagnosis of LyP. We present this case of LyP, which histologically mimics a B-cell proliferation with a plasmacytoid morphology arising in association with cutaneous amyloidosis to highlight the importance of clinicopathologic correlation, a thorough battery of immunohistochemical studies, and consideration for a second hematologic malignancy arising in the setting of LyP.

Clinically significant hemolytic disease of the newborn secondary to passive transfer of anti-D from maternal RhIG.

BACKGROUND: RhIG is used worldwide to reduce the incidence of alloimmunization to D during pregnancy. We report a case of clinically significant neonatal hemolysis mediated by maternally administered RhIG. CASE REPORT: A 25-year-old, O-, primigravid mother with a negative antenatal antibody screen delivered a 6-lb 4-oz, blood group A, D+ baby girl at 36.5 weeks' gestation. Prenatal care included a dose of intramuscular RhIG at 28 weeks' gestation. At delivery, the newborn was markedly jaundiced with a total bilirubin of 6.3 mg/dL, which reached more than 20 mg/dL after 6 days. The newborn's lactate dehydrogenase (LDH) was 485 U/L (normal, <226 U/L) and further laboratory studies revealed reticulocytosis (17.2%; normal range, 0.36%-1.9%) and a hemoglobin (Hb) of 14.3 g/dL (normal for age range, 13.4-19.8 g/dL) that decreased to 11.5 g/dL (normal for age range, 13.5-22.6 g/dL) by Day-of-life 7. Although the maternal antibody screen was negative, the newborn's direct antiglobulin test (DAT) was positive for immunoglobulin (Ig)G, with an anti-D identified by elution studies. The possibility of hemolytic disease of the newborn (HDN) due to anti-A was considered, but ultimately ruled out by the absence of anti-A1 in the eluate. The newborn's hyperbilirubinemia was adequately managed with phototherapy. Analysis of the mother's plasma 10 days postpartum revealed an anti-D titer of 8. Two months after birth, the child's laboratory studies, DAT, antibody screen, and peripheral smear were unremarkable. CONCLUSION: In the context of neonatal anemia, elevated LDH, and reticulocytosis, a positive IgG DAT with anti-D identified in the eluate suggests RhIG-mediated HDN. This appears to be a rarely reported event.

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage phi29 tail.

The small bacteriophage phi29 must penetrate the approximately 250-A thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage phi29 is noncontractile and approximately 380 A long. A 1.8-A resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the phi29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13(-) mutants with the phi29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.

The contribution of human papilloma virus infection to cutaneous squamous cell carcinoma in patients with chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL), a B-cell malignancy characterized by impaired humoral and cellular immunity, are at increased risk of developing cutaneous squamous cell carcinoma (cSCC). Human papilloma virus (HPV) is the most common sexually transmitted infection worldwide and it has been associated with various malignancies, including cSCC. Impaired cell-mediated immunity is considered a primary risk factor in HPV-induced cSCC. We examined cSCC lesions from CLL patients with consensus review and HPV genetic analysis to further characterize the relationship between HPV and prevalence of cutaneous malignancy in this population. Eleven patients with CLL contributed 35 cSCCs. Treatment with chemotherapy shortened the latency time to first cSCC. HPV was detected in 54% of the lesions. Among the HPV-positive cSCC lesions, 84% of the lesions contained alpha-genus HPV, 42% contained beta-genus HPV, and 26% of the lesions contained both genera. There was a significant association between HPV-containing lesions and peritumoral lymphocytic inflammation, suggesting this as a future area for further characterization. The majority of the lesions, including those with alpha-genus HPV, occurred in sun-exposed areas, such as the scalp and face. These findings may lead to practice-changing recommendations for skin cancer, including the use of vaccinations to reduce HPV-associated skin cancer.

Normal Respiratory Flora as a Cause of Community-Acquired Pneumonia.

BACKGROUND: Intensive studies have failed to identify an etiologic agent in >50% cases of community-acquired pneumonia (CAP). Bacterial pneumonia follows aspiration of recognized bacterial pathogens (RBPs) such as Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus after they have colonize the nasopharynx. We hypothesized that aspiration of normal respiratory flora (NRF) might also cause CAP. METHODS: We studied 120 patients hospitalized for CAP who provided a high-quality sputum specimen at, or soon after admission, using Gram stain, quantitative sputum culture, bacterial speciation by matrix-assisted laser desorption ionization time-of-flight, and viral polymerase chain reaction. Thresholds for diagnosis of bacterial infection were ≥105 colony-forming units (cfu)/mL sputum for RBPs and ≥106 cfu for NRF. RESULTS: Recognized bacterial pathogens were found in 68 of 120 (56.7%) patients; 14 (20.1%) of these had a coinfecting respiratory virus. Normal respiratory flora were found in 31 (25.8%) patients; 10 (32.2%) had a coinfecting respiratory virus. Infection by ≥2 RBPs occurred in 10 cases and by NRF together with RBPs in 13 cases. Among NRF, organisms identified as Streptococcus mitis, which share many genetic features of S pneumoniae, predominated. A respiratory virus alone was found in 16 of 120 (13.3%) patients. Overall, an etiologic diagnosis was established in 95.8% of cases. CONCLUSIONS: Normal respiratory flora, with or without viral coinfection, appear to have caused one quarter of cases of CAP and may have played a contributory role in an additional 10.8% of cases caused by RBPs. An etiology for CAP was identified in >95% of patients who provided a high-quality sputum at, or soon after, the time of admission.

Corrigendum to: Normal Respiratory Flora as a Cause of Community-Acquired Pneumonia.

[This corrects the article DOI: 10.1093/ofid/ofaa307.].

A simultaneous presentation of drug-induced lupus with drug-induced ANCA vasculitis secondary to hydralazine use in a patient with sarcoidosis.

Frequently used in the management of hypertension and heart failure, hydralazine is associated with the development of adverse rheumatologic side effects. The authors highlight a unique case of drug-induced lupus and drug-induced anti-neutrophil cytoplasmic antibody (ANCA) vasculitis from hydralazine use in a 50-year-old man with sarcoidosis and hypertension.

Genomic landscape of a metastatic malignant proliferating tricholemmal tumor and its response to PI3K inhibition.

Proliferating tricholemmal tumors (PTTs) are rare benign neoplasms that arise from the outer sheath of a hair follicle. Occasionally, these PTTs undergo malignant transformation to become malignant proliferating tricholemmal tumors (MPTTs). Little is known about the molecular alterations, malignant progression, and management of MPTTs. Here, we describe the case of a 58-year-old female that had a widely metastatic MPTT that harbored an activating PIK3CA mutation and was sensitive to the PI3K inhibitor, alpelisib (BYL719). We review the available literature on metastatic MPTT, detail the patient's course, and present a whole genome analysis of this rare tumor.

Secondary structure and compliance of a predicted flexible domain in kinesin-1 necessary for cooperation of motors.

Although the mechanism by which a kinesin-1 molecule moves individually along a microtubule is quite well-understood, the way that many kinesin-1 motor proteins bound to the same cargo move together along a microtubule is not. We identified a 60-amino-acid-long domain, termed Hinge 1, in kinesin-1 from Drosophila melanogaster that is located between the coiled coils of the neck and stalk domains. Its deletion reduces microtubule gliding speed in multiple-motor assays but not single-motor assays. Hinge 1 thus facilitates the cooperation of motors by preventing them from impeding each other. We addressed the structural basis for this phenomenon. Video-microscopy of single microtubule-bound full-length motors reveals the sporadic occurrence of high-compliance states alternating with longer-lived, low-compliance states. The deletion of Hinge 1 abolishes transitions to the high-compliance state. Based on Fourier transform infrared, circular dichroism, and fluorescence spectroscopy of Hinge 1 peptides, we propose that low-compliance states correspond to an unexpected structured organization of the central Hinge 1 region, whereas high-compliance states correspond to the loss of that structure. We hypothesize that strain accumulated during multiple-kinesin motility populates the high-compliance state by unfolding helical secondary structure in the central Hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple-motor situations.

TCF7L1 promotes skin tumorigenesis independently of ß-catenin through induction of LCN2.

The transcription factor TCF7L1 is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in skin tumorigenesis has not yet been defined. Here we document TCF7L1 upregulation in skin squamous cell carcinoma (SCC) and demonstrate that TCF7L1 overexpression increases tumor incidence, tumor multiplicity, and malignant progression in the chemically induced mouse model of skin SCC. Additionally, we show that downregulation of TCF7L1 and its paralogue TCF7L2 reduces tumor growth in a xenograft model of human skin SCC. Using separation-of-function mutants, we show that TCF7L1 promotes tumor growth, enhances cell migration, and overrides oncogenic RAS-induced senescence independently of its interaction with ß-catenin. Through transcriptome profiling and combined gain- and loss-of-function studies, we identified LCN2 as a major downstream effector of TCF7L1 that drives tumor growth. Our findings establish a tumor-promoting role for TCF7L1 in skin and elucidate the mechanisms underlying its tumorigenic capacity.

Contribution of Beta-HPV Infection and UV Damage to Rapid-Onset Cutaneous Squamous Cell Carcinoma during BRAF-Inhibition Therapy.

PURPOSE: BRAF-inhibition (BRAFi) therapy for advanced melanoma carries a high rate of secondary cutaneous squamous cell carcinoma (cSCC) and risk of other cancers. UV radiation and α-genus human papillomavirus (HPV) are highly associated with SCC, but a novel role for ß-genus HPV is suspected in BRAFi-cSCC. Cutaneous ß-HPV may act in concert with host and environmental factors in BRAFi-cSCC. EXPERIMENTAL DESIGN: Primary BRAFi-cSCC tissue DNA isolated from patients receiving vemurafenib or dabrafenib from two cancer centers was analyzed for the presence of cutaneous oncogenic viruses and host genetic mutations. Diagnostic specimens underwent consensus dermatopathology review. Clinical parameters for UV exposure and disease course were statistically analyzed in conjunction with histopathology. RESULTS: Twenty-nine patients contributed 69 BRAFi-cSCC lesions. BRAFi-cSCC had wart-like features (BRAFi-cSCC-WF) in 22% of specimens. During vemurafenib therapy, BRAFi-cSCC-WF arose 11.6 weeks more rapidly than conventional cSCC when controlled for gender and UV exposure (P value = 0.03). Among all BRAFi-cSCC, ß-genus HPV-17, HPV-38, HPV-111 were most frequently isolated, and novel ß-HPV genotypes were discovered (CTR, CRT-11, CRT-22). Sequencing revealed 63% of evaluated BRAFi-cSCCs harbored RAS mutations with PIK3CA, CKIT, ALK, and EGFR mutations also detected. CONCLUSIONS: We examined clinical, histopathologic, viral, and genetic parameters in BRAFi-cSCC demonstrating rapid onset; wart-like histomorphology; ß-HPV-17, HPV-38, and HPV-111 infection; UV damage; and novel ALK and CKIT mutations. Discovered ß-HPV genotypes expand the spectrum of tumor-associated viruses. These findings enhance our understanding of factors cooperating with BRAF inhibition that accelerate keratinocyte oncogenesis as well as broaden the knowledge base of multifactorial mediators of cancer in general.

The ambiguous boundary between EBV-related hemophagocytic lymphohistiocytosis and systemic EBV-driven T cell lymphoproliferative disorder.

Epstein Barr virus (EBV)-related hemophagocytic lymphohistiocytosis (EBV-HLH) is a form of acquired, infection-related HLH which typically represents a fulminant presentation of an acute EBV infection of CD8+ T cells with 30-50% mortality rate. Systemic EBV-positive lymphoproliferative disease of childhood (SE-LPD) is a rare T cell lymphoproliferative disorder predominantly arising in the setting of acute EBV infection, often presenting with HLH. Since both entities have been associated with clonal T cell populations, the discrimination between these diseases is often ambiguous. We report a unique case of a 21 years old female who presented with clinical and laboratory findings of florid HLH in the setting of markedly elevated EBV titers (>1 million) and an aberrant T cell population shown to be clonal by flow cytometry, karyotype, and molecular studies. This case raises the differential of EBV-HLH versus SE-LPD. Review of the literature identified 74 cases of reported EBV-HLH and 21 cases of SE-LPD with associated HLH in 25 studies. Of those cases with available outcome data, 62 of 92 cases (67%) were fatal. Of 60 cases in which molecular clonality was demonstrated, 37 (62%) were fatal, while all 14 cases (100%) demonstrating karyotypic abnormalities were fatal. Given the karyotypic findings in this sentinel case, a diagnosis of SE-LPD was rendered. The overlapping clinical and pathologic findings suggest that EBV-HLH and SE-LPD are a biologic continuum, rather than discrete entities. The most clinically useful marker of mortality was an abnormal karyotype rather than other standards of clonality assessment.

Severe cutaneous and neurologic toxicity in melanoma patients during vemurafenib administration following anti-PD-1 therapy.

Immune checkpoint inhibitors such as ipilimumab and targeted BRAF inhibitors have dramatically altered the landscape of melanoma therapeutics over the past few years. Agents targeting the programmed cell death-1/ligand (PD-1/PD-L1) axis are now being developed and appear to be highly active clinically with favorable toxicity profiles. We report two patients with BRAF V600E mutant melanoma who were treated with anti-PD-1 agents as first-line therapy without significant toxicity, followed by vemurafenib at disease progression. Both patients developed severe hypersensitivity drug eruptions with multi-organ injury early in their BRAF inhibitor treatment course. One patient subsequently developed acute inflammatory demyelinating polyneuropathy (AIDP) and the other developed anaphylaxis upon low-dose vemurafenib rechallenge. Further investigation of the immune response during combination or sequences of melanoma therapeutics is warranted. Furthermore, clinicians should maintain a high index of suspicion for these toxicities when vemurafenib is administered following an anti-PD-1 agent.

Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy.

In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.

Shared catalysis in virus entry and bacterial cell wall depolymerization.

Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide-cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active-site residues and a structural pattern of beta-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage varphi29. gp13 activity on PG and muropeptides was assayed using high-performance liquid chromatography, and gp13 was found to be a d,d-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile-bond activation and that His247 is oriented to mediate nucleophile generation. To our knowledge, this is the first model of a Zn(2)(+) metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn(2)(+) binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle-induced X-ray emission spectroscopy showed that the general protein folding and Zn(2)(+) binding were maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model in which the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage-entry enzymes have a shared chemical mechanism of action.