Supplementation with concentrated milk protein in patients undergoing hematopoietic stem cell transplantation

Accepted Manuscript Supplementation with concentrated milk protein in patients undergoing Hematopoietic Stem Cell Transplantation Ana Carolina Amaral de São José Perrone, Thaís Rodrigues Barbosa, Fernanda Lopes da Silva, Ítalo Tuler Perrone, Antônio Fernandes de Carvalho, Rodrigo Stephani, Kelli Borges dos Santos, Ângelo Atalla, Abrahão Elias Hallack Neto PII:

S0899-9007(16)30232-5

DOI:

10.1016/j.nut.2016.10.010

Reference:

NUT 9861

To appear in:

Nutrition

Received Date: 25 January 2016 Revised Date:

3 October 2016

Accepted Date: 8 October 2016

Please cite this article as: Perrone ACAdSJ, Barbosa TR, Silva FLd, Perrone ÍT, Carvalho AFd, Stephani R, Santos KBd, Atalla Â, Hallack Neto AE, Supplementation with concentrated milk protein in patients undergoing Hematopoietic Stem Cell Transplantation, Nutrition (2016), doi: 10.1016/j.nut.2016.10.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Supplementation with concentrated milk protein in patients undergoing

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Hematopoietic Stem Cell Transplantation

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Ana Carolina Amaral de São José Perrone1, Thaís Rodrigues Barbosa1, Fernanda Lopes da Silva2, Ítalo Tuler Perrone2, Antônio Fernandes de Carvalho2, Rodrigo Stephani3, Kelli Borges dos Santos4, Ângelo Atalla5, Abrahão Elias Hallack Neto 1,6 1

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Department of Hematology, Hemotherapy and Bone Marrow Transplantation, Federal University of Juiz de Fora, Minas Gerais, Brazil 2 Department of Food Technology, Federal University of Viçosa, Minas Gerais, Brazil 3 Department of Chemistry, Federal University of Juiz de Fora, Minas Gerais, Brazil 4 Department of Basic Nursing, Federal University of Juiz de Fora, Minas Gerais, Brazil 5 Monte Sinai Hospital, Division of Bone Marrow Transplantation and Departament of Clinical Medical, Federal University of Juiz de Fora, Minas Gerais, Brazil 6 Departament of Clinical Medical, Federal University of Juiz de Fora, Minas Gerais, Brazil

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ABSTRACT

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Objective: The aim of this study was to analyze the influence of dietary

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supplementation with whey protein concentrate (WPC) in the incidence of oral

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mucositis (OM) in patients undergoing Hematopoietic Stem Cell Transplantation

22

(HSCT). Methods: Patients were supplemented with a daily intake of WPC delivering

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50% of the daily protein requirements (DPR) according to the Dietary Reference

24

Intakes and classified later based on the amount of ingested supplement until OM

25

median onset. Results: 73 patients were evaluated. Forty-three were part of the

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historical control and 30 were supplemented with WPC. The OM had a mean

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duration of 5.3 days (SD: 4.5), ranging from the day of the infusion of stem cells until

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the 17th day after infusion and a median of 5 days after infusion. OM duration was

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influenced by the conditioning protocol (p<0.01) and WPC (p=0.01). Patients who

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consumed the WPC in an amount equal to or greater than 40% of DPR had a 35%

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reduction in duration of OM and the incidence of OM grades 3 and 4 was 11 times

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smaller. Body mass index, serum albumin and adverse reactions, such as diarrhea,

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nausea and vomiting, dysphagia, dry mouth and drooling, showed no statistically

34

significant differences. Conclusion: The WPC intake greater than or equal to 40% of

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DPR helped to reduce the severity and duration of OM. The use of WPC in patients

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undergoing HSCT was shown to be safe, encouraging new studies in this population

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in order to assess its action mechanism.

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KEYWORDS: Mucositis; Bone marrow transplantation; Recommended daily protein

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intake; Dairy product; Conditioning protocol.

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1.INTRODUCTION

43 Mucositis is the commonest toxicity effect of anticancer drugs during

45

Hematopoietic Stem Cell Transplantation (HSCT). It occurs in approximately 80% of

46

patients receiving high dose chemotherapy during conditioning for transplantation [1]

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and it has a significant impact on their quality of life [2].

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It is characterized by inflammatory lesions of the gastrointestinal mucosa. Its

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pathophysiologic process involves complex molecular and cellular events, which are

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described in five phases: initiation, primary damage response, amplification,

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ulceration and scarring [3].

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Mucositis has a significant impact on nutritional status, an important factor in

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indication of parenteral nutrition, use of opioids, increased length of hospitalization

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and costs [4,5]. HSCT patients showing a higher degree of mucositis (using OMAS

55

scale - Oral Mucositis Assessment Scale) lead to an approximately 45% increase in

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hospital costs [5].

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Whey is obtained during the production of cheese or casein. It comprises

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substances such as lactoferrin, insulin like growth factors I and II (IGF-I and IGF-II),

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acidic and basic stimulating fibroblasts growth factors (aFGF, bFGF), transforming

60

growth factors beta (TGF-β), and several others. WPC is obtained by whey

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processing in the dairy industry [6]. Transforming growth factor beta is the name of a family of polypeptides with

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several biological functions, such as stimulating proliferation of some cell types,

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especially in the connective tissue, while inhibiting others, like lymphocytes and

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epithelial cells [7]. The predominant form found in bovine milk is TGF-β2 (85%). It

66

has attracted the interest of the scientific community as a potential bioactive

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ingredient, which may be used in the production of functional foods [8,9], with various

68

clinical applications [7]. Several scientific papers on the effect of TGF-β

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supplementation on the incidence and severity of OM have been published [10-14].

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The aim of this study was to evaluate the effect of whey protein concentrate

71

(WPC) intake on the incidence and severity of oral mucositis in patients undergoing

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autologous and allogeneic HSCT.

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2. PATIENTS AND METHODS

74 A prospective and quantitative study was carried out in order to evaluate the

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influence of dietary supplementation with Whey Proteins Concentrate (WPC) on OM

77

in patients undergoing autologous and allogeneic HSCT at the Hospital of the

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Federal University of Juiz de Fora (HU-UFJF) and Monte Sinai Hospital.

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The study was approved by the Ethics and Research Committee of the HUUFJF according to protocol 15456513.5.1001.

Patients included were older than 12 years, agreed to participate in the

82

research on an Informed Consent Term and had not started the conditioning phase.

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Patients who were allergic to milk proteins or lactose intolerant were not included, as

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well as those who had oral feeding suspended by a physician during treatment or

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passed away during the data collection period.

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The control group was formed by patients who underwent HSCT at HU-UFJF

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from January 2012 to June 2013. The exclusion criteria were lack of information or

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inaccuracy in the records jeopardizing data collection, death during the transplant

89

period and being younger than 12 years old.

The OM prevention protocols include low-level laser and glutamine (4 joules

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each 48h and 30g/day respectively). Both procedures were maintained while using

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WPC, so the intervention group differed from the control group only by the

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supplement administration.

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The conditioning protocol used for patients diagnosed with multiple myeloma

95

(MM) was Melphalan (Mel) 200 mg/kg and Mel 140mg/kg for those over 65 years old.

96

For Hodgkin's Lymphoma (HL) or Non-Hodgkin's Lymphoma (NHL), the protocol was

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CBV (Cyclophosphamide 6 mg/kg, Carmustine 300 mg/kg, Etoposide 1200mg/kg) or

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LEAM (Lomustine 300 mg/kg, Etoposide 1000mg/kg, Cytarabine 4000mg/kg and

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Melfanan 140mg/kg). For Acute Myeloid Leukemia (AML), it was BuCy (Busulfan

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16mg/kg, Cyclophosphamide 120 mg/kg) or FLUMEL (Flubarabine 125mg/m2,

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Melphalan 140mg/m2).

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WPC with 80% of protein concentration was donated by Gemacon Tech in

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partnership with the Dairy Laboratory of the Food Technology Department of the

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Federal University of Viçosa (INOVALEITE/UFV). INOVALEITE also performed

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composition determination and microbiological analyzes ensuring the integrity and

106

quality of the product. The supplements were in accordance with the microbiological

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standards established by RDC nº 12 of January 2001 [15], the National Health

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Surveillance Agency (ANVISA) regulation for the Food Group 26 (Food for Specific

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Population Groups), Item B (food for immunosuppressed and immunocompromised)

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and Item C (powdered enteral diets, powdered nutrients modules for enteral feeding

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composition). WPC supplementation was taken once a day, from the first day of conditioning

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until the day of bone marrow engraftment. It was administered in liquid form after

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dilution in milk or processed juice, according to the preference of the patient. The

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temperature never exceeded 50°C, so that there was no denaturing of proteins.

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The amount offered represented 50% of the daily protein needs (DPN) in

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accordance with the age of the patient and with the Dietary Reference Intakes -

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Institute of Medicine, 2002-2005 [16].

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The evaluation of the patients was performed daily from the day of admission

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to the day of engraftment. The National Cancer Institute (NCI) criteria version 4.0 [17]

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was used for grading of adverse events during the study. The evaluated adverse

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reactions were nausea, emesis, dysphagia, dyspepsia, diarrhea and xerostomia. The World Health Organization (WHO) [18] oral toxicity scale was used to

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evaluate the degree of mucositis. Each patient was categorized according to the

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highest level reached during the evaluation period.

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To analyze the data, WPC supplemental feeding was considered until the day of OM median onset.

The intervention group was stratified by WPC consumption percentage in

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order to determine the influence of the supplement on the incidence and severity of

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OM. The cutoff points of the groups were determined to ensure homogeneity of the

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distribution of individuals in relation to gender, age, conditioning protocol, diagnosis

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and type of transplant.

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Statistical analysis

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The collected data were analyzed using R Commander program. Categorical

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data was described using frequencies and percentages, and associations with OM

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were verified by the Chi-square test or Fisher's exact test. Continuous data was

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presented using means, medians, standard deviations, ranges and univariate

139

analysis were performed with the T test or Mann-Whitney. Poisson regression

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analysis was used to identify multivariate associations to oral mucositis. The

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statistical tests were two-sided at a significance level of 5%.

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3.RESULTS

144 Eighty-five patients were evaluated, 37 in the supplemented group and 48 in

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the control group. Seven patients who fit the exclusion criteria were excluded from

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the supplemented group and 5 from the control group. Table 1 shows the distribution

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of patients in both groups.

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Table 1 - Descriptive statistics of the supplemented and control group.

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Sex Male Female Age Median Min-Max Diagnosis Multiple Myeloma

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Mean ± Standard Devation

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Supplemented (n = 30)

Characteristics

Control (n = 43)

n

%

n

%

24

80.0

25

58.1

06

20.0

18

41.9

45.5±14.4

45.6±11.6

47.5

49.0

16-68

16-63

15

50.0

21

48.8

06

20.0

08

18.6

Non- Hodgkin Lymphoma

05

16.7

08

18.6

Acute Myelogenous Leukemia

04

13.3

06

14.0

Autologous

27

90.0

40

93.0

Allogeneic

03

10.0

03

7.0

Melphalan

14

46.7

21

48.8

LEAM

09

30.0

10

23.3

CBV

02

6.7

05

11.6

BuCy

02

6.7

03

7.0

Other

03

9.9

04

9.3

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Transplant Type

Conditioning Protocol

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Twenty-four patients (32.9%) had Body Mass Index (BMI) adequate for their

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age, 3 had low body-weight (4.1%), 23 were overweight (31.5%) and 22 obese

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(30.1%). The mean serum albumin was 3.8 g/dl (SD=0.6), ranging from 2.9 g/dl to

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6.7 g/dl and with a median of 3.7 g/dl.

156 157 158 159

3.1 Supplemented Group versus Control Group There was no statistically significant difference between supplemented and control group regarding gender (p=0.76), diagnosis (p=1.00), conditioning protocol

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(p=0.96), transplantation type (p=0.69), age (p=0.98), albumin (p=0.12) and BMI pre-

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TCTH (p=0.65).

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The overall incidence of OM was 63.0% (n=46). It was 45.7% (n=21) in the

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supplemented group and 54.3% (n=25) in the control group (p=0.335). Among the

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supplemented patients, 23.3% (n=7) had severe oral mucositis (SOM), while among

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the control group it was 9.3% (n=4) (p=0.182). The overall average duration of OM

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was 4.81 ± 4.75 days (range: 0-17 days), while the average duration was 5.33 ± 4.54

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(range: 0-17 days) for patients receiving WPC and 4.44 ± 4.92 (range: 0-17 days) for

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patients in the control group (p=0.434). Supplemented and control group showed no

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statistically significant differences concerning the incidence and duration of OM and

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incidence of SOM.

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3.2 WPC Supplemented Group

Among those who had OM, the mean and the median OM onset day were the 5th day after infusion of hematopoietic stem cells (D+ 5) (SD: 1.70), ranging from the

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first to the eighth day after infusion (95%CI: 4.22-5.78).

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The average intake of WPC up to D+5 was 77.9% (95%CI: 68.2-87.6) of the

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amount offered (SD=26.0), ranging from 0 to 100% (CI: 68.2 to 87.6).

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The supplemented group was divided into two subgroups according to the

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amount ingested until D+5. Group 1 was composed of patients who consumed up to

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79.9% of WPC and group 2 of those who consumed 80% or more of the offered

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dose.

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WPC average consumption in the first group was 50.9% (SD=23.4; 95%CI:

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35.1-66.6), ranging from 0 to 75.0%. In the second group, the average was 94.5%

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(SD=5.8; 95%CI: 91.7-97.3), with the lowest consumption being 81.3% and the

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highest 100.0%.

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There was no statistically significant difference between the groups regarding sex, diagnosis, conditioning protocol, type of transplantation and the use of G-CSF.

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Nutritional status, evaluated by BMI and serum albumin, assessed at the first

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conditioning day, showed no statistically significant difference (p=0.763 and p=0.06,

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respectively). The reasons given by patients for WPC consumption of 50% of the offered

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amount or less were mostly nausea (46.7%), lack of appetite (16.7%) and vomiting

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(10%).

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3.2.1 Effects of supplementation

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The average length of hospitalization was similar in both groups (p=0.886),

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with an overall average of 17.1 days (SD=3.3; 95%CI: 15.9-18.3), a minimum of 14

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and maximum of 28 days.

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The overall mean duration of neutropenia was 9.87±6.80 (range: 6-40; 95%CI:

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7.33-12.41). For consumption 0-79%, the mean was 11.73 ± 10.13 (range 6-40;

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95%CI: 4.92-18.53) and for consumption 80-100% 8.79 ± 3.77 (range 6-22; 95%CI:

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6.97-10.60) (p = 0.549).

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The incidence of OM compared to supplementation showed no statistical

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difference between the two groups (p=0.419). Using Poisson regression, no

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associations between key variables and incident OM were identified.

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There was a predominance in group 1 of patients classified as grade III (45%),

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followed by grade I (27%). In group 2, more than 90% were classified between

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grades 0 and II and there were no patients classified as grade IV (p=0.008) (Figure

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1).

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Group 1 213

Group 2

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Figure 1 – Oral Mucositis classification in the supplementation groups. Grade 2 was

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not detected in group 1 and grade 4 in group 2.

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OM day of onset was on average 5.00 ± 1.70 (range 1-8; 95%CI: 4.22-5.78), with no statistically significant difference between the two groups (p=0.50). Considering only the patients with OM (grade 1-4), the OM mean length in

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group 1 was 8.4 days (SD=3.50; 95%CI: 5.75-11.14), with a minimum of 3 days and

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maximum of 16. In group 2, the mean length of OM was 7.0 days (SD=3.4; 95%CI:

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4.87-9.13), with a minimum of 4 and a maximum of 17 days (p=0.35) (Figure 2).

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Figure 2 – Variation of duration of Oral Mucositis considering grades 1-4 for the

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supplemented groups.

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However, when evaluating the whole hospital stay to identify the OM

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representation in the total duration of HSCT (grade 0-4), it was found that the overall

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average length of OM was 5.33 ± 4.54 (range: 0-17; 95%CI: 3.64 to 7.03). It was 6.75

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± 4.45 in Group 1 (range: 0-16; 95%CI: 3.79 to 10.02) and 4.39 ± 4.47 (range: 0-17;

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95%CI: 2.32 to 6.52) in Group 2 (p=0.007; RR=0.65) (figure 3).

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Figure 3 - Variation of the duration of Oral Mucositis considering grades 0-4 for supplemented groups

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Figure 4 depicts the mean hospitalization times of the patients in each group

G4

0.0 2.4

G3

0.7

GROUP 2 GROUP 1

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for OM degrees according to WHO classification.

OM Classification

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64.6 30

40

50

60

70

75.3 80

Duration (%)

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Figure 4 - Average percentage duration of each degree of OM during the hospital

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stay.

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Group 1 subjects had increased OM duration in grades 3 and 4. By contrast,

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Group 2 on average spent more than 70% of the hospitalization time with no OM,

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followed by mild degrees of it.

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Multivariable analysis using Poisson regression showed that the reduction in

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OM duration was only influenced by the conditioning protocol used and the

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consumption of WPC in the range of 80 to 100% of the offered dosage (Table 2).

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TABLE 2 – Determinants of the duration of Oral Mucositis (n = 30). VARIABLES

ESTIMATED PARAMETER

P-value

INCIDENCE RATE

95%CI

Protocol -1.38

< 0.001

0.25

0.16-0.39

LEAM

-1.22

< 0.001

0.29

0.18-0.48

CBV

-1.19

< 0.001

0.30

0.14-0.59

2.99

< 0.001

19.83

13.50-28.04

-0.84

0.003

0.43

0.24-0.75

-0.41

0.012

0.66

0.48-0.91

BUCY OTHER

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MEL

Group 2

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WPC

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The determinants of incident SOM were investigated through a multivariable

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analysis,

including

the following variables: gender, age, diagnosis, conditioning

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protocol, BMI prior to transplantation, albumin prior to transplantation, day of

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administration of granulocyte stimulating factor and consumption of WPC up to D+5.

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The only variable associated to incident SOM was WPC intake (p=0.035). The SOM mean incidence in the supplemented group was 11times lower in

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the group who ingested between 80 and 100% of the offered WPC compared to the

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group who ingested between 0 and 79% WPC (p=0.04, 95%CI: 0.005 to 0.65).

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In relation to nutritional status, there was no statistically significant differences

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in the initial and after-transplantation rates concerning BMI and albumin in groups 1

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and 2.

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Emesis degree was associated with the consumption of WPC (p=0.011). In the

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group with the lowest consumption, patients were generally classified as grades II

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and III. In Group 2, there was a predominance of grade 0, followed by grade I.

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More than 60% of patients in Group 1 showed mild dysphagia (grade I and II),

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whereas in Group 2 there was a predominance of those who did not present

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dysphagia (p=0.025).

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Despite the fact that the degree of emesis and dysphagia were associated to

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WPC consumption, a causal relationship of these variables with low consumption

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was not identified (p=0.260 and p=0.406, respectively).

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4.DISCUSSION

276 277

There was no statistically significant difference between the control group and

278

the supplemented concerning the analyzed variables. We attribute this fact to the use

279

of a retrospective control. Comparing the supplemented groups, the analysis of adverse reactions did

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not show statistically significant results to characterize them as causal factors for

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supplementation smaller than 79.9%. In contrast, previous studies show that food

283

intake is significantly compromised during the transplant due to side effects of the

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conditioning protocol [19-21]. Symptoms such as nausea, vomiting and mucositis

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may have affected the supply of transplant patients [22].

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In our study, among all the analyzed variables, only the conditioning protocol

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and WPC consumption affected the mucositis duration. A prospective study found

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that the conditioning protocol was the only determinant of the evolution of OM for

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patients undergoing myeloablative protocols [23]. The diagnosis of NHL, the use of

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etoposide in the mobilization phase and prior radiation therapy were the factors that

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influenced OM in patients undergoing autologous HSCT [24]. Both the factors related

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to cancer therapy and patient-related factors may influence OM [25].

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The mean and the median of the onset of mucositis were the 5th day after

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infusion of hematopoietic stem cells. The average OM duration was 5 days, 7 days

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for group 1 and 4 days for group 2. Comparing to the literature, the reduction in OM

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duration was mainly in patients supplemented above 80%. Studies shows that OM

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initiates between the end of the conditioning regimen and the four subsequent days

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[26, 27]. The average duration of OM ranges from 5 to 9 days, reaching up to 12

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days in patients undergoing allogeneic HSCT [28-31].

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Most clinical trials report the maximum toxicity grade throughout the course of

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therapy. Murphy reports that the duration of OM is relevant to the evaluation of

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treatment toxicity. Oral mucositis should not be classified only by degree of injury,

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because it is also associated with function loss [32].

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Previous studies proposed another assessment method of OM incorporating

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both classification of injury and loss of function. Consequently, they also evaluated

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the duration of each stage. The results showed that patients remained about half the

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evaluation time in grade 0 (52%), followed by grade II (21%) [33]. Better results were

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found in our study, since the group of patients who accepted above 80% of offered

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WPC spent on average 75.3% of hospitalization time in score 0 and 17.4% in score I.

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Therefore, patients remained almost the entire time without OM or on early stages,

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enabling satisfactory food intake and lower risk of complications from OM. Our analysis indicate that the use of WPC was associated with reduction of

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both severity and duration of OM. We believe that this reduction may be related to

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the presence of TGF-β in the supplement.

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In the scientific literature, references relate TGF-β to controlling of intestinal

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inflammation and mucositis in rats, the healing of mucous membranes and notable

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clinical applications in Crohn's patients, as well as reducing intestinal damage and

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facilitating regeneration after mucosal injury [34-38]. TGF-β stimulates cell growth,

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especially of the connective tissue, participates in the formation of bone and

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cartilage, in controlling of the immune system and in wound healing [39]. In 80% of

321

mice supplemented with TGF-β2, there was a significant reduction in the degree of

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intestinal mucosal injury induced by methotrexate compared with animals receiving

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placebo, and in one specific case accelerated repair of the intestinal mucosa was

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observed [34].

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The ability of dairy products to modulate inflammatory processes in humans is

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under study and is an important but unresolved issue [40]. Maternal milk has the

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ability to support the development of the immune system, to inhibit bacterial growth

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and to deliver anti-oxidative protection (e.g. glutathione) [41]. A long and detailed

329

review suggests that dairy products, in particular fermented products, have anti-

330

inflammatory properties in humans not suffering from allergy to milk, in particular in

331

subjects with metabolic disorders [40]. The milk proteins and the milk biopeptides are

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involved in anti-inflammatory mechanisms on the intestine of newborns, combats

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obesity and Type 2 diabetes, promotes the enhancement of mineral absorption,

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immunomodulation, opioid, antihypertensive and antimicrobial activities [42-48].

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Although the aim of this study was not to evaluate quality of life, the reduction

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of 35% in the number of days with mucositis and the reduction of 11times in the

337

incidence of SOM are undoubtedly important results, especially regarding the

338

consequences of OM.

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SOM is an important factor associated with infection in patients undergoing

340

HSCT [49]. The disruption of the mucosal barrier caused by cytotoxic agents affects

341

the capacity of blocking the entry of microorganisms into the bloodstream [50].

342

Several interventions to avoid or minimize oral lesions and its consequences

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have been studied. Low-power laser therapy showed analgesic effects and aided in

344

wound healing [30, 51], cryotherapy reduced the incidence of SOM [52] and

345

palifermin (recombinant human keratinocyte growth factor) is the only drug currently

346

approved by the FDA reducing the incidence and duration of SOM [53, 54]. Current guidelines MASCC/ISOO [1] recommend (level of evidence I and II)

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only low-power laser and palifermin for the prevention of OM in HSCT. More research

349

on new therapies is desirable, especially of low cost interventions such as the

350

consumption of WPC.

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351 5. CONCLUSION

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WPC consumption of 80% or more (40% of DRP) reduced by 35% the time of

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OM and contributed to a reduction of 11 times in the incidence of the more severe

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forms of mucositis among patients undergoing HSCT. Further studies are needed to

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assess the action mechanism of WPC in preventing SOM.

358 ACKNOWLEDGEMENTS

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The authors is grateful for the financial support of CAPES and FAPEMIG.

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