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Thrombocytosis Following Pancreatectomy with Islet Autotransplantation in Children: Cincinnati Children’s Hospital Experience

A thesis submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Master of Science

in Clinical & Translational Research

In the Department of Environmental Health

Division of Epidemiology

of the College of Medicine

January, 2018

Juan Pablo Gurria Juarez

Doctor of Medicine, Anahuac University, October, 2009

Committee Chair: Erin Haynes, DrPH

ABSTRACT: Background:

Total pancreatectomy with islet autotransplantation (TPIAT) is a surgical option for patients with debilitating chronic pancreatitis. Pancreatic islets are isolated and autotransplanted into the liver via the portal vein. A significant and sustained postoperative thrombocytosis is common, but poorly understood. The degree and duration of thrombocytosis are not typical of a reactive postsplenectomy process. Given that hepatocytes represent a major source of thrombopoietin (TPO), we hypothesized that increased TPO production drives post-TPIAT thrombocytosis.

Methods:

We performed a retrospective chart review of 13 patients (4/2015-12/2016) who underwent TPIAT (with splenectomy) with focus on platelet counts, thrombopoietin (TPO) levels, and hydroxyurea treatment.

Results:

There were 12 total and 1 subtotal pancreatectomies, median age 12.6 years, BMI 20.9 kg/m2, length of stay 26 days. All patients were discharged on basal and bolus insulin. TPO levels were normal preoperatively (mean 45 pg/mL, normal 7-99 pg/ml), then increased significantly reaching a mean of 219 pg/mL 4 days postoperatively (p<0.0001). Preoperative platelet counts were normal, and increased dramatically reaching a median of 835 K/l by postoperative day 3 (p<0.0001). Hydroxyurea, started on average 7.8 days postoperatively, significantly decreased the platelet count (p<0.0001). TPO levels remained elevated 20 days post-hydroxyurea (mean 82 pg/mL). There were no major surgical or thrombotic complications.

Conclusions:

All patients developed sustained and prolonged thrombocytosis after TPIAT. The significant rise in TPO postoperatively suggests an association between thrombocytosis and increased TPO production. Further studies are needed to explore the mechanism of thrombocytosis post-TPIAT.

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ACKNOWLEDGEMENTS:

Parts or all of this thesis were previously published: Pancreas. 2017, Nov; 46(1):1404.

I would like to acknowledge my co-authors:

Priscila Badia, MD - Cincinnati Children's Hospital Medical Center. The Cancer and Blood Diseases Institute.

Pediatric Hematology and Oncology. Clinical Fellow.

Lindsey Hornung, MS - Cincinnati Children's Hospital Medical Center. Division of Biostatistics and

Epidemiology.

Maisam Abu-El-Haija, MD - Cincinnati Children's Hospital Medical Center. Division of Pediatric

Gastroenterology. Medical Director, Pancreas Care Center. Assistant Professor, Department of Pediatrics,

University of Cincinnati College of Medicine.

Deborah A. Elder, MD - Cincinnati Children's Hospital Medical Center. Division of Pediatric Endocrinology.

Endocrinology Director, Pancreas Care Center. Associate Professor, Department of Pediatrics, University of Cincinnati College of Medicine.

Tom K. Lin, MD - Cincinnati Children's Hospital Medical Center. Division of Pediatric Gastroenterology.

Associate Director, Pancreas Care Center. Assistant Professor, Department of Pediatrics, University of

Cincinnati College of Medicine.

Lori Luchtman-Jones, MD - Cincinnati Children's Hospital Medical Center. The Cancer and Blood Diseases

Institute. Pediatric Hematology and Oncology. Medical Director, Hemostasis and Thrombosis. Professor,

Department of Pediatrics, University of Cincinnati College of Medicine.

Joseph S. Palumbo, MD - Cincinnati Children's Hospital Medical Center. The Cancer and Blood Diseases

Institute. Pediatric Hematology and Oncology. Director, Comprehensive Thrombophilia Center. Professor,

Department of Pediatrics, University of Cincinnati College of Medicine.

Jaimie D. Nathan, MD - Cincinnati Children's Hospital Medical Center. Division of General and Thoracic

Pediatric Surgery. Surgical Director, Pancreas Care Center, Kidney and Intestinal Transplant Program.

Associate Surgical Director, Liver Transplant Program. Associate Professor, Department of Surgery and

Pediatrics, University of Cincinnati College of Medicine.

TABLE OF CONTENTS

Introduction 1

Background 2

Patients & Methods 4

Results 5

Discussion 7

Conclusion 10

Tables and Figures 11

Bibliography 14

LIST OF TABLES AND FIGURES

Table 1. Etiology and Time with illness from diagnosis.

Table 2. Patient Characteristics

Figure 1 – TPO levels pre and post surgery

Figure 2 – Platelet count postoperative phase and before hydroxyurea

Figure 3- TPO levels postoperative phase.

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Thrombocytosis Following Pancreatectomy with Islet Autotransplantation in Children: Cincinnati Children’s Hospital Experience

Introduction:

Chronic pancreatitis (CP) represents a major debilitating medical condition both in the adult and pediatric populations. It is characterized by progressive and eventually irreversible parenchymal destruction that results in exocrine followed by endocrine impairment [1]. The incidence, etiology, classification, prognosis and management of both acute recurrent and CP in children, have substantially evolved in recent years

[2-4].

The diagnosis and management of pancreatitis in children has been increasing in recent years up to

13/100,000 [5]. Despite this rise, acute pancreatitis will usually resolve without complications when timely and adequate therapy is instituted [6]. The most common etiology of CP is genetic, including mutations of PRSS-1, SPINK-1, CFTR and CTRC [7, 8]. Genome-wide association studies have been able to analyze gene sequences including the CFTR gene in chromosome 7 and its most common single nucleotide polymorphism rs113993960, also known as delta F508, which has given tools for early diagnosis and future potential therapeutics [9]. Surgical therapy in children has evolved and novel approaches including total pancreatectomy have been used at a small number of specialized institutions with promising results. In this study we reviewed the experience of a large tertiary pediatric center and the treatment of CP with total pancreatectomy and islet autotransplantation (TPIAT) in children, and analyzed the associated thrombocytosis that follows this procedure.

Background:

TPIAT is a surgical option for patients with debilitating chronic pancreatitis that have failed medical, endoscopic or surgical treatment. TPIAT has shown to be safe and effective in providing pain relief and improvement in the quality of life of pediatric patients [10]. In order to mitigate diabetes, pancreatic islets are enzymatically and mechanically isolated, and autotransplanted into the liver via the portal vein. The autologous transplanted islets undergo ischemic and inflammatory changes that can impair the quality and hence the function of the cells [11].

An enigmatic, but consistent complication of TPIAT in children is a substantial and sustained postoperative thrombocytosis that despite being common is poorly understood. As of today, it has been thought to be a reactive thrombocytosis secondary to splenectomy; however, the level of thrombocytosis is far from the typical elevation seen in patients after splenectomy. It has been shown that these patients develop a hypercoagulable state with substantially increased platelet levels, increased fibrinogen, tissue plasminogen activator inhibitor and activated partial thromboplastin time, which persists for up to 3-5 months [12, 13], and is usually self-resolving [14]. Studies with thromboelastography have proven that the hypercoagulability starts as soon as 2 days after splenectomy, which along with the hemodynamic effects of the reduced flow through the portal vein after splenic vein ligation, increases substantially the risk of portal vein thrombosis post-operatively [15].

Patients that have undergone TPIAT, have platelet counts that typically rise to >1 million within 1 week of the procedure and remain elevated for up to a year or longer. Data from Cincinnati Children’s Hospital

Medical Center (CCHMC) suggests that it may be related to elevated levels of thrombopoietin (TPO). It is known that TPO is constantly produced by hepatocytes and it is the only physiologically substantial regulator of platelet and megakaryocyte production. It is bounded by high-affinity to TPO-receptors on

2 the platelets [16] promoting their activation. Studies have shown that TPO upregulates the production of early hematopoietic platelet progenitors and enhances the viability of peripheral thrombocytes [17].

Hydroxyurea has been successfully used to reduce platelet counts in the treatment of thrombocytosis secondary to splenectomy, inflammatory conditions and when present as a result of myeloproliferative disorders [18]. Its dosing is response dependent, has mild side effects, quick reversal and has shown to prevent thrombosis in high-risk patients [19]. Severe thrombocytosis may result in life threatening thrombotic events, and given its incidence in postsplenectomy patients up to 80% [14], early treatment is key in post TPIAT patients.

There is currently a lack of detailed understanding of the process behind the reactive and sustained thrombocytosis post TPIAT. Given that hepatocytes represent a major source of TPO, derangement of hepatocyte-derived TPO secretion is possible. We hypothesized that increased TPO production drives post-TPIAT thrombocytosis.

To date, no natural history studies of patients undergoing TPIAT with untreated thrombocytosis have been done. The current chronic antiplatelet therapy (including hydroxyurea and acetylsalicylic acid) recommended for this complication, could potentially be shifted and targeted towards counteracting TPO activity and the etiology of its thrombogenicity early in the inflammatory process, to prevent the potential prothrombotic complications of such elevated platelet counts like portal vein thrombosis in the case of

TPIAT.

Patients and Methods

We performed a retrospective chart review of 13 patients, from April 2015 to December 2016, who underwent total pancreatectomy with islet autotransplantation (TPIAT) or partial pancreatectomy with islet autotransplantation (IAT) at CCHMC. We evaluated the association of thrombocytosis with the levels of thrombopoietin (TPO) following surgery, and reviewed its management with hydroxyurea during the postoperative period. Each patient served as his/her own control by analyzing preoperative and postoperative laboratory values. Primary outcome was the association of TPO levels to platelet rise.

Secondary outcomes included: procedure time, surgical complications, glucose control, insulin requirements and length of stay.

Data collected included: patient’s age, sex, race, etiology of pancreatitis, interval time with pancreatitis, glucose levels in the perioperative period, comorbidities, post-operative infections, length of stay in ICU, invasive monitoring with central lines or arterial catheters, anticoagulation used postoperatively, procedure blood loss and nutrition therapy. Adjunct studies were also recorded for each patient in the postoperative period including: duplex ultrasound findings, and laboratory findings including: platelet levels, and liver function tests.

Statistical analysis

Data were analyzed using SAS®, version 9.4 (SAS Institute, Cary, NC). Due to the small sample size, continuous patient data was summarized as medians with 25th and 75th percentiles (Interquartile range:

IQR), while categorical data was summarized as frequency counts with percentages. Generalized linear mixed models with random effects (to account for multiple lab samples from the same subject) were used to analyze platelet count and TPO over time. P < 0.05 was considered statistically significant for all analyses.

Results

There were 12 total and 1 subtotal pancreatectomies (1 patient had had central pancreatectomy for pseudopapillary neoplasm with subsequent pancreatitis of distal segment from pancreaticojejunostomy anastomotic stricture). Etiology of pancreatitis and time with illness from diagnosis is described in Table

1. The median for age was 12.6 years, for BMI 20.9 kg/m2, for length of stay 26 days, and for operative time 13 hours (Table 2). The median for total Islet cell count infused was 447,000 (IQR: 357,000-603,000), with a median Islet equivalent per kilogram of 5,821 (IQR: 4,244-6,858). Insulin requirements were measured from day 0 until discharge. All patients were placed on insulin infusion while in the intensive care unit as to maintain perioperative euglycemia and maximize protection of islet cells. Median glucose levels at postoperative day 10 was 109 mg/dL (IQR: 107-118 mg/dL). All patients were discharged on basal and bolus insulin. TPO levels were normal preoperatively on all patients with a median of 45 pg/mL (IQR:

34-56), (normal 7-99 pg/mL). Postoperatively, TPO levels increased significantly reaching a median of 219 pg/mL (IQR: 171-303) 4 days postoperatively (p<0.0001) (Figure 1). On average the TPO level increased 39 pg/mL per day. Preoperative platelet counts were normal for all patients with a median of 216.5 K/l (IQR:

193.5-291.5), (normal for age 12 at our laboratory is 135-450 K/l), and increased exponentially between the first and second weeks, reaching a median of 835 K/l (IQR: 821-850) by postoperative day 9

(p<0.0001) (Figure 2). In our sample, from preoperative phase until 3 days postoperative, platelet count did not significantly increase; however, after 3 days postoperative, the platelet count significantly increased until hydroxyurea is started (p<0.0001). In contrast, TPO levels significantly increase in all patients early in the postoperative period from day 1 to a maximum level at 4 days postoperative (p=0.04)

(Figure 1).

Hydroxyurea was started on average 7.8 days postoperatively, the variation was due to the difference in timing of patients reaching abnormally high platelet counts. Therapy with hydroxyurea significantly

5 decreased the platelet count (p<0.0001) from a median of 835 at 9 days to a median of 382 K/l (IQR:

279-536) at around 34 days postoperative. TPO levels decreased as well and at 20 days on average post- hydroxyurea therapy were a median of 82 pg/mL (IQR: 45-128)(Figure 3).

There were no major surgical or thrombotic complications in any patient. Doppler ultrasonography was performed on all patients on day 0, 1, 7, with no patients showing signs of portal thrombosis. Fifteen percent (2/13) had sterile intraabdominal collections drained. Also 2 patients (15%) had superficial surgical site infections requiring incision and drainage. One patient developed non-clinically significant gastrointestinal bleeding through the nasogastric tube controlled with proton pump inhibitors.

Discussion

TPIAT in children is currently performed only in highly specialized centers in the United States given the perioperative challenge of its nature. One of the most common complications of this surgical procedure, that has high morbid potential, is the sustained and prolonged thrombocytosis that in our series, was developed by all patients. Our study is the first pediatric report that directly addresses the details of the reactive thrombocytosis associated to TPIAT. Current literature describes the splenectomy as the cause of the thrombocytosis; however, our findings point towards a more detailed inflammatory reaction that likely promotes the production of TPO by the hepatocytes that in turn, activates hematopoietic progenitors of platelets driving its levels exponentially. Some theories have proposed that interleukin 6

(IL-6) is the rate-limiting step in reactive thrombocytosis. A study performed in a cohort of cancer patients with reactive thrombocytosis that had been treated with IL-6, showed that increased IL-6 production results in an increase in TPO plasma levels from TPO-mRNA transcription by a specific hepatoblastoma- line hepatic cells. This increase in TPO subsequently promoted enhanced thrombopoiesis [20], and might support the theory behind the inflammatory mediators-related thrombocytosis.

The production of platelets is a complex process that is regulated by TPO and the TPO-receptor

(MPL)/JAK2 axis. This axis regulation responds to either intrinsic or extrinsic signaling pathways that increase or decrease the production of TPO. One study evaluated the genetic alterations involved in the regulation of this axis and shown that gain-of-function mutations lead to increased TPO synthesis and thrombocytosis [21]. Another group studied the effect of thrombopoietin on thrombocytosis and liver regeneration in an animal model, and showed that cases treated with TPO had platelet counts increased by 60% in the first week and accelerated liver regeneration through induced hepatocyte growth factor compared with controls [22]. It is known that hepatocytes constitutively express TPO genes regardless of the platelet count, and platelets in turn clear TPO levels through high-affinity TPO-receptors in the healthy

7 state; however, it has been shown that increase in plasma TPO and IL-6 secondary to an inflammatory cascade activation, causes reactive thrombocytosis [23].

Recent improvements in isolation and transplantation techniques during TPIAT have enhanced the quality and amount of transplanted cells and decreased the peri-transplant inflammatory reaction [24]; however, this immune reaction remains the rate-limiting step on the outcomes of this technique. Current surgical and laboratory techniques mandate preserving peri-transplantation normoglycemia, reducing ischemic time, and reducing the prothrombotic state, thought to be driven by tissue factor released from the endothelium, to improve outcomes of cell survival and engraftment [25-27]. Controlling these variables promotes a substantial reduction in blood-mediated and post-transplant inflammatory reaction after the infusion of islet cells. Studies have shown that this post-infusion reaction also activates the coagulation cascade increasing thrombin concentrations and promoting pro-inflammatory cytokines and complement activation leading to thrombin-induced apoptosis of islet cells [28, 29].

One of the key components of glycemic control is limiting beta cell death rate. One group described a specific marker of apoptosis in the form of unmethylated insulin DNA that can predict hyperglycemia post- transplant [30]. But despite major efforts to protect grafts from cell apoptosis, including the use of dipeptidyl peptidase 4 inhibitors and some other techniques [31], beta cell death continues to limit the success of TPIAT on insulin independence. Although with the novel contemporary methods of post- transplant islet protection, graft survival rates are showing promising results [32].

TPIAT in children has proven its early success in controlling the chronically debilitating pain, and improving the quality of life of patients with chronic or acute recurrent pancreatitis [34]. A recent study showed that early-onset acute recurrent and chronic pancreatitis strongly correlates with genetic PRSS1 or CTRC mutations [33], which correlates with our patient population. The management of the post-operative complications of this technique have been significantly improved and standardized in our center. The

8 treatment of thrombocytosis with hydroxyurea has been successful in our institution, and has prevented the potential thrombotic complications of the extremely high platelet levels.

The number of patients in our case series limits the capability of being able to fully understand and analyze the platelet and TPO relationship; however, the significant rise in TPO slightly preceding the platelet rise is suggestive of an association to the TPO levels. Prospective data is being currently collected to obtain more TPO samples to more accurately determine when the rise in TPO occurs, and whether the type of rise is linear or exponential. There seems to be a lag time of a couple days in between the TPO rise and the platelet rise and with more data collected the lag time relationship can be better determined.

Conclusion

All patients developed sustained and prolonged thrombocytosis after TPIAT as seen by an exponential increase in platelet count in this case series. The significant rise in TPO slightly preceding the significant rise in platelet count postoperatively, suggests an association between thrombocytosis and increased TPO production. Further studies are needed to explore the mechanism of thrombocytosis post-TPIAT and to better determine this relationship

Table 1. Etiology and Time with illness from diagnosis.

Patient ID Etiology Time with illness (months)

1 Abnormal pancreatic duct anatomy 18

2 SPINK1*/CFTR˜/Pancreas Divisum 42

3 CFTR 27

4 PRSS1ᶲ 50

5 Tumor - Pancreatico-jejunostomy 36 anastomotic stricture

6 PRSS1 17

7 PRSS1 108

8 CFTR/Pancreas Divisum 53

9 SPINK1 84

10 SPINK1/Pancreas Divisum 54 SPINK1/Pancreas Divisum 96 11 CFTR 23 Necrotizing pancreatitis 12 12

*SPINK1 – Serine Peptidase Inhibitor, Kazal Type 1

˜CFTR- Cystic Fibrosis Transmembrane Conductance Regulator

ᶲPRSS1- Protease, Serine 1/ human cationic trypsinogen

Table 2. Patient Characteristics Patient Characteristics TPIAT patients N=13 Age at surgery, years 12.6 (9.6 - 15.9) Sex, female 8 (62%) Race, White/Caucasian 12 (92%) BMI at surgery 20.9 (18.6 - 24.6) Length of stay, days 26.0 (22.0 - 32.0) Operative time, hours 13.0 (11.0 - 14.0) Time with illness, months 42.0 (23.0 - 54.0) Total Islet cell count 447,000 (357,000 - 603,000) Islet equivalent per kg 5821 (4244 - 6858) Glucose post op (mean day 10) mg/dL 109.0 (107.0 - 118.0) Insulin requirements (no. patients) Bolus/basal 8 (62%) Basal 2 (15%) None 3 (23%) Complications (no. patients) Hypotension POD 1 2 (15%) Intraabdominal collections (sterile) 2 (15%) SSI 2 (15%) AKI 1 (8%) Diabetes 1 (8%) Extended ICU stay, respiratory failure 1 (8%) GI bleed 1 (8%) None 3 (23%)

Figure 1 – TPO levels pre and post surgery

Figure 2 – Platelet count postoperative phase and before hydroxyurea

Figure 3- TPO levels postoperative phase.

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