Characteristics of isatuximab-derived interference in serum protein electrophoresis and immunofixation, and an absence of sustained in vivo interference due to belantamab mafodotin and denosumab.

OBJECTIVES: Some therapeutic monoclonal antibodies, like daratumumab and elotuzumab, produce interfering monoclonal bands on serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). Whether other common therapeutic antibodies also produce interference has not been systematically evaluated. DESIGN AND METHODS: SPEP/IFE from patients receiving isatuximab (48 patients), belantamab mafodotin (BM; 41), and denosumab (41) were retrospectively reviewed for therapeutic antibody interference. Cases exhibiting isatuximab interference were quantified and the maximum duration of isatuximab effect was evaluated. To characterize band position, neat human serum was spiked with BM or denosumab at supratherapeutic concentrations. Band migration patterns were compared on SPEP and IFE, with band position expressed relative to other constant protein fractions. RESULTS: Isatuximab-induced IFE interference was common (81.3 % of evaluated patients) with a maximum observed duration of 8 weeks. 10.4 % of isatuximab patients had IgG-kappa monoclonal gammopathies that co-migrated with the drug; this subset could benefit from HYDRASHIFT 2/4 isatuximab testing. 8.3 % of IFE cases were negative for an isatuximab band but showed large, endogenous M-spikes migrating elsewhere. All patients in this group expired within 1 year of this finding. We hypothesize that an inability to detect isatuximab in this setting corresponds to a large residual myeloma burden that reduces isatuximab serum concentration. This observation may serve as a negative prognostic factor. Spiking studies demonstrated that BM and denosumab produce interference in vitro, but sustained interference was not observed in >40 treated patients. CONCLUSIONS: Therapeutic antibody interference in patients receiving isatuximab is common, and can persist for at least 8 weeks after administration. >10 % of patients receiving isatuximab may benefit from HYDRASHIFT testing post-therapy. In contrast, BM and denosumab fail to produce sustained interference in treated patients.

Flow cytometry quantification of tumor-infiltrating lymphocytes to predict the survival of patients with diffuse large B-cell lymphoma.

Introduction: Our previous studies have demonstrated that tumor-infiltrating lymphocytes (TILs), including normal B cells, T cells, and natural killer (NK) cells, in diffuse large B-cell lymphoma (DLBCL) have a significantly favorable impact on the clinical outcomes of patients treated with standard chemoimmunotherapy. In this study, to gain a full overview of the tumor immune microenvironment (TIME), we assembled a flow cytometry cohort of 102 patients diagnosed with DLBCL at the Duke University Medical Center. Methods: We collected diagnostic flow cytometry data, including the proportion of T cells, abnormal B cells, normal B cells, plasma cells, NK cells, monocytes, and granulocytes in fresh biopsy tissues at clinical presentation, and analyzed the correlations with patient survival and between different cell populations. Results: We found that low T cell percentages in all viable cells and low ratios of T cells to abnormal B cells correlated with significantly poorer survival, whereas higher percentages of normal B cells among total B cells (or high ratios of normal B cells to abnormal B cells) and high percentages of NK cells among all viable cells correlated with significantly better survival in patients with DLBCL. After excluding a small number of patients with low T cell percentages, the normal B cell percentage among all B cells, but not T cell percentage among all cells, continued to show a remarkable prognostic effect. Data showed significant positive correlations between T cells and normal B cells, and between granulocytes and monocytes. Furthermore, we constructed a prognostic model based on clinical and flow cytometry factors, which divided the DLBCL cohort into two equal groups with remarkable differences in patient survival and treatment response. Summary: TILs, including normal B cells, T cells, and NK cells, are associated with favorable clinical outcomes in DLBCL, and flow cytometry capable of quantifying the TIME may have additional clinical utility for prognostication.

Pediatric Atypical Melanocytic Proliferations: Single-Site Retrospective Cohort Assessment of Treatment and Long-Term Follow-Up.

Atypical and malignant cutaneous tumors are understudied in the pediatric population, with limited data on long-term follow-up. This study examines pediatric (0-18 years) atypical melanocytic proliferations over a twenty-year period (January 2002-December2022) using the EPIC SlicerDicer at our institution. Over a twenty-year period, there were 55 cases of pediatric melanoma (53 patients). The median follow-up time was 8 years, 11 months. A proportion of 96% were treated with wide local excision (WLE), and 47% had a sentinel lymph node biopsy (SLNB) (35% positive rate). There were 101 atypical Spitz tumor cases (85% atypical Spitz tumors, 15% Spitz melanoma), with a median follow-up duration of 9 years. A proportion of 77% were treated with WLE (with one patient dying of metastatic disease). There were 10 cases of atypical melanocytic proliferations not otherwise specified, including 5 pigmented epithelioid melanocytomas (PEM), 4 deep-penetrating nevi, and 1 atypical cellular blue nevus. This study adds to the growing body of knowledge on pediatric atypical cutaneous melanocytic proliferations, aligning with many described characteristics such as disease location and overall survival rates, with distinct exceptions (higher melanoma positive SLNB rate, lower atypical Spitz tumor WLE rate, and a case of fatal metastatic atypical Spitz tumor).

Renal biopsy in systemic infections: expect the unexpected.

Infection-related glomerulonephritis is well recognized in patients with ongoing infections. It can be missed, however, if the infection is unusual or undetected. We present three cases where the renal biopsy findings prompted the identification or treatment of systemic infections.Case 1: A 84-year-old male presented with acute kidney injury (AKI) and IgA vasculitis on skin biopsy. A renal biopsy showed active glomerulonephritis with abundant neutrophils and predominantly mesangial immune complex deposits containing IgA. The findings prompted an infectious workup which was positive for COVID-19, suggesting exacerbation of IgA nephropathy by recent COVID-19 infection. Case 2: A 31-year-old female status post kidney transplant for granulomatosis with polyangiitis (GPA) had recent pregnancy with preterm delivery, disseminated herpes simplex virus (HSV) infection with HSV hepatitis, E. coli on urine culture, and AKI. A renal biopsy showed proliferative glomerulonephritis with subendothelial and mesangial immune complex deposits containing IgG and C3. The findings were most consistent with infection-related immune complex glomerulonephritis, most likely HSV-related. Case 3: A 78-year-old female presented with AKI, proteinuria, hematuria, and positive p-ANCA. Clinically, ANCA vasculitis was suspected, and renal biopsy did show focal, segmental, necrotizing glomerulonephritis. However, immunofluorescence and electron microscopy showed IgM-rich deposits in the mesangium. The unusual presentation prompted an infectious workup including a Bartonella antibody panel which showed very high titers, suggesting Bartonella endocarditis.Infection-related glomerulonephritis has a wide variety of presentations histologically and clinically. The three cases we present here emphasize the importance of recognizing these entities to help guide treatment and improve patient care.

Reconciling Discordance Between Prostate Biopsy Histology and Magnetic Resonance Imaging Suspicion - Implementation of a Quality Improvement Protocol of Imaging Re-review and Reverse-fusion Target Analysis.

There is uncertainty with how to proceed when targeted prostate biopsy of suspicious multiparametric magnetic resonance imaging (mpMRI) lesions return without clinically significant prostate cancer (csPCa). While possible, there are error sources that could contribute to such discordance including the mpMRI read, mpMRI-ultrasound fusion, biopsy technique, and histologic classification. Consequences are potentially significant; mistakenly missing csPCa can lead to delays in curative treatment. Conversely, in cases of incorrect mpMRI interpretation, the patient may be subjected to unnecessary workup/burden. At our institution, we implemented a quality improvement (QI) initiative triggered after a discordant case occurs. This multidisciplinary review process incorporates mpMRI re-review and assessment of accurate lesion-sampling, termed "reverse-fusion." Herein, we describe the protocol, present sample cases, and discuss clinical implications.

Targeting Protein Arginine Methyltransferase 5 Suppresses Radiation-induced Neuroendocrine Differentiation and Sensitizes Prostate Cancer Cells to Radiation.

Prostate cancer remains the second leading cause of cancer death among American men. Radiotherapy is a potentially curative treatment for localized prostate cancer, and failure to control localized disease contributes to the majority of prostate cancer deaths. Neuroendocrine differentiation (NED) in prostate cancer, a process by which prostate adenocarcinoma cells transdifferentiate into neuroendocrine-like (NE-like) cells, is an emerging mechanism of resistance to cancer therapies and contributes to disease progression. NED also occurs in response to treatment to promote the development of treatment-induced neuroendocrine prostate cancer (NEPC), a highly aggressive and terminal stage disease. We previously demonstrated that by mimicking clinical radiotherapy protocol, fractionated ionizing radiation (FIR) induces prostate cancer cells to undergo NED in vitro and in vivo. Here, we performed transcriptomic analysis and confirmed that FIR-induced NE-like cells share some features of clinical NEPC, suggesting that FIR-induced NED represents a clinically relevant model. Furthermore, we demonstrated that protein arginine methyltransferase 5 (PRMT5), a master epigenetic regulator of the DNA damage response and a putative oncogene in prostate cancer, along with its cofactors pICln and MEP50, mediate FIR-induced NED. Knockdown of PRMT5, pICln, or MEP50 during FIR-induced NED and sensitized prostate cancer cells to radiation. Significantly, PRMT5 knockdown in prostate cancer xenograft tumors in mice during FIR prevented NED, enhanced tumor killing, significantly reduced and delayed tumor recurrence, and prolonged overall survival. Collectively, our results demonstrate that PRMT5 promotes FIR-induced NED and suggests that targeting PRMT5 may be a novel and effective radiosensitization approach for prostate cancer radiotherapy.

Pathologic Characteristics Associated With Local Recurrence of Atypical Meningiomas Following Surgical Resection.

BACKGROUND: Optimal management of grade II meningiomas following resection remains controversial, owing mostly to the heterogeneity of post-operative (post-op) recurrence patterns across studies. Improved risk stratification of these patients would ensure that only those most at risk of recurrence would undergo appropriate post-op radiation therapy (RT). METHODS: Medical records from patients who underwent resection for grade II meningiomas were retrospectively reviewed. Demographic, disease characteristics, treatment, and clinical course data were retrospectively collected. Logistic regression, Cox proportional hazards modeling, and Kaplan-Meier curves with log rank testing were conducted to describe any potential relationships with time of recurrence. RESULTS: Of the 49 patients identified, 18 (36.7%) suffered a local recurrence following resection with a median follow-up of 3.1 years (range: 0.23 - 17.1 years). Past recurrence of the meningioma (P = 0.002) and extent of resection (P = 0.02) were significantly associated with local recurrence. On multivariable analysis, only prior meningioma recurrence was associated with time to local failure (P = 0.021). No histopathologic factors were found to be associated with the initial local failure. Of those who suffered a local recurrence, the presence of bone invasion (hazard ratio: 0.069, P = 0.008) and lack of salvage RT (P = 0.02) were associated with subsequent local failure. CONCLUSIONS: Currently considered histopathologic factors appear not to be helpful in guiding initial treatment course. History of prior local failure and bone invasion appear to be associated with multiple recurrences. Optimal surgical resection is critical to improving outcomes, and salvage RT may reduce subsequent local failure.

Dynamic human MutSα-MutLα complexes compact mismatched DNA.

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

Potassium-sparing effects of furosemide in mice on high-potassium diets.

In individuals on a regular "Western" diet, furosemide induces a kaliuresis and reduction in plasma K concentration by inhibiting Na reabsorption in the thick ascending limb of Henle's loop, enhancing delivery of Na to the aldosterone-sensitive distal nephron. In the aldosterone-sensitive distal nephron, the increased Na delivery stimulates K wasting due to an exaggerated exchange of epithelial Na channel-mediated Na reabsorption of secreted K. The effects of furosemide are different in mice fed a high-K, alkaline (HK) diet: the large-conductance Ca-activated K (BK) channel, in conjunction with the BK ß4-subunit (BK-α/ß4), mediates K secretion from intercalated cells (IC) of the connecting tubule and collecting ducts. The urinary alkaline load is necessary for BK-α/ß4-mediated K secretion in HK diet-fed mice. However, furosemide acidifies the urine by increasing vacuolar ATPase expression and acid secretion from IC, thereby inhibiting BK-α/ß4-mediated K secretion and sparing K. In mice fed a low-Na, high-K (LNaHK) diet, furosemide causes a greater increase in plasma K concentration and reduction in K excretion than in HK diet-fed mice. Micropuncture of the early distal tubule of mice fed a LNaHK diet, but not a regular or a HK diet, reveals K secretion in the thick ascending limb of Henle's loop. The sites of action of K secretion in individuals consuming a high-K diet should be taken into account when diuretic agents known to waste K with low or moderate K intakes are prescribed.

Furosemide reduces BK-αß4-mediated K+ secretion in mice on an alkaline high-K+ diet.

Special high-K diets have cardioprotective effects and are often warranted in conjunction with diuretics such as furosemide for treating hypertension. However, it is not understood how a high-K diet (HK) influences the actions of diuretics on renal K+ handling. Furosemide acidifies the urine by increasing acid secretion via the Na+-H+ exchanger 3 (NHE3) in TAL and vacuolar H+-ATPase (V-ATPase) in the distal nephron. We previously found that an alkaline urine is required for large conductance Ca2+-activated K+ (BK)-αß4-mediated K+ secretion in mice on HK. We therefore hypothesized that furosemide could reduce BK-αß4-mediated K+ secretion by acidifying the urine. Treating with furosemide (drinking water) for 11 days led to decreased urine pH in both wild-type (WT) and BK-ß4-knockout mice (BK-ß4-KO) with increased V-ATPase expression and elevated plasma aldosterone levels. However, furosemide decreased renal K+ clearance and elevated plasma [K+] in WT but not BK-ß4-KO. Western blotting and immunofluorescence staining showed that furosemide treatment decreased cortical expression of BK-ß4 and reduced apical localization of BK-α in connecting tubules. Addition of the carbonic anhydrase inhibitor, acetazolamide, to furosemide water restored urine pH along with renal K+ clearance and plasma [K+] to control levels. Acetazolamide plus furosemide also restored the cortical expression of BK-ß4 and BK-α in connecting tubules. These results indicate that in mice adapted to HK, furosemide reduces BK-αß4-mediated K+ secretion by acidifying the urine.

Net K+ secretion in the thick ascending limb of mice on a low-Na, high-K diet.

Because of its cardio-protective effects, a low-Na, high-K diet (LNaHK) is often warranted in conjunction with diuretics to treat hypertensive patients. However, it is necessary to understand the renal handling of such diets in order to choose the best diuretic. Wild-type (WT) or Renal Outer Medullary K channel (ROMK) knockout mice (KO) were given a regular (CTRL), LNaHK, or high-K diet (HK) for 4-7 days. On LNaHK, mice treated with either IP furosemide for 12 hrs, or given furosemide in drinking water for 7 days, exhibited decreased K clearance. We used free-flow micropuncture to measure the [K+] in the early distal tubule (EDT [K+]) before and after furosemide treatment. Furosemide increased the EDT [K+] in WT on CTRL but decreased that in WT on LNaHK. Furosemide did not affect the EDT [K+] of KO on LNaHK or WT on HK. Furosemide-sensitive Na+ excretion was significantly greater in mice on LNaHK than those on CTRL or HK. Patch clamp analysis of split-open TALs revealed that 70-pS ROMK exhibited a higher open probability (Po) but similar density in mice on LNaHK, compared with CTRL. No difference was found in the density or Po of the 30 pS K channels between the two groups. These results indicate mice on LNaHK exhibited furosemide-sensitive net K+ secretion in the TAL that is dependent on increased NKCC2 activity and mediated by ROMK. We conclude that furosemide is a K-sparing diuretic by decreasing the TAL net K+ secretion in subjects on LNaHK.

Maintaining K+ balance on the low-Na+, high-K+ diet.

A low-Na+, high-K+ diet (LNaHK) is considered a healthier alternative to the "Western" high-Na+ diet. Because the mechanism for K+ secretion involves Na+ reabsorptive exchange for secreted K+ in the distal nephron, it is not understood how K+ is eliminated with such low Na+ intake. Animals on a LNaHK diet produce an alkaline load, high urinary flows, and markedly elevated plasma ANG II and aldosterone levels to maintain their K+ balance. Recent studies have revealed a potential mechanism involving the actions of alkalosis, urinary flow, elevated ANG II, and aldosterone on two types of K+ channels, renal outer medullary K+ and large-conductance K+ channels, located in principal and intercalated cells. Here, we review these recent advances.

Deficient acid handling with distal RTA in the NBCe2 knockout mouse.

In many circumstances, the pathogenesis of distal renal tubular acidosis (dRTA) is not understood. In the present study, we report that a mouse model lacking the electrogenic Na(+)-HCO3 (-) cotransporter [NBCe2/Slc4a5; NBCe2 knockout (KO) mice] developed dRTA after an oral acid challenge. NBCe2 expression was identified in the connecting tubule (CNT) of wild-type mice, and its expression was significantly increased after acid loading. NBCe2 KO mice did not have dRTA when on a standard mouse diet. However, after acid loading, NBCe2 KO mice exhibited complete features of dRTA, characterized by insufficient urinary acidification, hyperchloremic hypokalemic metabolic acidosis, and hypercalciuria. Additional experiments showed that NBCe2 KO mice had decreased luminal transepithelial potential in the CNT, as revealed by micropuncture. Further immunofluorescence and Western blot experiments found that NBCe2 KO mice had increased expression of H(+)-ATPase B1 in the plasma membrane. These results showed that NBCe2 KO mice with acid loading developed increased urinary K(+) and Ca(2+) wasting due to decreased luminal transepithelial potential in the CNT. NBCe2 KO mice compensated to maintain systemic pH by increasing H(+)-ATPase in the plasma membrane. Therefore, defects in NBCe2 can cause dRTA, and NBCe2 has an important role to regulate urinary acidification and the transport of K(+) and Ca(2+) in the distal nephron.

Increased Epithelial Sodium Channel Activity Contributes to Hypertension Caused by Na+-HCO3- Cotransporter Electrogenic 2 Deficiency.

The gene SLC4A5 encodes the Na(+)-HCO3 (-) cotransporter electrogenic 2, which is located in the distal nephron. Genetically deleting Na(+)-HCO3 (-) cotransporter electrogenic 2 (knockout) causes Na(+)-retention and hypertension, a phenotype that is diminished with alkali loading. We performed experiments with acid-loaded mice and determined whether overactive epithelial Na(+) channels (ENaC) or the Na(+)-Cl(-) cotransporter causes the Na(+) retention and hypertension in knockout. In untreated mice, the mean arterial pressure was higher in knockout, compared with wild-type (WT); however, treatment with amiloride, a blocker of ENaC, abolished this difference. In contrast, hydrochlorothiazide, an inhibitor of Na(+)-Cl(-) cotransporter, decreased mean arterial pressure in WT, but not knockout. Western blots showed that quantity of plasmalemmal full-length ENaC-α was significantly higher in knockout than in WT. Amiloride treatment caused a 2-fold greater increase in Na(+) excretion in knockout, compared with WT. In knockout, but not WT, amiloride treatment decreased plasma [Na(+)] and urinary K(+) excretion, but increased hematocrit and plasma [K(+)] significantly. Micropuncture with microelectrodes showed that the [K(+)] was significantly higher and the transepithelial potential (Vte) was significantly lower in the late distal tubule of the knockout compared with WT. The reduced Vte in knockout was amiloride sensitive and therefore revealed an upregulation of electrogenic ENaC-mediated Na(+) reabsorption in this segment. These results show that, in the absence of Na(+)-HCO3 (-) cotransporter electrogenic 2 in the late distal tubule, acid-loaded mice exhibit disinhibition of ENaC-mediated Na(+) reabsorption, which results in Na(+) retention, K(+) wasting, and hypertension.

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.

Long-range architecture in a viral RNA genome.

We have developed a model for the secondary structure of the 1058-nucleotide plus-strand RNA genome of the icosahedral satellite tobacco mosaic virus (STMV) using nucleotide-resolution SHAPE chemical probing of the viral RNA isolated from virions and within the virion, perturbation of interactions distant in the primary sequence, and atomic force microscopy. These data are consistent with long-range base pairing interactions and a three-domain genome architecture. The compact domains of the STMV RNA have dimensions of 10-45 nm. Each of the three domains corresponds to a specific functional component of the virus: The central domain corresponds to the coding sequence of the single (capsid) protein encoded by the virus, whereas the 5' and 3' untranslated domains span signals essential for translation and replication, respectively. This three-domain architecture is compatible with interactions between the capsid protein and short RNA helices previously visualized by crystallography. STMV is among the simplest of the icosahedral viruses but, nonetheless, has an RNA genome with a complex higher-order structure that likely reflects high information content and an evolutionary relationship between RNA domain structure and essential replicative functions.