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Graft-versus-Host Disease

Major salivary gland damage in allogeneic hematopoietic progenitor cell transplantation assessed by scintigraphic methods

Abstract

Salivary gland dysfunction is a common sequela of hematopoietic progenitor cell transplantation (HPCT). The investigation of major salivary gland dysfunction with sodium pertechnetate scintigraphy is a non-invasive method that provides images of the parotid and submandibular glands. In this prospective trial, 20 HPCT patients were submitted to scintigraphic study with 99mTc-pertechenate and 67Ga in order to evaluate the major salivary glands early involvement following HPCT. Major salivary glands were evaluated prior to HCPT as well as at Days +30, +60 and +100 post transplant. Major salivary glands uptake and clearance of 99mTc-pertechenate results did not demonstrate any functional differences between pre- versus post transplant periods. Results of the 67Ga scan revealed inflammatory infiltration following HPCT, primarily in submandibular glands, suggest a persistent involvement of major salivary glands up to Day +100 after HPCT.

Introduction

Salivary gland dysfunction is a common complication in the hematopoietic progenitor cell transplantation (HPCT) patient.1, 2 Salivary secretion rates can be substantially reduced during the conditioning regimen phase, likely due to high-dose therapy (HDT) plus total body irradiation (TBI) and other drugs utilized for supportive care.2, 3, 4

Effects of chemotherapy on minor salivary glands are well documented.5, 6 For example, a pilot study of patients with hematologic malignant diseases and who received chemotherapy demonstrated reduced secretion from the minor salivary glands compared to secretion from the major salivary glands.6 In addition, effects of radiation therapy in salivary glands are also well documented, primarily in head–neck cancer patients.7, 8

Recently, salivary gland scintigraphy has been refined such that quantitative data regarding glandular function after parenchymal insult can be obtained. Sodium pertechnetate scintigraphy is a minimally invasive procedure and provides images of the parotid and submandibular glands.9 Another radioactive isotope, gallium-67 (67Ga), has been widely utilized for the imaging study of various malignant tumors.10 Accumulation of 67Ga can be detected in inflammatory lesions that facilitate help on diagnosis of different major salivary gland diseases, as Sjögren's Syndrome.10

However, few studies regarding major salivary glands damage using scintigraphy were published. Most reports are directed to pediatric populations.11 In the current study, we performed a prospective evaluation of the major salivary glands up to +100 days post-HPCT in order to further study the early in the major salivary glands.

Patients and methods

This study enrolled 20 consecutive patients, 12 (60%) males and eight (40%) females, from March 2002 until November 2003 who were scheduled to receive allogeneic HPCT at BMT Unit of State University of Campinas (Unicamp) – Brazil. Table 1 shows the patients’ and donors’ characteristics, follow-up and the cell source. Seventeen out of 20 (85%) had chronic myelogenous leukemia (CML) and three (15%) severe aplastic anemia (SAA). Overall median age was 39 years old (range: 30–55). The study subjects had not received multi-chemotherapy regimens prior the study. CML patients were treated previously with hydroxyurea alone.

Table 1 Patients’ and donors’ characteristics, follow-up and cell source

Research ethics committee approval

Research Ethics Committee of Medical School – State University of Campinas approved the study protocol. All participants signed written informed consent.

HPCT features, conditioning regimen and GVHD prophylaxis

All transplants were allogeneic myeloablative and HLA-related full match according to Institutional practice. The conditioning regimen for CML included cyclophosphamide (CY) 120 mg/kg and busulfan (BU) 16 mg/kg. For SAA, the conditioning regimen consisted of CY 200 mg/kg plus BU 4 mg/kg.12 Cyclosporine-A and methotrexate were administered for GVHD prophylaxis, in accordance with current protocols at the University Hospital.

Supportive care

Supportive care was provided in single-bedrooms with high-efficiency particulate air (HEPA) filtration. Ceftazidime was administered to non-febrile patients whenever the neutrophils counts dropped below 0.5 × 109/l; the antibiotic regimen was modified according to the clinical course and maintained until bone marrow recovery. During neutropenia, all patients received fluconazole (200 mg/day) as antifungal prophylaxis.13 Pre-emptive ganciclovir therapy was administered for patients presenting two consecutive positive PCR assays and/or antigenemia for CMV.

Oral clinical examination

The oral mucosa, dentition, periodontium and orthopantographic radiograph were evaluated by the Dental Ambulatory Service prior to and during 100 days following HPCT. Acute dental infections were treated prior HPCT by the Dental Ambulatory Service.

Measurement of unstimulated salivary flow rate (USSFR)

An USSFR sample was collected prior to HPCT and again on Days +30, +60 and +100 following HPCT, 30 min after most recent dental brushing by the patient.14 The procedure consisted of a 5 min salivary expectoration into a sterile previously weighted glass bottle. Patients were asked not to swallow during the collection period. Salivary flow rates were calculated based of saliva collected per minute and converted to ml/min.3, 15 A USSFR 0.1 ml/min was considered low.3

Clinical features of xerostomia

Clinical signs of xerostomia subjectively assessed via (i) absence of the sublingual salivary pooling, (ii) adherence of a wooden tongue depressor to the buccal mucosa, (iii) dry lips and (iv) tongue snap.14

Major salivary glands scintigraphy

Major salivary gland scintigraphy was performed in order to assess: (1) major salivary glands uptake and salivary clearance using 99mTc-pertechenate scintigraphy, and (2) inflammatory activity using 67Ga scan. Scintigraphic studies were performed prior to HPCT and then Days +30, +60 and +100 days after transplant. 99mTc-pertechenate scintigraphy was obtained after intravenous injection of 370 MBq of 99mTc-pertechenate. Head and neck blood flow continuous images were acquired every 2 s during 80 s in the anterior projection. Static images with 500 000 counts in the anterior, right anterior oblique and left anterior oblique projections of the head were acquired 20 min after injection (prior stimulation images) and repeated in the same projections 2 min after stimulation with citric acid (two drops of lemon juice) for the same acquisition time of pre-stimulation images.

Regions of interest (ROI) were demarcated relative to submandibular and parotid glands and in adjacent areas for background count calculation. After acquisition, 99mTc-pertechnetate uptake by the major salivary glands was evaluated by two Nuclear Medicine physicians who were blinded to other study data and visually compared to thyroid uptake and considered as normal or decreased. In case of discordance, the final decision was obtained by consensus.

99mTc-pertechnetate salivary gland clearance was calculated for left parotid, right parotid, left submandibular and right submandibular by the following equation:

Demonstration of clearance of <50% was considered as evidence of impaired salivary gland function.

67Ga scan was performed 48 h after venous injection of 111 MBq of 67Ga citrate. Images were also acquired in the anterior, right anterior oblique and left anterior oblique projections of the head for 600 s. After acquisition, 67Ga uptake in the salivary glands was qualitatively evaluated by the two blinded Nuclear Medicine physicians and considered as physiologic or increased. In case of discordance, the final decision was obtained by consensus.

Statistical analysis

Fischer's exact test was utilized to compare major salivary glands scintigraphic data following HPCT to prior transplant and Wilcoxon rank-sum test was used to compare salivary flow rates following HPCT to rates observed prior transplant.

Results

Oral clinical evaluation

Two out of 16 patients on Day +30, six out of 15 on Day +60 and six out of 14 on Day +100 presented mucosal erythema and xerostomia following HPCT. One patient developed cGVHD previously Day +100. Two patients developed clinically and microbiologically documented oral candidiasis between Days+30 and +60; these patients were treated with oral Fluconazole, according to Institutional protocol.

Clinical evaluation of xerostomia on Day +100

Seven out of 14 patients complained of xerostomia and all exhibited associated clinical features such as difficulties with food intake and oral erythema. One patient developed cGVHD and presented severe hyposalivation prior to Day +100.

Unstimulated salivary flow rates

Prior to HPCT::

median 0.76 ml/min (range: 0.69–4.72 ml/min);

Day +30::

median 0.69 ml/min (range: 0.46–1.96 ml/min);

Day +60::

median 0.67 ml/min (range: 0.30–1.68 ml/min);

Day +100::

median 0.65 ml/min (range: 0.30–120 ml/min).

99mTc-pertechnetate uptake prior and following HPCT

Prior to HPCT::

Three patients showed decreased uptake in the parotid glands and four patients in the submandibular glands;

Day +30::

Three patients showed decreased of uptake in the parotid glands and eight patients in the submandibular glands;

Day +60::

Two patients showed decreased of uptake in the parotid glands and five patients in the submandibular glands;

Day +100::

Two patients showed decreased uptake in the parotid glands and two patients in the submandibular glands.

Four patients died before Day +30, more 1 before Day +60 and more one before Day +100. Overall six patients were not evaluated during the study.

Range of 99mTc-pertechnetate clearance prior and following HPCT

Prior HPCT::

the clearance ranged between 50 and 60% (left parotid: 60%; right parotid: 56.5%; left submandibular 51.16% and right submandibular: 50%);

Day +30::

the clearance ranged between 40.3 and 60% (left parotid: 66%; right parotid: 59.5%; left submandibular 40.34% and right submandibular: 41.5%);

Day +60::

the clearance between 37 and 66% (left parotid: 66%; right parotid: 62.45%; left submandibular: 37% and right submandibular: 37%);

Day +100::

the clearance ranged between 43 and 64% (left parotid: 61.66%; right parotid: 64%; left submandibular: 43% and right submandibular: 45.5%).

67Ga scan

Table 2 shows the uptake of 67Ga prior and following HPCT.

Prior to HPCT::

one patient showed increased uptake in the submandibular glands, while no increase was observed in the parotid glands;

Day +30::

Three patients showed increased uptake in the parotid glands and six patients demonstrated an uptake increase in the submandibular glands;

Day +60::

Three patients showed increased uptake in parotid glands and seven patients in the submandibular glands;

Day +100::

Five patients showed increased uptake in parotid glands and eight patients in the submandibular glands.

Table 2 Results of uptake of 67Ga in major salivary glands

No statistical significance was observed in any of the comparative analyses.

Discussion

Intact salivary gland function is essential to preserving oral health.16, 17 Hyposalivation refers to decreased measurable output of saliva; patients typically but not always complain of the symptom of dry mouth.17 It has not been clearly determined whether the subjective complaint of xerostomia in adults reflects actual salivary gland functional compromise.3

Adequate delivery to oral tissue minimizes or prevents xerostomia if the flow rate exceeds the rate of fluid loss by mucosal absorption and evaporation.18 Oral dryness occurred when the total salivary flow rate has been reduced to just <50% of normal.18 In the current study, although seven out of 14 (50%) of our patients complained xerostomia at Day +100, this study was not able to confirm the reduction of salivary flow rate.

The study data suggest an alteration in major salivary gland function following HPCT with a possible recovery on Day +100. These findings were reported previously.2 Patients in the current study presented a decreased unstimulated salivary output approximately Day +30. Despite that, apparent functional recovery was observed, on Days +60 and +100. However, no statistical significance was evident.

Functional compromise of the major salivary gland can be difficult to evaluate clinically. In transplanted patients it is a critical issue and may have influence in quality of life and infections complications. Trials, particularly in pediatric HPCT using 99mTc-pertechnetate were published.11, 19, 20 The scintigraphy may help us to clarify damage to the major salivary gland. However, few published studies have utilized salivary gland scintigraphy to assess salivary gland function in the early period following HPCT. Dynamic scintigraphy utilizes acquisition of images throughout the period of the scan reflecting blood flow to head and neck, perfusion, concentration, activation and clearance of salivary glands. 99mTc-pertechnetate uptake and clearance by major salivary glands correlate with salivary flow rates.21 It is like that 99mTc-pertechnetate uptake can be changed whether there is secretor unit damage. Concurrently, clearance may be reflecting a non-secretory ability, due to any drug use or compromised central nervous system stimulation. In this study, no difference was observed for uptake and/or clearance of 99mTc-pertechnetate in major salivary glands when compared to salivary flow rate. Moreover, submandibular glands presented evidence of major dysfunction characterized by 99mTc-pertechnetate and 67Ga scintigraphic images. This compromise of glandular function could quite possibly be responsible for the xerostomia as one of the major patients’ complaint following HPCT.2, 22

67Ga scans revealed an increase of inflammatory cell infiltrate on the submandibular glands when compared with findings associated with the parotid glands. The conditioning regimen can cause the release of inflammatory mediators IL-10, IL-6 and IL-2 in tissue.23 IL-2 may be responsible to decrease of function of parotid and submandibular glands.24 Nagler et al. using IL-2-based immunotherapy following HPCT demonstrated a major salivary gland dysfunction similar to chronic GVHD.24 In the current study, the increased uptake of 67Ga was observed on Day +30 and may be persistent up to Day +100. These data suggest a cumulative effect of conditioning regimen that may be responsible to patients’ complaint of xerostomia.

Salivary gland dysfunction is more pronounced in patients developing clinical GVHD; furthermore, degree of impairment positively correlated with severity of GVHD.2, 25, 26, 27, 28, 29, 30 Only one patient in the present study developed chronic GVHD, showing images patterns like described previously.19, 27, 28, 31, 32, 33 Major salivary glands showed uptake and clearance decreased, in association with the severe inflammatory process.

Results of USSFR suggest that on Day +100 the USSFR continue to decrease comparing to prior HPCT and may be due to persistent inflammatory cell infiltrate as demonstrated with 67Ga scans.

Owing to the relatively small sample size of this study, further research is needed to fully address the questions concerning the relationship between early major salivary glands dysfunction and HPCT.

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Acknowledgements

We gratefully acknowledge the support and contributions of the staff of the Nuclear Medicine Service of University Hospital – Campinas State University, HPCT Unit. The expert data management by Mrs Eliana Miranda and Mrs Amanda Margelo is also sincerely appreciated. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP-(The State of São Paulo Research Foundation) proc. 00/12188-4.

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Correspondence to C A De Souza.

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Coracin, F., Pizzigatti Correa, M., Camargo, E. et al. Major salivary gland damage in allogeneic hematopoietic progenitor cell transplantation assessed by scintigraphic methods. Bone Marrow Transplant 37, 955–959 (2006). https://doi.org/10.1038/sj.bmt.1705351

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Keywords

  • major salivary glands
  • HPCT
  • 99mTc-pertechnetate and gallium-67 scintigraphy

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