A large-scale retrospective study enabled deep-learning based pathological assessment of frozen procurement kidney biopsies to predict graft loss and guide organ utilization.

Lesion scores on procurement donor biopsies are commonly used to guide organ utilization for deceased-donor kidneys. However, frozen sections present challenges for histological scoring, leading to inter- and intra-observer variability and inappropriate discard. Therefore, we constructed deep-learning based models to recognize kidney tissue compartments in hematoxylin & eosin-stained sections from procurement needle biopsies performed nationwide in years 2011-2020. To do this, we extracted whole-slide abnormality features from 2431 kidneys and correlated with pathologists' scores and transplant outcomes. A Kidney Donor Quality Score (KDQS) was derived and used in combination with recipient demographic and peri-transplant characteristics to predict graft loss or assist organ utilization. The performance on wedge biopsies was additionally evaluated. Our model identified 96% and 91% of normal/sclerotic glomeruli respectively; 94% of arteries/arterial intimal fibrosis; 90% of tubules. Whole-slide features of Sclerotic Glomeruli (GS)%, Arterial Intimal Fibrosis (AIF)%, and Interstitial Space Abnormality (ISA)% demonstrated strong correlations with corresponding pathologists' scores of all 2431 kidneys, but had superior associations with post-transplant estimated glomerular filtration rates in 2033 and graft loss in 1560 kidneys. The combination of KDQS and other factors predicted one- and four-year graft loss in a discovery set of 520 kidneys and a validation set of 1040 kidneys. By using the composite KDQS of 398 discarded kidneys due to "biopsy findings", we suggest that if transplanted, 110 discarded kidneys could have had similar survival to that of other transplanted kidneys. Thus, our composite KDQS and survival prediction models may facilitate risk stratification and organ utilization while potentially reducing unnecessary organ discard.

Honoring the gift: The transformative potential of transplant-declined human organs.

For decades, transplantation has been a life-saving treatment for those fortunate enough to gain access. Nevertheless, many patients die waiting for an organ and countless more never make it onto the waitlist because of a shortage of donor organs. Concurrently, thousands of donated organs are declined for transplant each year because of concerns about poor outcomes post-transplant. The decline of any donated organ-even if medically justified-is tragic for both the donor family and potential recipients. In this Personal Viewpoint, we discuss the need for a new mindset in how we honor the gift of organ donation. We believe that the use of transplant-declined human organs in translational research has the potential to hasten breakthrough discoveries in a multitude of scientific and medical areas. More importantly, such breakthroughs will allow us to properly value every donated organ. We further discuss the many practical challenges that such research presents and offer some possible solutions based on experiences in our own research laboratories. Finally, we share our perspective on what we believe are the necessary next steps to ensure a future where every donated organ realizes its full potential to impact the lives of current and future patients.

A digital pathology tool for quantification of color features in histologic specimens.

In preclinical research, histological analysis of tissue samples is often limited to qualitative or semiquantitative scoring assessments. The reliability of this analysis can be impaired by the subjectivity of these approaches, even when read by experienced pathologists. Furthermore, the laborious nature of manual image assessments often leads to the analysis being restricted to a relatively small number of images that may not accurately represent the whole sample. Thus, there is a clear need for automated image analysis tools that can provide robust and rapid quantification of histologic samples from paraffin-embedded or cryopreserved tissues. To address this need, we have developed a color image analysis algorithm (DigiPath) to quantify distinct color features in histologic sections. We demonstrate the utility of this tool across multiple types of tissue samples and pathologic features, and compare results from our program to other quantitative approaches such as color thresholding and hand tracing. We believe this tool will enable more thorough and reliable characterization of histological samples to facilitate better rigor and reproducibility in tissue-based analyses.

Inactive status is an independent predictor of liver transplant waitlist mortality and is associated with a transplant centers median meld at transplant.

BACKGROUND: Approximately 30% of patients on the liver transplant waitlist experience at least one inactive status change which makes them temporarily ineligible to receive a deceased donor transplant. We hypothesized that inactive status would be associated with higher mortality which may differ on a transplant centers' or donor service areas' (DSA) Median MELD at Transplant (MMaT). METHODS: Multi-state models were constructed (OPTN database;06/18/2013-06/08/2018) using DSA-level and transplant center-level data where MMaT were numerically ranked and categorized into tertiles. Hazards ratios were calculated between DSA and transplant center tertiles, stratified by MELD score, to determine differences in inactive to active transition probabilities. RESULTS: 7,625 (30.2% of sample registrants;25,216 total) experienced at least one inactive status change in the DSA-level cohort and 7,623 experienced at least one inactive status change in the transplant-center level cohort (30.2% of sample registrants;25,211 total). Inactive patients with MELD≤34 had a higher probability of becoming re-activated if they were waitlisted in a low or medium MMaT transplant center or DSA. Transplant rates were higher and lower re-activation probability was associated with higher mortality for the MELD 26-34 group in the high MMaT tertile. There were no significant differences in re-activation, transplant probability, or waitlist mortality for inactivated patients with MELD≥35 regardless of a DSA's or center's MMaT. CONCLUSION: This study shows that an inactive status change is independently associated with waitlist mortality. This association differs by a centers' and a DSAs' MMaT. Prioritization through care coordination to resolve issues of inactivity is fundamental to improving access.

Implementing a pharmacist-led transition of care model for posttransplant hyperglycemia.

PURPOSE: The implementation of a pharmacist-managed transition of care program for kidney transplant recipients with posttransplant hyperglycemia (PTHG) is described. METHODS: In September 2015, a collaborative practice agreement between pharmacists and transplant providers at an academic medical center for management of PTHG was developed. The goal of the pharmacist-run service was to reduce hospitalizations by providing care to patients in the acute phase of hyperglycemia while they transitioned back to their primary care provider or endocrinologist. For continuous quality improvement, preimplementation data were collected from August 2014 to August 2015 and compared to postimplementation data collected from August 2017 to August 2018. The primary endpoint was hospitalizations due to hyperglycemia within 90 days post transplantation. Secondary endpoints included emergency department (ED) visits due to hypoglycemia and the number of interventions performed, number of encounters completed, and number of ED visits or admissions for hypoglycemia. A Fisher's exact test was used to compare categorical data, and a Student t test was used to compare continuous data. A P value of <0.05 was considered to be statistically significant. RESULTS: Forty-three patients in the preimplementation group were compared to 35 patients in the postimplementation group. There was a significant reduction in hospitalizations due to hyperglycemia in the postimplementation versus the preimplementation group (9 vs 1, P < 0.05); there was a reduction in ED visits due to hyperglycemia (5 vs 0, P = 0.06). There were no ED visits or hospitalizations due to hypoglycemia in either group. Clinical transplant pharmacists performed an average of 8.3 (SD, 4.4) encounters per patient per 90 days. CONCLUSION: A collaborative practice agreement was created and successfully implemented. A pharmacist-managed PTHG program could be incorporated into the standard care of kidney transplant recipients to help minimize rehospitalizations due to hyperglycemia.

Expansion of Patient Education Programming Regarding Live Donor Liver Transplantation via Virtual Group Encounters During the COVID-19 Pandemic.

During the coronavirus 2019 pandemic we converted our liver transplant waitlist candidate education and support program to a virtual format and expanded it to include ongoing engagement sessions aimed to educate and empower patients to maximize opportunity for live donor liver transplantation. Over a period of 6 months from April 2020 to Sept 2020 we included 21 patients in this pilot quality improvement program. We collected data regarding patient response and potential donor referral activity. Overall, patient response was positive, and some patients saw progress toward live donor liver transplantation by fostering inquiry of potential live liver donors. Optimization of logistical aspects of the program including program flow, technology access, and utilization is required to enhance patient experience. Long-term follow-up is needed to assess impact on the outcome of transplantation rates. Future data collection and analysis should focus on assessment of any potential disparity that may result from utilization of virtual programming. Herein we provide a framework for this type of virtual program and describe our experience.

Lysis of cold-storage-induced microvascular obstructions for ex vivo revitalization of marginal human kidneys.

Thousands of kidneys from higher-risk donors are discarded annually because of the increased likelihood of complications posttransplant. Given the severe organ shortage, there is a critical need to improve utilization of these organs. To this end, normothermic machine perfusion (NMP) has emerged as a platform for ex vivo assessment and potential repair of marginal organs. In a recent study of 8 transplant-declined human kidneys on NMP, we discovered microvascular obstructions that impaired microvascular blood flow. However, the nature and physiologic impact of these lesions were unknown. Here, in a study of 39 human kidneys, we have identified that prolonged cold storage of human kidneys induces accumulation of fibrinogen within tubular epithelium. Restoration of normoxic conditions-either ex vivo during NMP or in vivo following transplant-triggered intravascular release of fibrinogen correlating with red blood cell aggregation and microvascular plugging. Combined delivery of plasminogen and tissue plasminogen activator during NMP lysed the plugs leading to a significant reduction in markers of renal injury, improvement in indicators of renal function, and improved delivery of vascular-targeted nanoparticles. Our study suggests a new mechanism of cold storage injury in marginal organs and provides a simple treatment with immediate translational potential.

Metabolic Disorders with Kidney Transplant.

Metabolic disorders are highly prevalent in kidney transplant candidates and recipients and can adversely affect post-transplant graft outcomes. Management of diabetes, hyperparathyroidism, and obesity presents distinct opportunities to optimize patients both before and after transplant as well as the ability to track objective data over time to assess a patient's ability to partner effectively with the health care team and adhere to complex treatment regimens. Optimization of these particular disorders can most dramatically decrease the risk of surgical and cardiovascular complications post-transplant. Approximately 60% of nondiabetic patients experience hyperglycemia in the immediate post-transplant phase. Multiple risk factors have been identified related to development of new onset diabetes after transplant, and it is estimated that upward of 7%-30% of patients will develop new onset diabetes within the first year post-transplant. There are a number of medications studied in the kidney transplant population for diabetes management, and recent data and the risks and benefits of each regimen should be optimized. Secondary hyperparathyroidism occurs in most patients with CKD and can persist after kidney transplant in up to 66% of patients, despite an initial decrease in parathyroid hormone levels. Parathyroidectomy and medical management are the options for treatment of secondary hyperparathyroidism, but there is no randomized, controlled trial providing clear recommendations for optimal management, and patient-specific factors should be considered. Obesity is the most common metabolic disorder affecting the transplant population in both the pre- and post-transplant phases of care. Not only does obesity have associations and interactions with comorbid illnesses, such as diabetes, dyslipidemia, and cardiovascular disease, all of which increase morbidity and mortality post-transplant, but it also is intimately inter-related with access to transplantation for patients with kidney failure. We review these metabolic disorders and their management, including data in patients with kidney transplants.

Association of Racial Disparities With Access to Kidney Transplant After the Implementation of the New Kidney Allocation System.

Importance: Inactive patients on the kidney transplant wait-list have a higher mortality. The implications of this status change on transplant outcomes between racial/ethnic groups are unknown. Objectives: To determine if activity status changes differ among races/ethnicities and levels of sensitization, and if these differences are associated with transplant probability after implementation of the Kidney Allocation System. Design, Setting, and Participants: A multistate model was constructed from the Organ Procurement and Transplantation Network kidney transplant database (December 4, 2014, to September 8, 2016). The time interval followed Kidney Allocation System implementation and provided at least 1-year follow-up for all patients. The model calculated probabilities between active and inactive status and the following competing risk outcomes: living donor transplant, deceased donor transplant, and death/other. This retrospective cohort study included 42 558 patients on the Organ Procurement and Transplantation Network kidney transplant wait-list following Kidney Allocation System implementation. To rule out time-varying confounding from relisting, analysis was limited to first-time registrants. Owing to variations in listing practices, primary center listing data were used for dually listed patients. Individuals listed for another organ or pancreatic islets were excluded. Analysis began July 2017. Main Outcome and Measures: Probabilities were determined for transitions between active and inactive status and the following outcome states: active to living donor transplant, active to deceased donor transplant, active to death/other, inactive to living donor transplant, inactive to deceased donor transplant, and inactive to death/other. Results: The median (interquartile range) age at listing was 55.0 (18.0-89.0) years, and 26 535 of 42 558 (62.4%) were men. White individuals were 43.3% (n = 18 417) of wait-listed patients, while black and Hispanic individuals made up 27.8% (n = 11 837) and 19.5% (n = 8296), respectively. Patients in the calculated plasma reactive antibody categories of 0% or 1% to 79% showed no statistically significant difference in transplant probability among races/ethnicities. White individuals had an advantage in transplant probability over black individuals in calculated plasma reactive antibody categories of 80% to 89% (hazard ratio [HR], 1.8 [95% CI, 1.4-2.2]) and 90% or higher (HR, 2.4 [95% CI, 2.1-2.6]), while Hispanic individuals had an advantage over black individuals in the calculated plasma reactive antibody group of 90% or higher (HR, 2.5 [95% CI, 2.1-2.8]). Once on the inactive list, white individuals were more likely than Hispanic individuals (HR, 1.2 [95% CI, 1.17-1.3]) or black individuals (HR, 1.4 [95% CI, 1.3-1.4]) to resolve issues for inactivity resulting in activation. Conclusions and Relevance: For patients who are highly sensitized, there continues to be less access to kidney transplant in the black population after the implementation of the Kidney Allocation System. Health disparities continue after listing where individuals from minority groups have greater difficulty in resolving issues of inactivity.

Hepatoid Carcinoma of the Pancreas: Case Report, Next-Generation Tumor Profiling, and Literature Review.

Fewer than 25 cases of hepatoid carcinoma of the pancreas have been reported in the literature. We present a case in a 61-year-old male with a remote history of Hodgkin's lymphoma and gastric neuroendocrine cell hyperplasia. On surveillance endoscopic ultrasound, an 8 × 5 mm cystic lesion was seen in the tail of the pancreas. MRI showed a focal pancreatic duct cut-off with mild ductal dilation. Fine needle aspiration was performed, which was concerning for acinar cell carcinoma. The patient underwent distal pancreatectomy and recovered uneventfully. Final pathology demonstrated a 1.3-cm hepatoid carcinoma of the pancreas, with a final clinicopathological stage of T1N0M0. Next-generation nucleic acid sequencing of the tumor did not suggest a viable adjuvant chemotherapeutic agent, and no adjuvant therapy was administered. The patient has no evidence of disease 6 months following resection. A further characterization and description of the outcomes of these rare tumors is warranted to help guide providers and counsel patients.

Prognostic factors in kidney transplantation in the septuagenarian: a multicenter analysis.

Kidney transplant (KT) programs have extended recipient eligibility to those who were previously excluded due to advanced age. We aimed to determine the outcomes of the patients ≥70 years undergoing KT and investigate factors predicting survival. Two thousand six hundred and twenty-four KT patients between 2003 and 2013 at two institutions were divided into two groups; those ≥70 years (n=300) and those <70 years (n=2324) at the time of KT. Patient survival at 1, 3, and 5 years was 95%, 86%, and 77% in ≥70 years of age group and 98%, 95%, and 90% in the <70 years group (P<.001). When graft loss due to death was censored, graft survival was not significantly different between the two groups (P=.18). On multivariable analysis, the significant predictors of inferior survival in patients ≥70 years included: body mass index (BMI)>30 kg/m(2) (hazard ratio [HR] 1.07; P=.01), panel reactive antibody (PRA)>20% (HR 2.38; P=.01), previous coronary artery bypass grafting (CABG; HR 1.95; P=.03) and peripheral vascular disease (PVD; HR 2.60; P=.04). Acceptable outcomes can be achieved in KT recipients ≥70 years. Caution should be used when listing these patients if they have BMI>30 kg/m(2) , PRA>20%, CABG or PVD.

Metastatic Fibrolamellar Hepatocellular Carcinoma to the Pancreas.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of hepatocellular carcinoma, usually presenting in the younger population (<40 years) without underlying liver disease. Although it has a better prognosis than hepatocellular carcinoma, it has a high rate of recurrence months to years after primary resection. While sites of recurrence usually involve the liver, regional lymph nodes, peritoneum, and lung, metastasis to the pancreas is extremely rare, with only 2 other cases reported in the literature. We present the case of a 46-year-old patient with metastatic FL-HCC to the pancreas 30 years after diagnosis and 26 years since his last resected liver recurrence.

Role of ERCP in pediatric blunt abdominal trauma: a case series at a level one pediatric trauma center.

BACKGROUND: There is no consensus regarding the appropriate use of endoscopic retrograde cholangiopancreatography (ERCP) in pediatric trauma. We report our experience with ERCP for management of pediatric pancreatic and biliary injury following blunt abdominal trauma. METHODS: A retrospective chart review was performed for pediatric patients with blunt abdominal trauma from July 2008 through December 2012 at our pediatric trauma center. For patients who underwent ERCP, demographics, injury characteristics, diagnostic details, procedures performed, length of stay, total parenteral nutrition use, and complications were reviewed. RESULTS: There were 532 patients identified: 115 hepatic injuries, 25 pancreatic injuries and one gall bladder injury. Nine patients (mean age 7.8 years) underwent ERCP. Seven (78%) had pancreatic injuries, while two (22%) had bilateral hepatic duct injuries. The median time to diagnosis was one day (range, 0-12). Diagnostic ERCP only was performed in three patients, two of which proceeded to distal pancreatectomy. Five patients had stents placed (two biliary and three pancreatic) and four sphincterotomies were performed. Despite pancreatic stenting, one patient required distal pancreatectomy for persistent leak. Median length of stay was 11 days. CONCLUSIONS: Pediatric pancreatic and biliary ductal injuries following blunt abdominal trauma are uncommon. ERCP can safely provide definitive treatment for some patients.

Clinical inertia during postoperative management of diabetes mellitus: relationship between hyperglycemia and insulin therapy intensification.

OBJECTIVE: Our objective was to assess the application of insulin regimens in surgical postoperative patients with diabetes. METHODS: A chart review was conducted of patients with diabetes who were hospitalized postoperatively between January 1 and April 30, 2011. Analysis was restricted to patients hospitalized for ≥3 days and excluded cases with an endocrinology consult. Insulin regimens were categorized as "basal plus short acting," "short acting only," or "none," and the pattern of use was evaluated by hyperglycemia severity according to tertiles of both mean glucose and the number of glucose measurements >180 mg/dl. RESULTS: Among cases selected for analysis (n = 119), examination of changes in insulin use based on tertiles of mean glucose showed that use of basal plus short-acting insulin increased from 10% in the lowest tertile (mean glucose, 120 mg/dl) to 18% in the highest tertile (mean glucose, 198 mg/dl; p < .01); however, 70% of patients in the highest tertile continued to receive short-acting insulin only, with 12% receiving no insulin. Intensification of insulin to a basal plus short-acting regimen was also seen when changes were evaluated by the number of measurements >180 mg/dl (p < .01), but 70% and 12% of patients in the highest tertile still remained only on short-acting insulin or received no insulin, respectively. CONCLUSIONS: Use of basal plus short-acting insulin therapy increased with worsening hyperglycemia, but many cases did not have therapy intensified to the recommended insulin regimen--evidence of clinical inertia. Strategies should be devised to overcome inpatient clinical inertia in the treatment of postoperative patients with diabetes.

Perioperative management of patients with diabetes undergoing ambulatory elective surgery.

OBJECTIVE: The objective was to assess processes of care for patients with diabetes undergoing elective surgery. METHODS: A retrospective review of medical records was conducted to determine frequency of perioperative glucose monitoring, changes in glucose control, and treatment of intraoperative hyperglycemia. RESULTS: A total of 268 patients underwent 287 elective procedures. Mean age was 67 years, 63% were men, 97% had type 2 diabetes, and most (57%) were treated with oral hypoglycemic agents. Average perioperative time was approximately 8 h. Mean preoperative hemoglobin A1c was 7.0%; however, this value was checked in only 52% of cases. A glucose measurement was obtained in 89% of cases in the preoperative area and in 87% in the postanesthesia care unit, but in only 33% of cases did a value get checked intraoperatively. Average glucose was 139 mg/dl preoperatively, increasing to 166 mg/dl postoperatively (p <.001). Glucose levels increased regardless of type of outpatient medical therapy used to treat hyperglycemia, except for those on combination oral agents plus insulin (p =.06). CONCLUSIONS: These data indicate suboptimal documentation of outpatient hemoglobin A1c. Intraoperative glucose monitoring seldom occurred, despite prolonged periods under anesthesia and perioperative deterioration of glycemic control. Standards need to be developed and interventions are needed to enhance management of diabetes patients undergoing elective procedures.

The impact of obesity on breast cancer: a retrospective review.

BACKGROUND: Obesity has been linked to many adverse health consequences, including breast cancer; however, the impact on clinical presentation, tumor characteristics, and survival outcomes has yet to be clearly defined. METHODS: Retrospective review of a prospectively collected database of patients treated at a single institution for invasive breast cancer from 2000-2008 comparing two groups: nonobese (body mass index of <30) and obese (body mass index of ≥ 30) patients. Continuous variables, categorical variables, and survival data were analyzed. RESULTS: Of 1352 total patients, 76% were classified as nonobese and 24% were obese. When comparing age, obese patients presented less frequently than nonobese patients <50 years old (10% vs. 90%), and when comparing patients >50 years old (18% vs. 82%, P = 0.0019). Obese patients were more likely to present with disease detected by imaging when compared to nonobese patients (67% vs. 56%, P = 0.0006). Obese patients had larger tumors (1.7 cm vs. 1.4 cm, P < 0.001) and higher rates of lymph node (LN) metastases (31% vs. 25%, P = 0.026). On multivariate analysis, obesity was associated with nonpalpable tumors, larger tumors, a higher incidence of LN metastasis, lower incidence of Her2 positivity, lower incidence of multifocality, and less likely to undergo reconstruction after mastectomy. CONCLUSIONS: Obese patients clinically present at older ages with mammographically detected breast cancer at more advanced stages than nonobese patients. Strategies to encourage screening among the obese patient population are important.

A significant number of women present with palpable breast cancer even with a normal mammogram within 1 year.

BACKGROUND: Although mammography is the gold standard for breast cancer screening, clinical breast examination (CBE) and self breast examination (SBE) are important adjuncts whose utility has been questioned. METHODS: A retrospective review of invasive breast cancer patients from 2000 to 2008 was performed. We compared 3 groups: breast cancer detected by (1) imaging only (nonpalpable) or palpable mass with a normal mammogram (2) ≥ 1 year (mammogram ≥ 1 year) or (3) <1 year (mammogram <1 year). RESULTS: Of 1,222 women, presentation included 67% nonpalpable, 21% mammogram ≥ 1 year, and 13% mammogram <1 year. Patients presenting with palpable masses on SBE or CBE even with a normal mammogram within 1 year tended to have more aggressive tumors (larger size, lymph node positive, and triple-negative disease) resulting in more aggressive therapy (a higher mastectomy rate and a greater likelihood of chemotherapy). CONCLUSION: A significant number of women present with palpable breast cancer within 1 year of a normal mammogram, many with an aggressive cancer. Therefore, we continue to advocate SBE and CBE for breast cancer screening.

Pituitary tumors arising from glycoprotein hormone alpha-subunit-deficient mice contain transcription factors and receptors present in thyrotropes.

Glycoprotein-hormone alpha-subunit deficient (alphaSUnull) mice are hypothyroid and hypogonadal due to the absence of functional TSH, LH and FSH, despite normal production of the corresponding beta subunits. Pituitary tumors spontaneously developing in alphaSUnull mice were propagated in hypothyroid mice. The purpose of the current studies was to compare the gene expression profile of these alphaSUnull tumors with previously characterized TtT-97 thyrotropic tumors. A group of animals bearing each tumor type was treated with thyroid hormone (T4) prior to tumor removal. Both tumor types equally expressed TSHbeta mRNA, which significantly decreased when exposed to T4, whereas alpha-subunit mRNA was absent in alphaSUnull tumors. Northern blot analysis was performed using cDNA probes for the following transcription factors: Pit1, GATA2, pLIM, Msx1, Ptx1 and Ptx2. Both tumors were found to contain identical transcripts with similar responses to T4, with the exception of Pit1. In contrast to the signal pattern seen in TtT-97, only two bands were seen in alphaSUnull tumors, which were similar in size to those in alphaTSH cells, a thyrotropic cell line that lacks TSHbeta-subunit expression and Pit1 protein. However, western blot analysis revealed a protein band in the alphaSUnull tumors consistent with Pit1, while this signal was absent in alphaTSH cells. Northern blot analysis was also performed with specific cDNA probes for the following receptors: TRbeta1, TRbeta2, TRalpha1, non-T3 binding alpha2, RXRgamma and Sst5. Similarly-sized transcripts were found in both types of tumor, although the signal for Sst5 was seen in T4-treated alphaSUnull tumors only with a more sensitive RT-PCR analysis. The overall similarity between the two tumor types renders the alphaSUnull tumor as a suitable thyrotropic tumor model.

The proliferative status of thyrotropes is dependent on modulation of specific cell cycle regulators by thyroid hormone.

In this report we have examined changes in cell growth parameters, cell cycle effectors, and signaling pathways that accompany thyrotrope growth arrest by thyroid hormone (TH) and growth resumption after its withdrawal. Flow cytometry and immunohistochemistry of proliferation markers demonstrated that TH treatment of thyrotrope tumors resulted in a reduction in the fraction of cells in S-phase that is restored upon TH withdrawal. This is accompanied by dephosphorylation and rephosphorylation of retinoblastoma (Rb) protein. The expression levels of cyclin-dependent kinase 2 and cyclin A, as well as cyclin-dependent kinase 1 and cyclin B, were decreased by TH, and after withdrawal not only did these regulators of Rb phosphorylation and mitosis increase in their expression but so too did the D1 and D3 cyclins. We also noted a rapid induction and subsequent disappearance of the type 5 receptor for the growth inhibitor somatostatin with TH treatment and withdrawal, respectively. Because somatostatin can arrest growth by activating MAPK pathways, we examined these pathways in TtT-97 tumors and found that the ERK pathway and several of its upstream and downstream effectors, including cAMP response element binding protein, were activated with TH treatment and deactivated after its withdrawal. This led to the hypothesis that TH, acting through increased type 5 somatostatin receptor, could activate the ERK pathway leading to cAMP response element binding protein-dependent decreased expression of critical cell cycle proteins, specifically cyclin A, resulting in hypophosphorylation of Rb and its subsequent arrest of S-phase progression. These processes are reversed when TH is withdrawn, resulting in an increase in the fraction of S-phase cells.