A novel method to assess copy number variations in melanocytic neoplasms: Droplet digital PCR for precise quantitation of MYC and MYB genes.

INTRODUCTION: While most melanocytic neoplasms can be classified as either benign or malignant by histopathology alone, ancillary molecular diagnostic tests can be necessary to establish the correct diagnosis in challenging cases. Currently, the detection of copy number variations (CNVs) by fluorescence in situ hybridization and chromosomal microarray (CMA) are the most popular methods, but remain expensive and inaccessible. We aim to develop a relatively inexpensive, fast, and accessible molecular assay to detect CNVs relevant to melanoma using droplet digital polymerase chain reaction (ddPCR) technology. METHODS: In this proof-of-concept study, we evaluated CNVs in MYC and MYB genes from 73 cases of benign nevi, borderline melanocytic lesions, and primary and metastatic melanoma at our institution from 2015 to 2022. A multiplexed ddPCR assay and CMA were performed on each sample, and the results were compared. RESULTS: Concordance analysis of ddPCR with CMA for quantification of MYC and MYB CNVs revealed a sensitivity and specificity of 89% and 86% for MYC and 83% and 74% for MYB, respectively. CONCLUSION: We demonstrate the first use of a multiplexed ddPCR assay to identify CNVs in melanocytic neoplasms. With further improvement and validation, ddPCR may represent a low-cost and rapid tool to aid in the diagnosis of histopathologically ambiguous melanocytic tumors.

A Novel Method to Detect Copy Number Variation in Melanoma: Droplet Digital PCR for Quantitation of the CDKN2A Gene, a Proof-of-Concept Study.

ABSTRACT: A definitive diagnosis of nevus or melanoma is not always possible for histologically ambiguous melanocytic neoplasms. In such cases, ancillary molecular testing can support a diagnosis of melanoma if certain chromosomal aberrations are detected. Current technologies for copy number variation (CNV) detection include chromosomal microarray analysis (CMA) and fluorescence in situ hybridization. Although CMA and fluorescence in situ hybridization are effective, their utilization can be limited by cost, turnaround time, and inaccessibility outside of large reference laboratories. Droplet digital polymerase chain reaction (ddPCR) is a rapid, automated, and relatively inexpensive technology for CNV detection. We investigated the ability of ddPCR to quantify CNV in cyclin-dependent kinase inhibitor 2A ( CDKN2A ), the most commonly deleted tumor suppressor gene in melanoma. CMA data were used as the gold standard. We analyzed 57 skin samples from 52 patients diagnosed with benign nevi, borderline lesions, primary melanomas, and metastatic melanomas. In a training cohort comprising 29 randomly selected samples, receiver operator characteristic curve analysis revealed an optimal ddPCR cutoff value of 1.73 for calling CDKN2A loss. In a validation cohort comprising the remaining 28 samples, ddPCR detected CDKN2A loss with a sensitivity and specificity of 94% and 90%, respectively. Significantly, ddPCR could also identify whether CDKN2A losses were monoallelic or biallelic. These pilot data suggest that ddPCR can detect CDKN2A deletions in melanocytic tumors with accuracy comparable with CMA. With further validation, ddPCR could provide an additional CNV assay to aid in the diagnosis of challenging melanocytic neoplasms.

Transfusion Avoidance in Severely Anemic Total Hip and Total Knee Arthroplasty Patients: An Analysis of Risk.

Background: Allogenic blood transfusions increase the risk of multiple complications. We evaluated the influence of restricting transfusions in adults with osteoarthritis that underwent total hip or knee arthroplasty (THA/TKA) with severe postoperative anemia. Material and methods: Patients that underwent THA/TKA for osteoarthritis with postoperative hemoglobin (Hb) ≤ 8 g/dl were retrospectively identified. We evaluated characteristics and adverse postoperative outcomes of patients not transfused and compared them to those of patients who received postoperative transfusion. Adverse outcomes were 90-day readmission, reoperation, infection, and falls, as well as inpatient cardiovascular events and deaths. Results: One thousand eighty-seven patients meeting inclusion criteria underwent THA and TKA. The 399 patients (36.7%) who did not undergo transfuion were younger (67.4 vs 69.5 years, P = .008), healthier (American Society of Anesthesiologist ≤ 2: 64.2% vs 56%, P = .006), comprised a lower proportion of cardiovascular disease patients (13.8% vs 24.7%, P < .001), a lower proportion of patients with Medicare/Medicare Managed Care (57.2% vs 65.5%, P = .05), received tranexamic acid more frequently (66.4% vs 52.9%, P < .01), had a shorter procedure time (92.7 vs 103.1, P < .01), a lower postoperative drop in Hb (4.0 vs 4.2 g/dl, P = .022), a later drop in Hb (2.6 vs 2.2 days, P = .003), and a shorter length of stay (3.5 vs 4.8, P < .01). TKA patients underwent transfusion more frequently than THA patients (67.5% vs 59%, P = .004). There were no postoperative deaths. Adverse events were similar between the 2 groups. Conclusion: Findings suggest that younger and healthier patients that have lower Hb later during their hospital stay need not undergo transfusion solely based on Hb levels. Routine transfusion triggers can be avoided even in more anemic patients.

Survivorship and Risk Factors for Re-Revision after Aseptic Revision Total Hip Arthroplasty in Patients Aged ≤55 Years.

BACKGROUND: There is a relative paucity of literature on the outcomes after revision total hip arthroplasty (rTHA) in young patients. This study reports the survivorship and risk factors for re-revision in patients aged ≤55 years. METHODS: We identified 354 patients undergoing aseptic nononcologic rTHA at mean follow-up of 5 years after revision, with mean age of 48 years, body mass index of 28 kg/m2, and 64% female. Thirty-five (10%) patients underwent at least 1 previous rTHA. The main indications for rTHA included wear/osteolysis (21%), adverse local tissue reaction (21%), recurrent instability (20%), acetabular loosening (16%), and femoral loosening (7%); and included acetabular component-only rTHA in 149 patients (42%), femoral component-only rTHA in 46 patients (13%), both component rTHA in 44 patients (12%), and head/liner exchanges in patients 115 (33%). The Kaplan-Meier method was used to measure survivorship free from re-revision THA, and multivariate regression was used to identify risk factors for re-revision THA. RESULTS: Sixty-two patients (18%) underwent re-revision THA at the mean time of 2.5 years, most commonly for instability (37%), aseptic loosening (27%), and prosthetic joint infection (15%). The rTHA survivorship from all-cause re-revision and reoperation was 83% and 79% at 5 years, respectively. Multivariate analysis demonstrated that patients undergoing femoral component only (hazard ratio 4.8, P = .014) and head/liner exchange rTHA (hazard ratio 2.5, P = .022) as risk factors for re-revision THA. CONCLUSION: About 1 in 5 patients aged ≤55 years undergoing rTHA required re-revision THA at 5 years, most commonly for instability. The highest risk group included patients undergoing head/liner exchanges and isolated femoral component revisions.

Contemporary Distal Femoral Replacements for Supracondylar Femoral Fractures Around Primary and Revision Total Knee Arthroplasties.

BACKGROUND: There is a paucity of data on the outcomes of distal femoral replacements (DFRs) in patients with total knee arthroplasty (TKA) periprosthetic fractures. We sought to characterize these patients' survivorship free from rerevision. METHODS: We retrospectively identified 49 patients, including 34 after primary TKA (primary cohort), 9 after revision TKA, and 6 conversions for failed open reduction and internal fixation (revision cohort) that underwent DFR for a periprosthetic femur fracture. The mean age was 76 years, and 40 patients (82%) were female. The mean follow-up was 4 years. Femoral fixation included 44 cemented stems (90%) and 5 cementless stems (10%). Survivorship free from rerevision was characterized by the Kaplan-Meier method; cox proportional regression was used to analyze the risk factors for rerevision. RESULTS: Survivorship free from any rerevision at 5 years in the primary and revision cohort was 93% and 18%, respectively. The revision cohort had a 5.3× higher risk of re-revision (P = .008). Survivorship free from re-revision for aseptic loosening at 5 years in the primary and revision cohort was 93% and 53%, respectively. Two of the 3 patients with cementless stems in the primary cohort underwent early rerevision for aseptic loosening, but patients with prior primary TKAs treated with cemented femoral fixation (n = 31) had a 97% 5-year survivorship free from re-revision. CONCLUSION: Patients with periprosthetic fractures around prior primary TKAs treated with DFRs with cemented femoral fixation had a 97% 5-year survivorship free from any re-revision. DFRs for periprosthetic femur fractures around revision TKAs or conversions of failed open reduction and internal fixations have a 5× increased risk of rerevision.

High Rate of Re-Revision in Patients Less Than 55 Years of Age Undergoing Aseptic Revision Total Knee Arthroplasty.

BACKGROUND: There are limited data on the outcomes of revision total knee arthroplasty in young patients. We sought to characterize the re-revision-free survival and risk factors for re-revision in patients less than 55 years who underwent aseptic revision TKA. METHODS: We retrospectively reviewed 197 revision TKAs at a mean follow-up of 5 years. Mean age was 49 years; mean body mass index was 31 kg/m2. Twenty-seven (14%) patients had at least 1 prior revision TKA. The most common indications for revision included instability (29%), arthrofibrosis (26%), and aseptic loosening (24%). Constraint included the following: 59 posterior-stabilized (30%), 123 varus-valgus constrained (62%), and 15 hinged (8%). Components revised included the following: 93 femur/tibia (47%), 68 polyethylene-only (35%), 19 femur-only (10%), and 17 other (9%). Survivorship free from re-revision was calculated via the Kaplan-Meier method and a multivariate Cox proportional regression was utilized to identify risk factors for re-revision. RESULTS: Survivorship free from any re-revision at 5 years was 80%. In the multivariate analysis, patients with a prior revision (hazard ratio [HR] = 2.78, P = .02), an isolated polyethylene exchange (HR = 3.0, P = .004), and a hinged prosthesis (HR = 3.47, P = .05) were significant risk factors for lower revision-free survival. Forty-two patients (21%) underwent re-revision, most commonly for periprosthetic joint infection (7%), instability (6%), and aseptic loosening (5%). Re-revision occurred in 18/68 (26%) patients undergoing an isolated polyethylene exchange. CONCLUSION: Patients less than 55 years undergoing revision TKA have a modest 5-year revision-free survival of 80%. Patients with prior revision TKAs (HR = 2.78), hinge type prostheses (HR = 3.47), and polyethylene-only revisions (HR = 3.0) had higher revision rates.

Dual-Mobility Constructs in Primary Total Hip Arthroplasty in High-Risk Patients With Spinal Fusions: Our Institutional Experience.

BACKGROUND: Prior spinal fusion significantly increases the risk of dislocation in patients after total hip arthroplasty (THA). Owing to these high risks, surgeons may use dual-mobility (DM) constructs in these patients to optimize hip stability. However, there is a paucity of data on the outcomes of DM constructs in patients who underwent prior spinal fusions. METHODS: We retrospectively identified 80 patients (86 THAs) who underwent a spinal arthrodesis and a subsequent posterior approach THA with a DM construct. The median number of levels fused was 4, with 59 (74%) patients having 2 or more levels fused; in addition, 50 (63%) patients were fused to the sacrum. Ninety percent and 55% of THAs were within the Lewinnek safe zone for inclination and anteversion, respectively. Patients were evaluated for any episode of hip instability, complications, and patient reported outcome measures. RESULTS: At 3-year mean follow-up, no patients sustained a postoperative dislocation or intraprosthetic dislocation (0%). Overall, there were 6 (7.5%) complications during the study period leading to reoperation in 3 (4%) patients, none related to the acetabular component or instability. Hip Injury and Osteoarthritis Outcome Score, Joint Replacement scores significantly improved from a mean of 50 preoperatively to 87 postoperatively (P < .001), and the Veterans Rand 12 Item Health Survey physical score improved from a mean of 31 preoperatively to 44 postoperatively (P < .001). CONCLUSION: In a high-risk series of patients who underwent prior spinal fusion, posterolateral primary THA with a DM construct demonstrated no dislocations at mean 3-year follow-up. Although these early data are clearly encouraging, more patients with longer term follow-up are needed.

Early Peri-operative Outcomes Were Unchanged in Patients Undergoing Spine Surgery During the COVID-19 Pandemic in New York City.

BACKGROUND: Healthcare resources have been greatly limited by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic halting non-essential surgical cases without clear service expansion protocols. QUESTIONS/PURPOSES: We sought to compare the peri-operative outcomes of patients undergoing spine surgery during the SARS-CoV-2 pandemic to a matched cohort prior to the pandemic. METHODS: We identified a consecutive sample of 127 adult patients undergoing spine surgery between March 9, 2020, and April 10, 2020, corresponding with the state of emergency declared in New York and the latest possible time for 1-month surgical follow-up. The study group was matched one-to-one based on age, gender, and body mass index with eligible control patients who underwent similar spine procedures prior to the SARS-CoV-2 outbreak. Surgeries performed for infectious or oncologic indications were excluded. Intra- and post-operative complication rates, re-operations, hospital length of stay, re-admissions, post-operative visit format, development of post-operative fever and/or respiratory symptoms, and SAR-CoV2 testing. RESULTS: A total of 254 patients (127 SARS-CoV-2 pandemic, 127 matched controls) were included. One hundred fifty-eight were male (62%), and 96 were female (38%). The mean age in the pandemic group was 59.8 ± 13.4 years; that of the matched controls was 60.3 ± 12.3. All patients underwent general anesthesia and did not require re-intubation. There were no significant differences in 1-month post-operative complication rates (16.5% pandemic vs. 12.6% control). There was one death in the pandemic group. No patients tested positive for the virus. CONCLUSION: This study represents the first report of post-operative outcomes in a large group of spine surgical patients in an area heavily affected by the SARS-CoV-2 pandemic.

Dual suppression of inner and outer mitochondrial membrane functions augments apoptotic responses to oncogenic MAPK inhibition.

Mitogen-activated protein kinase (MAPK) pathway inhibitors show promise in treating melanoma, but are unsuccessful in achieving long-term remission. Concordant with clinical data, BRAFV600E melanoma cells eliminate glycolysis upon inhibition of BRAFV600E or MEK with the targeted therapies Vemurafenib or Trametinib, respectively. Consequently, exposure to these therapies reprograms cellular metabolism to increase mitochondrial respiration and restrain cell death commitment. As the inner mitochondrial membrane (IMM) is sub-organellar site of oxidative phosphorylation (OXPHOS), and the outer mitochondrial membrane (OMM) is the major site of anti-apoptotic BCL-2 protein function, we hypothesized that suppressing these critical mitochondrial membrane functions would be a rational approach to maximize the pro-apoptotic effect of MAPK inhibition. Here, we demonstrate that disruption of OXPHOS with the mitochondria-specific protonophore BAM15 promotes the mitochondrial pathway of apoptosis only when oncogenic MAPK signaling is inhibited. Based on RNA-sequencing analyses of nevi and primary melanoma samples, increased pro-apoptotic BCL-2 family expression positively correlates with high-risk disease suggesting a highly active anti-apoptotic BCL-2 protein repertoire likely contributes to worse outcome. Indeed, combined inhibition of the anti-apoptotic BCL-2 repertoire with BH3-mimetics, OXPHOS, and oncogenic MAPK signaling induces fulminant apoptosis and eliminates clonogenic survival. Altogether, these data suggest that dual suppression of IMM and OMM functions may unleash the normally inadequate pro-apoptotic effects of oncogenic MAPK inhibition to eradicate cancer cells, thus preventing the development of resistant disease, and ultimately, supporting long-term remission.