DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Primary anatomic double-bundle anterior cruciate ligament reconstruction: a preliminary 2-year prospective study.

BACKGROUND: The anterior cruciate ligament consists of 2 functional bundles, the anteromedial bundle and the posterolateral bundle. Single-bundle anterior cruciate ligament reconstruction is the current standard for the treatment of anterior cruciate ligament deficiency. However, a significant subset of patients continues to report residual symptoms of instability after such reconstruction. HYPOTHESIS: Anatomic double-bundle anterior cruciate ligament reconstruction may more closely restore normal kinematics of the knee by reproducing the native anatomy. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: We report the current clinical outcomes of the initial 100 consecutive patients who underwent anatomic double-bundle anterior cruciate ligament reconstruction with an average follow-up of 2.1 +/- 0.5 years. All patients were prospectively followed to document range of motion, ligamentous laxity, and functional strength, as well as activity and sports participation. RESULTS: Side-to-side difference in range of motion was 2 degrees +/- 3 degrees for extension and 2 degrees +/- 5 degrees for flexion. Sixty-five percent of patients had a normal Lachman test result, and 33% were nearly normal. For the pivot-shift test findings, 94% were normal, and 6% were nearly normal. The average side-to-side difference in the KT-2000 arthrometer test was 1.0 +/- 2.3 mm. There were 8 graft failures, 7 of which had subsequent revision surgery. No patients reported pain, swelling, or instability during activities of daily living, and 73% to 78% had no symptoms during very strenuous or strenuous sports activities. The scores of the International Knee Documentation Committee Subjective Knee Form, Activities of Daily Living, and Sports Activity Scores of the Knee Outcome Survey were 85.0, 91.8, and 87.0, respectively, and were similar compared with patients undergoing single-bundle anterior cruciate ligament reconstruction, which we have previously reported. Fifty-one percent described their current activity level as normal, and 35% reported it as nearly normal. CONCLUSION: Anatomic double-bundle anterior cruciate ligament reconstruction results in good restoration of joint stability and patient-reported outcomes when evaluated 2 years after surgery.