Clinical Utility and Concordance of Upper Urinary Tract Cytology and Biopsy in Predicting Clinicopathologic Features of Upper Urinary Tract Urothelial Carcinoma

5% of urothelial carcinoma occurs in the upper urinary tract (UUT), a challenging location to biopsy. We aim to evaluate concordance between biopsy, cytology, and resection specimens in a large upper tract urothelial carcinoma (UTUC) cohort.117 UTUC resections with UUT biopsy and/or cytology specimens from 2000–2016 were retrieved; pathologic material was re-reviewed, evaluated for concordance, and correlated with clinical information. 14% pre-operative biopsies, including 8 from renal pelvis and 6 from ureter, lacked neoplastic diagnoses. 77% diagnostic biopsies included subepithelial tissue; 11% demonstrated reclassification of grade and 30% demonstrated reclassification of invasion status. 26% of renal pelvis UTUC and 36% ureter UTUC were invasive only on resection. Of 18 UTUC reclassified from noninvasive high-grade papillary urothelial carcinoma (HGPUC) to invasive HGPUC, 39% had prior radical cystectomy (versus 8% invasive UTUC and 11% noninvasive UTUC with concordant biopsies). Most high-grade UTUC (88%) and some low-grade UTUC (58%) resections had abnormal cytology results. Biopsy-resection pairs with concordant invasion status and pairs with discordant invasion status showed similar rates of recurrence (38% versus 38%) and metastasis (25% versus 27%). 14% of UUT biopsies lacked diagnostic neoplastic material. Grade concordance between biopsy and resection was high (89%), but 30% of cases showed invasion only on resection. Subepithelial tissue was less commonly present in ureter biopsies, particularly from mid or proximal ureter. UTUC in patients with prior cystectomy were more likely to show invasion on resection but not biopsy

Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

<div><p>Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.</p></div

Mutations affecting the TPI dimer interface recapitulate <i>dTPI</i>^<i>M80T</i> phenotypes.

<p>(A) <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> homozygotes display severely reduced lifespans, n>150. (B) Dimer interface mutations exhibit severe mechanical stress at Day 1 and (C) thermal stress sensitivity at Day 2, n>30. Thermal stress paralysis times at 360 sec. represent wild type behavior, the assay was stopped at 6 min. (D) Both <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> homozygotes display reduced lysate isomerase activity, n≥3. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, and lifespans by a Log-rank (Mantel-Cox) survival test, ** indicated p<0.01, *** p<0.001.</p

TPI dimer interface substitutions do not alter NMJ development and morphology.

<p>(A) NMJ morphology of segment A2 muscle 6/7 was characterized for (B) bouton number and (C) branching. Boutons were defined as varicosities greater than 2 μm in diameter. Neither parameter showed significant differences elicited by the mutations, relative to either <i>dTPI</i>^<i>WT</i> or <i>dTPI</i>^{<i>WT-CFP</i>}<i>/TPI</i>^<i>WT</i>. CFP tags did not alter the behavioral deficits of the animals (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005941#pgen.1005941.s006" target="_blank">S6 Fig</a>). n = 10. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, <i>ns</i> indicated no significance. Scale bar = 10 μm.</p

Heterodimerization of inactive TPI and dimer interface mutations.

<p>(A) dTPI^Δcat-CFP interacts modestly with dTPI^M80T, dTPI^T73R, and dTPI^T73R,G74E, yet robustly with dTPI^G74E. Representative immunoprecipitation and input blots are shown with (B) IP:anti-GFP quantification n = 3. Quantification represents 25kD TPI IP signal, with negative control subtracted, normalized to the lysate β-tubulin loading control, and compared to WT. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, <i>ns</i> indicates no significance, ** p<0.01, and *** p<0.001.</p

Crystallographic Data collection and Refinement statistics.

<p>Crystallographic Data collection and Refinement statistics.</p

<i>dTPI</i>^<i>T73R</i> impairs NMJ synaptic vesicle dynamics.

<p>(A) An FM1-43 timecourse at the NMJ with loading times of 15, 30, and 60 sec., (B) with quantification of 60 sec. at 38°C, and (C) 60 sec. at room temperature (RT). (D) Representative images of <i>dTPI</i>^<i>WT</i>, <i>dTPI</i>^<i>T73R</i>, <i>dTPI</i>^{<i>T73R/Δcat</i>} and <i>Shi</i>^<i>ts1</i>, n = 6. (E) FM1-43 unloading is unchanged between <i>dTPI</i>^<i>WT</i> and <i>dTPI</i>^<i>T73R</i> at 38°C with animal replicates indicated, and (F) representative images. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, ***p<0.001. Scale bars = 5μm.</p

hTPI^M82T elicits a conformational change in TPI resulting in reduced dimerization.

<p>(A) Sequence alignment of <i>D</i>.<i>melanogaster</i> and <i>H</i>.<i>sapiens</i> TPI protein sequence with asterisks highlighting residues of interest. (B) The hTPI^M82T mutation confers a reduction in mean protein hydrodynamic radius as measured by dynamic light scattering. (C) Intensity correlation plots reveal a largely monodisperse hTPI^WT population and polydisperse hTPI^M82T population. (D) Gel filtration indicates a change in monomer:dimer ratios elicited by hTPI^M82T with relative quantification (inset). n≥3, comparisons were made using Student’s T test, *** indicates p<0.001.</p

hTPI^Δcat models predict that hTPI^Δcat::hTPI^G76E heterodimerization alters the TPI dimer interface.

<p>(A) The R75 side chain may insert itself into the active site in the context of a hTPI^Δcat::hTPI^T75R heterodimer. Shown is a superposition of hTPI^WT (grey), hTPI^Δcat (blue), and hTPI^Δcat::hTPI^T75R (green) obtained from Rosetta Backrub modeling. Loop3, containing T75R and G76E, is indicated in tan. For clarity, the mainchain of hTPI^Δcat::hTPI^T75R has been omitted. (B) Repositioning of E104 and R98 side chains assists the dimer interface to accommodate the G76E substitution in Rosetta modeling. Superposition of hTPI^Δcat structure (blue) with the hTPI^Δcat::hTPI^G76E model (green). In both structures, Loop 3 is shown in tan and selected active site pocket residues are indicated. (C) Modeling the effect of T75R and G76E substitutions in the context of hTPI^WT and hTPI^Δcat structures. Normalized scores for the best 10% of 50 simulations were averaged for each experimental structure with the indicated computational substitution. Higher scores indicate a resulting model that is less favorable using the Rosetta energy function.</p