pVACview: an interactive visualization tool for efficient neoantigen prioritization and selection.

Background: Neoantigen targeting therapies including personalized vaccines have shown promise in the treatment of cancers, particularly when used in combination with checkpoint blockade therapy. At least 100 clinical trials involving these therapies are underway globally. Accurate identification and prioritization of neoantigens is highly relevant to designing these trials, predicting treatment response, and understanding mechanisms of resistance. With the advent of massively parallel DNA and RNA sequencing technologies, it is now possible to computationally predict neoantigens based on patient-specific variant information. However, numerous factors must be considered when prioritizing neoantigens for use in personalized therapies. Complexities such as alternative transcript annotations, various binding, presentation and immunogenicity prediction algorithms, and variable peptide lengths/registers all potentially impact the neoantigen selection process. There has been a rapid development of computational tools that attempt to account for these complexities. While these tools generate numerous algorithmic predictions for neoantigen characterization, results from these pipelines are difficult to navigate and require extensive knowledge of the underlying tools for accurate interpretation. This often leads to over-simplification of pipeline outputs to make them tractable, for example limiting prediction to a single RNA isoform or only summarizing the top ranked of many possible peptide candidates. In addition to variant detection, gene expression and predicted peptide binding affinities, recent studies have also demonstrated the importance of mutation location, allele-specific anchor locations, and variation of T-cell response to long versus short peptides. Due to the intricate nature and number of salient neoantigen features, presenting all relevant information to facilitate candidate selection for downstream applications is a difficult challenge that current tools fail to address. Results: We have created pVACview, the first interactive tool designed to aid in the prioritization and selection of neoantigen candidates for personalized neoantigen therapies including cancer vaccines. pVACview has a user-friendly and intuitive interface where users can upload, explore, select and export their neoantigen candidates. The tool allows users to visualize candidates across three different levels, including variant, transcript and peptide information. Conclusions: pVACview will allow researchers to analyze and prioritize neoantigen candidates with greater efficiency and accuracy in basic and translational settings The application is available as part of the pVACtools pipeline at pvactools.org and as an online server at pvacview.org.

Reconstruction after Mohs Surgery for Digit Melanoma: Description of Techniques and Postoperative Limb Function.

Introduction: Mohs surgery and reconstruction has enabled tissue-preserving resection of cutaneous malignancies. The goal of our case series evaluation is to present reconstructive techniques and functional outcomes in patients undergoing digit-sparing treatment for primary melanoma. Materials and Methods: A chart review was performed to identify consecutive patients undergoing Mohs surgery and reconstruction for melanoma of the digits. Quality of life (QOL) survey was performed to assess function after the procedure. Results: Thirty-two patients (13 hand, 19 foot, Age: 65.03 +/-17.78 years) who were undergoing Mohs surgery were identified. No recurrence was identified with an average follow-up of 16.1 months (1-95 months). The average defect size was 5.79 +/-4.54 cm2. Reconstruction was performed 0-4 days after resection. The most common techniques included full-thickness skin graft (FTSG) (N = 7), collagen matrix + FTSG (N = 4), and volar advancement flap (N = 7). The reconstructive technique choice appears correlated with defect size (p = 0.0125). Neuro-QOL upper extremity survey results showed a difference that approached statistical significance between patients who underwent digit-sparing treatment (n = 7) versus direct to amputation controls (n = 5) (p = 0.072). No survey differences between digit-sparing treatment (n = 10) and amputation (n = 8) were identified in the lower extremity (p = 0.61). Conclusion: Our results show digit-sparing treatment can confirm clear surgical margins and a trend toward improvement in upper extremity function compared with immediate amputation.

Oncologic control and predictors of urologic reconstruction after Mohs micrographic surgery for low-risk penile malignancy.

PURPOSE: Mohs micrographic surgery (MMS) is a low-risk penile cancer management option. However, contemporary patients' short-term oncologic control and preoperative characteristics predicting reconstruction needs are undefined. This study assesses MMS's oncologic efficacy for low-risk penile cancer and identifies baseline predictors of post-resection reconstruction referral. METHODS: We retrospectively reviewed 73 adult males with 78 penile cutaneous malignancies treated with MMS from 2005 to 2019. Patients underwent MMS with or without surgical reconstruction. Demographic information, MMS operative details, lesion pathology, and short-term outcomes were recorded. Descriptive statistics for all variables were calculated, and logistic regression identified predictive factors for urologic referral for complex reconstruction. RESULTS: Seventy-three men with 78 lesions, all staged ≤ cT1a prior to MMS, were identified. Twenty-one men were found to have invasive SCC. Median follow-up was 2.0 years (IQR 0.8-5.2 years). MMS was able to clear the disease in 90.4% of cases. One patient had disease related death following progression. Dermatology closed primarily in 68% of patients. Twenty percent of patients had a complication, most commonly poor wound healing. On univariate and multivariate linear regression analysis, lesion size > 3 cm and involvement of the glans independently predicted the need for referral to a reconstructive surgeon. CONCLUSIONS: MMS for penile cancer appears to provide sound oncologic control in the properly selected patient. Involvement of a reconstructive surgeon may be needed for glandular and large lesions, necessitating early referral to a comprehensive multidisciplinary care team.

Antibiotic adherence in dermatologic surgery: a Multicenter prospective cohort study.

Understanding patient non-adherence to prescribed antibiotics can inform clinical practices, patient counseling, and antibiotic efficacy study design in dermatology. The primary objective was to determine the rate of and reasons for antibiotic non-adherence in the dermatologic surgery setting. The secondary objective was to test the applicability of previously studied survey questions for antibiotic non-adherence screening in the dermatologic surgery setting. Five academic outpatient dermatologic surgery centers across the United States conducted one multicenter prospective cohort study. Dermatologic surgery patients ≥ 18 years of age who were prescribed an antibiotic were included as part of this study. 15.2% (42/276) of patients did not adhere to their antibiotic regimen after dermatologic surgery. Most common reasons for incomplete antibiotic courses included forgotten antibiotics (42.9%,18/42) and side effects (28.6%, 12/42). Previously evaluated questions to identify and predict non-adherence had modest performance in the dermatologic surgery setting (Area under the curve of 0.669 [95% CI (0.583-0.754)]). Antibiotic non-adherence after skin surgery is prevalent and commonly due to reasons that physicians can address with patients.

Reconstructing Nasal Defects With Acellular Dermal Matrix After Mohs Micrographic Surgery: A 12-year Experience.

BACKGROUND AND OBJECTIVE: Large defects of the nose after Mohs surgery pose a significant reconstructive challenge to both dermatologic and reconstructive surgeons. The authors present their 12-year experience utilizing acellular dermal matrices for nasal reconstruction. METHODS: A retrospective review of patients undergoing Mohs surgery and alloplastic nasal reconstruction with acellular dermal matrices between 2010 and 2022 was performed. Patients who underwent single-stage reconstruction and dual-stage reconstruction with skin graft with at least 90 days of follow-up were included. RESULTS: Fifty-one patients met criteria with a median age of 77 years. Fifty-three lesions were reconstructed with acellular dermal matrices. The most common lesion location was nasal sidewall (50%) with a mean defect size of 10.8 cm 2 . 30.8% underwent same-day acellular dermal matrix reconstruction, with 69.2% undergoing two-stage reconstruction. Acellular dermal matrices successfully reconstructed acquired defects in 94.2% of lesions. Average time to re-epithelialization was 27.6 + 6.2 days. Average time to repigmentation was 145.35 + 86 days. No recurrences were recorded. Total complication rate was 9.62%. Average size for successful healing was 10.8 cm 2 . Average defect size for complication or failure was 14.7 cm 2 . Seven sites (13.46%) underwent aesthetic improvement procedures. CONCLUSION: Acellular bilayer wound matrix is an adequate reconstructive option for single or dual-stage reconstruction of the nose with low complication and revision rates.

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells.

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Frozen Section Histoanatomy of the Peritarsal Eyelid.

BACKGROUND: Mohs surgery of eyelid skin cancers requires detailed knowledge of anatomy for precise surgery and accurate evaluation of histology. OBJECTIVE: To review the histology of the peritarsal eyelid using frozen sections as encountered intraoperatively by Mohs surgeons. METHODS: The authors review the literature describing the anatomy and histology of the peritarsal eyelid from the lens of a Mohs surgeon. Histology from select Mohs cases is used to frame the discussion of the microanatomy of this region. RESULTS: The peritarsal eyelids contain a unique mixture of skin, muscle, tarsus, glandular tissue, and conjunctiva. The histologic appearance of many of these structures differs from skin found outside of this anatomic region. Tumors of the eyelid and periocular region may mimic normal histologic structures found within the peritarsal eyelid. CONCLUSION: The peritarsal eyelids have unique anatomy and associated histologic structures. Knowledge of the detailed histoanatomy is required for confident execution of Mohs surgery in this anatomic region.

Minimally Invasive vs Open Approach for First Metatarsophalangeal Joint Arthrodesis: Short Report of Early Results.

BACKGROUND: We aim to compare early surgical results between groups who underwent minimally invasive surgery (MIS) vs open first metatarsophalangeal (MTP) arthrodesis to treat end-stage hallux rigidus. METHODS: We conducted a retrospective cohort review of 65 patients who underwent a first MTP fusion procedure at an academic medical center between 2015 and 2023. Success of fusion was determined radiographically. Postoperative complications were identified through medical record review. RESULTS: Sixty-seven first MTP fusion surgeries (41 open and 26 MIS) were performed on 65 patients with a primary diagnosis of hallux rigidus. Open surgery and MIS groups had similarly high fusion rates: 95% (39/41) and 96% (25/26), respectively (P = .84). We identified no difference in overall complication rates: 20% for open surgery and 23% for MIS (P = .73). CONCLUSION: This retrospective analysis of 67 first MTP arthrodesis procedures showed no significant differences in fusion success or complications in the short term when comparing MIS to open surgery. Further studies are needed to elucidate potential differences between MIS vs open surgery. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Effect of sequential burr passes on minimally invasive akin and first metatarsal dorsiflexion osteotomies.

BACKGROUND: Minimally invasive surgical (MIS) osteotomies are increasing as a surgical option for treating midfoot and forefoot conditions. This study aimed to evaluate the impact of each burr pass on the degree of correction, gap size, and alignment in MIS Akin and first metatarsal dorsiflexion osteotomies (DFO). METHODS: MIS Akin and first metatarsal DFO were performed on ten cadaveric specimens. Fluoroscopic measurements included the metatarsal dorsiflexion angle (MDA), dorsal cortical length (MDCL), first phalangeal medial cortical length (PCML) and proximal to distal phalangeal articular angle (PDPAA). RESULTS: The average decrease in PCML with each burr pass was as follows: 1.53, 1.33, 1.27, 1.23 and 1.13 mm at the 1st to 5th pass, respectively. The MDCL sequentially decreased by 1.80, 1.59, 1.35, 0.75, and 0.60 mm. The MDA consistently decreased, and the PDPAA incrementally became more valgus oriented. CONCLUSION: On average, a first metatarsal dorsal wedge resection of 4.7 mm and first phalangeal medial wedge resection of 2.9 mm was achieved after 3 and 2 burr passes, respectively. This data may aid surgeons determine the optimal number of burr passes required to achieve the desired patient-specific surgical correction.

Rapid and accurate remethylation of DNA in Dnmt3a-deficient hematopoietic cells with restoration of DNMT3A activity.

Here, we characterize the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic deficiency of Dnmt3a or Dnmt3b (or both enzymes) or expressing the dominant-negative Dnmt3aR878H mutation [R882H in humans; the most common DNMT3A mutation found in acute myeloid leukemia (AML)]. Using these cells as substrates, we defined DNA remethylation after overexpressing wild-type (WT) DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive "chaperone" for DNMT3A and DNMT3B in early embryogenesis). Overexpression of DNMT3A for 2 weeks reverses the hypomethylation phenotype of Dnmt3a-deficient cells or cells expressing the R878H mutation. Overexpression of DNMT3L (which is minimally expressed in AML cells) also corrects the hypomethylation phenotype of Dnmt3aR878H/+ marrow, probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele. DNMT3L reactivation may represent a previously unidentified approach for restoring DNMT3A activity in hematopoietic cells with reduced DNMT3A function.

A revised nomenclature for the lemur family of protein kinases.

The lemur family of protein kinases has gained much interest in recent years as they are involved in a variety of cellular processes including regulation of axonal transport and endosomal trafficking, modulation of synaptic functions, memory and learning, and they are centrally placed in several intracellular signalling pathways. Numerous studies have also implicated role of the lemur kinases in the development and progression of a wide range of cancers, cystic fibrosis, and neurodegenerative diseases. However, parallel discoveries and inaccurate prediction of their kinase activity have resulted in a confusing and misleading nomenclature of these proteins. Herein, a group of international scientists with expertise in lemur family of protein kinases set forth a novel nomenclature to rectify this problem and ultimately help the scientific community by providing consistent information about these molecules.

Genital epithelial barrier function is conserved by intravaginal rings releasing etonogestrel and ethinyl estradiol.

The injectable progestin depot-medroxyprogesterone acetate (DMPA) is a popular contraceptive choice in sub-Saharan Africa although mouse models indicate it weakens genital epithelial integrity and barrier function and increases susceptibility to genital infection. The intravaginal ring NuvaRing® is another contraceptive option that like DMPA suppresses hypothalamic pituitary ovarian (HPO) axis function with local release of progestin (etonogestrel) and estrogen (ethinyl estradiol). As we previously reported that treating mice with DMPA and estrogen averts the loss of genital epithelial integrity and barrier function induced by DMPA alone, in the current investigation we compared genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and genital epithelial permeability in rhesus macaques (RM) treated with DMPA or a NuvaRing®re-sized for RM (N-IVR). While these studies demonstrated comparable inhibition of the HPO axis with DMPA or N-IVR, DMPA induced significantly lower genital DSG1 levels and greater tissue permeability to intravaginally administered low molecular mass molecules. By identifying greater compromise of genital epithelial integrity and barrier function in RM administered DMPA vs. N-IVR, our results add to the growing body of evidence that indicate DMPA weakens a fundamental mechanism of anti-pathogen host defense in the female genital tract.

Effect of sequential burr passes on osteotomy magnitude and calcaneal morphology in minimally invasive Zadek osteotomy.

PURPOSE: This study aimed to evaluate the impact of each burr pass on degree of correction, gap size and calcaneal morphology in MIS Zadek osteotomy. METHODS: MIS Zadek osteotomy was performed on ten cadaveric specimens using a 3.1 mm Shannon burr. After each burr pass, the osteotomy gap was manually closed, and the subsequent burr passes were carried out with the foot held in dorsiflexion, which was repeated five times. Lateral X-rays were taken before and after each burr pass. Two independent reviewers measured the dorsal calcaneal length after each burr passage, as well as changes in several calcaneal parameters including X/Y ratio, Fowler Philip angle, and Böhler angle. RESULTS: The average decrease in dorsal calcaneal cortical length with each burr pass was as follows: 2.6 ± 0.9 mm at the 1st pass, 2.4 ± 1 mm at the 2nd pass, 2 ± 1 mm at the 3rd pass, 1.6 ± 1 mm at the 4th pass, and 1.4 ± 0.7 mm at the 5th pass. The Fowler Philip and Böhler angles consistently decreased while the X/Y ratio consistently increased following each consecutive burr pass. Interobserver reliability analysis demonstrated good agreement for all parameters. CONCLUSION: The results revealed the trends of length and anatomical changes in the calcaneus with each burr pass. On average, a dorsal wedge resection of 10 mm was achieved after 5 burr passes. This data can aid surgeons in determining the optimal number of burr passes required for a particular amount of resection, ensuring the attainment of the desired patient-specific surgical outcome.

Identifying critical quality metrics in Mohs Surgery: A national expert consensus process.

BACKGROUND: Amid a movement toward value-based healthcare, increasing emphasis has been placed on outcomes and cost of medical services. To define and demonstrate the quality of services provided by Mohs surgeons, it is important to identify and understand the key aspects of Mohs micrographic surgery (MMS) that contribute to excellence in patient care. OBJECTIVE: The purpose of this study is to develop and identify a comprehensive list of metrics in an initial effort to define excellence in MMS. METHODS: Mohs surgeons participated in a modified Delphi process to reach a consensus on a list of metrics. Patients were administered surveys to gather patient perspectives. RESULTS: Twenty-four of the original 66 metrics met final inclusion criteria. Broad support for the initiative was obtained through physician feedback. LIMITATIONS: Limitations of this study include attrition bias across survey rounds and participation at the consensus meeting. Furthermore, the list of metrics is based on expert consensus instead of quality evidence-based outcomes. CONCLUSION: With the goal of identifying metrics that demonstrate excellence in performance of MMS, this initial effort has shown that Mohs surgeons and patients have unique perspectives and can be engaged in a data-driven approach to help define excellence in the field of MMS.

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID-interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

Using Integra for Reconstruction of Facial Defects after Mohs Micrographic Surgery.

Background: We aimed to identify how Integra bilayer wound matrix has expanded facial reconstruction options after Mohs surgery due to its reliability in both single- and dual-stage reconstruction. Methods: A retrospective review of patients undergoing Mohs surgery and alloplastic facial reconstruction with Integra between 2012 and 2022 was performed. Patients who underwent single-stage reconstruction and dual-stage reconstruction with skin graft with at least 90 days of follow-up were included. Results: One hundred thirty patients with a median age of 76 years were included. Basal cell carcinoma was the most common malignancy (39%). One hundred forty-two lesions were treated and reconstructed same-day with Integra. Lesions most commonly involved the nose (34%) and forehead (22%). The mean postoperative defect size was 26.9 cm2. An estimated 45.5% (n = 60) of defect sites underwent single-stage reconstruction with healing by secondary intention, whereas 54.5% (n = 72) underwent dual-stage reconstruction with skin graft. Integra success rate was 90.2%. Average time to re-epithelialization was 32.2 + 7.3 days. Average time to repigmentation was 169.5 + 14.6 days. The complication rate was 12.8% (n = 17), with 12 undergoing debridement, three needing new Integra graft, and seven needing new skin grafts. Average size for successful healing without complication was 26.6 cm2. Nineteen sites (13.2%) underwent aesthetic improvement procedures, with the majority occurring after dual-stage reconstruction (n = 13). Conclusions: Integra is a reliable outpatient reconstructive option for facial Mohs defects that can increase the threshold for autologous tissue harvesting and successfully reconstruct large defects of 26.6 cm2 on average with low complication and reoperation rates.

Ultra-Deep Sequencing Reveals the Mutational Landscape of Classical Hodgkin Lymphoma.

The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to approximately 1,000× median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single-nucleotide variants (SNV) in single-nuclei RNA sequencing (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of patients with cHL. SIGNIFICANCE: Our data demonstrate the utility of ultra-deep exome sequencing in uncovering somatic variants in Hodgkin lymphoma, creating new opportunities to define the genes that are recurrently mutated in this disease. We also show for the first time the successful application of snRNA-seq in Hodgkin lymphoma and describe the expression profile of a putative cluster of HRS cells in a single patient.