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Immunosuppression and lung cancer of donor origin after bilateral lung transplantation
Lung Cancer 76 (2012) 118–122
Contents lists available at SciVerse ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Case report
Immunosuppression and lung cancer of donor origin after bilateral lung transplantation Lotta von Boehmer a,∗,1 , Alice Draenert b,1 , Wolfgang Jungraithmayr b , Ilhan Inci b , Schäfer Niklaus a , Annette Boehler c , Markus Hofer c , Rolf Stahel a , Alex Soltermann d , Maries van den Broek a , Walter Weder b,1 , Alexander Knuth a,1 a
Medical Oncology, University Hospital Zurich, Switzerland Thoracic Surgery, University Hospital Zurich, Switzerland c Pneumology, University Hospital Zurich, Switzerland d Surgical Pathology, University Hospital Zurich, Switzerland b
a r t i c l e
i n f o
Article history: Received 18 June 2011 Received in revised form 29 September 2011 Accepted 2 October 2011 Keywords: Lung transplantation Immunosuppression Non-small cell lung cancer Immunosurveillance Tumor infiltrating lymphocytes
a b s t r a c t Analysis of databases from transplant recipients revealed a 3–5 fold higher risk to develop de novo malignancies under continued immunosuppression. The underlying mechanisms are poorly understood. Here we describe a patient who received a bilateral lung transplantation for end-stage ‘Usual Interstitial Pneumonia’ (UIP) resulting in idiopathic lung fibrosis. The recipient presented with a non-small cell lung carcinoma (NSCLC) in the donor lung 7 months later. Molecular and immunological typing of the tumor revealed a cancer of donor origin with a prominent intratumoral immune cell infiltrate without detectable effector function. This is a unique case of de novo outgrowth of a NSCLC of donor origin under continued immunosuppression, supporting the concept of tumor immunosurveillance in vivo. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Advances in transplantation medicine have resulted in improved overall survival of organ transplant recipients. With longer survival, chronic complications, including the development of secondary malignancies, are more likely to occur. Analysis of databases from transplant recipients showed that the risk to develop de novo malignancies is 3–5 fold higher in organ transplant recipients under continued immunosuppression [1–3]. Over the past 20 years, non-small cell lung carcinoma (NSCLC) development has been described in the setting of single lung transplantation. The carcinoma was found in the recipient’s native lung [4]. After bilateral lung transplantation, there is one report of de novo NSCLC of recipient’s origin [6]. A case of donor acquired small cell lung carcinoma (SCLC) has been described in the setting of bilateral lung transplantation [5].
Here we describe a patient, who received a bilateral lung transplant as treatment for end-stage ‘Usual Interstitial Pneumonia’ (UIP) resulting in ‘Idiopathic Lung Fibrosis’. The recipient developed a non-small cell lung carcinoma in the donor lung 7 months later. Molecular and immunological typing of the tumor revealed a cancer of donor origin with a prominent intratumoral immune cell infiltrate deficient of any detectable effector function. 2. Materials and methods 2.1. Laboratory parameters Retrospective analysis of hospital electronic laboratory records was performed. Hematological, biochemical and coagulation samples were obtained. 2.2. Molecular typing
∗ Corresponding author at: Medical Oncology, University Hospital Zurich, Raemistrasse 100, 8091 Zürich, Switzerland. Tel.: +41 44 255 97 79; fax: +41 44 255 97 80. E-mail address: [email protected] (L. von Boehmer). 1 Equally contributed. 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.10.001
An representative paraffin block containing tumor tissue was selected for analysis after reviewing the hematoxilin-eosin (HE) stained slides. The tumor content of the slides was evaluated by an experienced pathologist. Punch extracted tissue was then used for DNA extraction using Qiagen QIAamp DNA Mini Kit. DNA yield and purity was determined by standard methods and stored at 4 ◦ C
L. von Boehmer et al. / Lung Cancer 76 (2012) 118–122
until amplification by SSP. Tissue-typing by SSP was done using the GenoVision Kit according to the manufacturer’s instructions. The amplified products were visualized on 2% agarose gel in 0.5 × TBE buffer.
2.3. Immunohistochemistry Three m thick sections of formalin-fixed, paraffin-embedded tissues were mounted on glass slides, deparaffinized, rehydrated and stained with hematoxylin-eosin using standard histological techniques. For immunohistochemical staining, the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, AZ) and Ventana reagents were used. After deparaffinization in xylene, slides were rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase was blocked using the Ventana endogenous peroxidase blocking kit after a rinse with distilled water. For antigen retrieval, slides were heated with cell conditioning solution (CC1, Ventana) according to the manufacturer’s instructions. Primary antibodies against CD4 (clone 1F6, dilution: 1:30, Novocastra, Newcastle Upon Tyne, UK), CD8 (clone C8/114B, dilution: 1:100, DAKO), perforin (clone 5B10, dilution: 1:20, Novocastra Laboratories LTD), granzyme (clone Gr B-7, dilution: 1:25, DAKO), FoxP3 (code ab10563, dilution: 1:400, Abcam) and MHC-I (2-microglobulin) (clone 2l, dilution: 1:1000, RDI Research Diagnostic Inc.) were applied adjusted to the Ventana Benchmark system after performing titrations. iVIEW-DAB was used as chromogen. For each section, sixteen high power fields (16 × 100 m2 ) were identified randomly. The amount of stained cells in the sixteen fields was averaged. Areas of normal lung, scar tissue, necrosis and clusters of anthracotic pigment were excluded from the analysis.
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3. Results 3.1. Patient’s history A 61 year-old Caucasian woman with end-stage UIP underwent bilateral, sequential lung transplantation through bilateral anterio-lateral thoracotomy in September 2008. The patient had a 20 pack-year history of smoking, suspended twenty years earlier, a history of tuberculosis lymphadenitis at the age of 26, treated by 6 months of tuberculostatic drugs and an early stage cervical cancer at the age of 41, cured by surgery. The donor lung was obtained from a 60 year-old Caucasian woman, a non-smoker, who was healthy except for type II diabetes mellitus. The arterial pO2 prior to explantation of the donor lung was on 100% oxygen up to 400 mmHg and preoperative imaging showed no abnormalities. The recipient’s clinical course after transplantation was without any complications. She was extubated one day after surgery and was transferred from the ICU to the regular ward on the second day after transplantation. She had a low-risk status with regard to CMV, EBV and Toxoplasma, as determined by standard procedures. Immunosuppression was started with cyclosporine, mycophenolat and steroids and she was discharged from the hospital 4 weeks after transplantation. Surveillance bronchoscopy before discharge showed unremarkable anastomoses and mild signs of acute rejection (ISHLT-classification A1, B0 [7]). There was no evidence of pathologic microorganisms in the bronchoalveolar lavage fluid. Surveillance bronchoscopy, transbronchial biopsy and lung function tests were carried out once per month for about 6 months and revealed unremarkable results. Signs of rejection (ISHLT A0, B0) had resolved, and no dysplasia or malignancy was seen, except for a generalised epithelial metaplasia and mild inflammatory reaction.
Fig. 1. PET-CT. Seven months after bilateral lung transplantation a PET-CT analysis was performed. (a,b) Two intrapulmonary nodules in the middle lobe (red arrows). (c) The large right-sided paracarinar lymph node with high FDG accumulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Post-transplantation bronchitis was diagnosed after 6.5 months and treated with antibiotics. One month later the patient still showed a slightly decreased lung function (FEV1) as compared to previous values. At that time the patient refused a bronchoscopic biopsy, but agreed to perform a CT scan to detect any indications of chronic rejection. The CT scan revealed a large right-sided paracarinar lymph node of 6 cm in diameter and two small intrapulmonary nodules in the middle lobe, all of which suggested a malignant process. The following PET-CT scan showed strong FDG positivity in the lymph node region with a SUV (standardized uptake value) max of 10.7 (Fig. 1). Histopathology revealed a large cell carcinoma (LC) with partial sarcomatoid differentiation, which was staged as cT4 based on re-evaluation of the CT scan. At that time the immunosuppression consisted of cyclosporine with a target CO /C2 level of 100/700 g/l, mycophenolat 2 × 1 g and prednisone 7.5 mg. We decided with the patient to start neoadjuvant chemotherapy with pemetrexed (500 mg/m2 ) combined with carboplatin (75 mg/m2 ), as a precaution due to the nephrotoxic effect of cisplatin and cyclosporine. Under chemotherapy immunosuppression was reduced to the cyclosporine target level of 100/600 g/l, mycophenolat to 2 × 250–500 mg and prednisone was increased to 10 mg. After 3 cycles the SUV in the PET-CT decreased to a maximum of 6.7 with a partial metabolic response and stable morphology. The patient was subjected to an extended right-sided pneumonectomy with partial resection of the vena cava superior. The primary postoperative FEV1 was 0.91 l. The postsurgical recovery was uneventful except for weight loss of 6 kg over 3 months and a reduced physical performance status (Karnofsky 80%). The pathological staining after the resection was ypT4 ypN2 (2/7), stage IIIA. A follow-up PET-CT after 4 months revealed multiple metastatic lesions in the bone and a new lesion in the superior lingua of the left lung. The patient died 7 months after initial diagnosis.
Fig. 2. SSP Genovision analysis of tumor DNA. HLA haplotype analysis of the tumor DNA revealed expression of HLA-DR11 (shown here), which was only expressed by the donor. The graft recipient expressed HLA-A2, -A29(19), -B7, -B44(12), -DR14(6), -DR7, whereas the donor expressed HLA-A1, -A23(9), -B8, -B49(21), -DR3, -DR11(5).
B8, -B49(21), -DR3, -DR11(5). HLA haplotype analysis of the tumor DNA revealed expression of HLA-DR11, which was only expressed by the donor (Figs. 2 and 3). 3.3. Laboratory parameters Laboratory findings included normal blood values for AST, ALT, gGT and bilirubin, the creatinine remained stable at a high level of 140 mol/l (Ref.: 44–80 mol/l) before and after transplantation. The haemoglobin stabilized to 10 g/dl (Ref.: 11.7–15.3 g/dl) with a persisting lymphopenia (<1.5 × 103 lymphocytes/l) after transplantation. 3.4. Peri- and intratumoral immune infiltration Immunohistochemical analysis of the tumor revealed a dense peri- and intratumoral infiltration by CD8+ and CD4+ T cells (Fig. 4a and b). The tumor expressed MHC class I (Fig. 4c), which is a prerequisite for recognition by CD8+ T cells. The CD8+ T cells, however, did not express the effector molecules perforin and granzyme B (Figs. 4d, e and 5) and thus appeared deficient of T lymphocyte effector function. FoxP3+ T cells were present in the tumor tissue (Fig. 4f).
3.2. HLA haplotype analysis 4. Discussion The tumor was typed by HLA haplotype analysis (SSP, GenoVision, Oslo, Norway) and was identical with the organ donor. The graft recipient expressed HLA-A2, -A29(19), -B7, -B44(12), -DR14(6), -DR7, whereas the donor expressed HLA-A1, -A23(9), -
There is accumulating evidence for immunological editing and control of cancer in the non-immunocompromised host [11,12]. Immunosuppression in association with organ transplantation is
Fig. 3. Absolute lymphocyte count. Lymphocyte count was determined by standard methods over time.
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Fig. 4. Immunohistochemical analysis of the tumor. (a) Cytotoxic CD8+ T cells staining, 20X, (b) helper CD4+ T cells staining, 20X, (c) MHC-I staining, MHC-I is required for CD8+ T cell activation, 20X, (d) perforin staining, perforin is produced in CD8+ T cells upon activation, 20X, (e) granzyme B staining, granzyme B also is produced in CD8+ T cells upon activation, 20X, and (f) FoxP3 staining, FoxP3 is expressed in CD4+ regulatory T cells, 20X.
Fig. 5. Quantification of CD8-, perforin- and granzyme B- positive cells in the tumor. For each section, sixteen random high power fields (HPF, 16 × 100 m2 ) were evaluated and the average number ± SD of positive cells per HPF is shown. Areas of normal lung, scar tissue, necrosis and clusters of anthracotic pigment were excluded from the analysis.
required to protect the graft from rejection but concomitantly compromises immunological control mechanisms against infectious diseases and cancer [8,9]. This report describes a patient, who received a bilateral lung transplantation and developed NSCLC 7 months later. The tumor was found to be donor-derived as determined by HLA haplotype analysis. During chemotherapy and thereafter the immunosuppression was reduced first to reduce the risk of infection due to additional immunosuppression arising through chemotherapy and second to attempt a host versus tumor effect. In the PET-CT the patient had no objective morphological tumor response but a moderate reduction in FDG-uptake after 3 cycles of neoadjuvant chemotherapy. In the surgical specimen, despite a prominent immune infiltrate in the tumor, the latter was not rejected, likely to be explained by the detected lack of effector function of infiltrating CD8+ T cells as well as the presence of regulatory FoxP3+ T cells. In most cases, cancer in immunosuppressed organ transplant recipients arises from cells of recipient origin (most commonly skin cancer or lymphoma). Donor-derived cancers arising in the host have been described in the literature [5,13–15]. In such cases, it is suggested that before transplantation outgrowth of occult tumor cells was controlled by the donor’s uncompromised immune system and under immunosuppressive therapy minimal residual disease, early stage cancer or premalignant conditions may have an advantage for outgrowth in the recipient.
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Cancer chemotherapy, radiotherapy and surgery are standard treatment options for patients with NSCLC. The treatment of NSCLC in transplanted patients has not been investigated. Post-transplant lymphoproliferative disease (PTLD) is a common heterogeneous group of lymphoid neoplasms following solid organ transplantation. Standard treatment for early lesions of PTLD is reduction of immunosuppression. Recommendations for the treatment of donor-related melanoma also includes withdrawal of immunosuppression and explantation of the allograft. In cancer patients with life-sustaining organ transplantation, withdrawal of immunosuppression and graft removal is not feasible. Reduction of immunosuppression, urgent retransplantation and/or standard cancer treatment are the only possible salvage strategies. In clinical practice success with standard treatment, e.g. radiotherapy or chemotherapy, in immunosuppressed cancer patients is rare. In mouse models it has been shown that the efficacy of radiotherapy [16] and chemotherapy [17] depends on intact immune effector functions. This report supports that the lack of efficacy may be explained by the missing help from the immune system to effectively control cancer. Current protocols of nontargeted immunosuppression are clearly insufficient for prevention and treatment of cancer in vital organ transplanted patients. 4 classes of immunosuppressive agents are used in lung transplant patients: corticosteroids, calcineurin inhibitors, inhibitors of nucleotide biosynthesis and mTOR inhibitors. Interestingly, mTOR inhibitors have both immunosuppressive and anti-cancer effects [10]. They have shown promise in maintenance immunosuppressive regimens in lung transplantation but further studies are needed to identify the optimal way to use these agents. Conflict of interest The authors state no conflict of interest. Acknowledgment We thank Ms. Barbara Rüsi for technical assistance. This work was supported by Grants from the Cancer Research Institute, the Science Foundation for Oncology (SFO, Zurich), the Hanne
Liebermann Foundation (Zurich) and the Dr. Leopold and Carmen Ellinger Foundation Zurich. References [1] Hoover R, Fraumeni Jr JF. Risk of cancer in renal-transplant recipients. Lancet 1973;2:55–7. [2] Kinlen LJ, Sheil AG, Peto J, Doll R. Collaborative United Kingdom – Australasian study of cancer in patients treated with immunosuppressive drugs. Br Med J 1979;2:1461–6. [3] Lindelof B, Sigurgeirsson B, Gabel H, Stern RS. Incidence of skin cancer in 5356 patients following organ transplantation. Br J Dermatol 2000;143:513–9. [4] Picard C, Grenet D, Copie-Bergman C, Martin N, Longchampt E, Zemoura L, et al. Small-cell lung carcinoma of recipient origin after bilateral lung transplantation for cystic fibrosis. J Heart Lung Transplant 2006;25:981–4. [5] De Soyza AG, Dark JH, Parums DV, Curtis A, Corris PA. Donor-acquired small cell lung cancer following pulmonary transplantation. Chest 2001;120:1030–1. [6] Beyer EA, DeCamp MM, Smedira NG, Farver C, Mehta A, Warshawsky I. Primary adenocarcinoma in a donor lung: evaluation and surgical management. J Heart Lung Transplant 2003;22:1174–7. [7] Stewart S, Fishbein MC, Snell GI, Berry GJ, Boehler A, Burke MM, et al. Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant 2007;26:1229–42. [8] Soulillou JP, Giral M. Controlling the incidence of infection and malignancy by modifying immunosuppression. Transplantation 2001;72:S89–93. [9] Dantal J, Hourmant M, Cantarovich D, Giral M, Blancho G, Dreno B, et al. Effect of long-term immunosuppression in kidney-graft recipients on cancer incidence: randomised comparison of two cyclosporin regimens. Lancet 1998;351:623–8. [10] Campistol JM, Gutierrez-Dalmau A, Torregrosa JV. Conversion to sirolimus: a successful treatment for posttransplantation Kaposi’s sarcoma. Transplantation 2004;77:760–2. [11] Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 2002;3:991–8. [12] Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 2011;331:1565–70. [13] Myron Kauffman H, McBride MA, Cherikh WS, Spain PC, Marks WH, Roza AM. Transplant tumor registry: donor related malignancies. Transplantation 2002;74:358–62. [14] Wilson RE, Hager EB, Hampers CL, Corson JM, Merrill JP, Murray JE. Immunologic rejection of human cancer transplanted with a renal allograft. N Engl J Med 1968;278:479–83. [15] MacKie RM, Reid R, Junor B. Fatal melanoma transferred in a donated kidney 16 years after melanoma surgery. N Engl J Med 2003;348:567–8. [16] Burnette BC, Liang H, Lee Y, Chlewicki L, Khodarev NN, Weichselbaum RR, et al. The efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity. Cancer Res 2011;71:2488–96. [17] Zitvogel L, Kepp O, Kroemer G. Immune parameters affecting the efficacy of chemotherapeutic regimens. Nat Rev Clin Oncol 2011;8:151–60.
Contents lists available at SciVerse ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Case report
Immunosuppression and lung cancer of donor origin after bilateral lung transplantation Lotta von Boehmer a,∗,1 , Alice Draenert b,1 , Wolfgang Jungraithmayr b , Ilhan Inci b , Schäfer Niklaus a , Annette Boehler c , Markus Hofer c , Rolf Stahel a , Alex Soltermann d , Maries van den Broek a , Walter Weder b,1 , Alexander Knuth a,1 a
Medical Oncology, University Hospital Zurich, Switzerland Thoracic Surgery, University Hospital Zurich, Switzerland c Pneumology, University Hospital Zurich, Switzerland d Surgical Pathology, University Hospital Zurich, Switzerland b
a r t i c l e
i n f o
Article history: Received 18 June 2011 Received in revised form 29 September 2011 Accepted 2 October 2011 Keywords: Lung transplantation Immunosuppression Non-small cell lung cancer Immunosurveillance Tumor infiltrating lymphocytes
a b s t r a c t Analysis of databases from transplant recipients revealed a 3–5 fold higher risk to develop de novo malignancies under continued immunosuppression. The underlying mechanisms are poorly understood. Here we describe a patient who received a bilateral lung transplantation for end-stage ‘Usual Interstitial Pneumonia’ (UIP) resulting in idiopathic lung fibrosis. The recipient presented with a non-small cell lung carcinoma (NSCLC) in the donor lung 7 months later. Molecular and immunological typing of the tumor revealed a cancer of donor origin with a prominent intratumoral immune cell infiltrate without detectable effector function. This is a unique case of de novo outgrowth of a NSCLC of donor origin under continued immunosuppression, supporting the concept of tumor immunosurveillance in vivo. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Advances in transplantation medicine have resulted in improved overall survival of organ transplant recipients. With longer survival, chronic complications, including the development of secondary malignancies, are more likely to occur. Analysis of databases from transplant recipients showed that the risk to develop de novo malignancies is 3–5 fold higher in organ transplant recipients under continued immunosuppression [1–3]. Over the past 20 years, non-small cell lung carcinoma (NSCLC) development has been described in the setting of single lung transplantation. The carcinoma was found in the recipient’s native lung [4]. After bilateral lung transplantation, there is one report of de novo NSCLC of recipient’s origin [6]. A case of donor acquired small cell lung carcinoma (SCLC) has been described in the setting of bilateral lung transplantation [5].
Here we describe a patient, who received a bilateral lung transplant as treatment for end-stage ‘Usual Interstitial Pneumonia’ (UIP) resulting in ‘Idiopathic Lung Fibrosis’. The recipient developed a non-small cell lung carcinoma in the donor lung 7 months later. Molecular and immunological typing of the tumor revealed a cancer of donor origin with a prominent intratumoral immune cell infiltrate deficient of any detectable effector function. 2. Materials and methods 2.1. Laboratory parameters Retrospective analysis of hospital electronic laboratory records was performed. Hematological, biochemical and coagulation samples were obtained. 2.2. Molecular typing
∗ Corresponding author at: Medical Oncology, University Hospital Zurich, Raemistrasse 100, 8091 Zürich, Switzerland. Tel.: +41 44 255 97 79; fax: +41 44 255 97 80. E-mail address: [email protected] (L. von Boehmer). 1 Equally contributed. 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.10.001
An representative paraffin block containing tumor tissue was selected for analysis after reviewing the hematoxilin-eosin (HE) stained slides. The tumor content of the slides was evaluated by an experienced pathologist. Punch extracted tissue was then used for DNA extraction using Qiagen QIAamp DNA Mini Kit. DNA yield and purity was determined by standard methods and stored at 4 ◦ C
L. von Boehmer et al. / Lung Cancer 76 (2012) 118–122
until amplification by SSP. Tissue-typing by SSP was done using the GenoVision Kit according to the manufacturer’s instructions. The amplified products were visualized on 2% agarose gel in 0.5 × TBE buffer.
2.3. Immunohistochemistry Three m thick sections of formalin-fixed, paraffin-embedded tissues were mounted on glass slides, deparaffinized, rehydrated and stained with hematoxylin-eosin using standard histological techniques. For immunohistochemical staining, the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, AZ) and Ventana reagents were used. After deparaffinization in xylene, slides were rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase was blocked using the Ventana endogenous peroxidase blocking kit after a rinse with distilled water. For antigen retrieval, slides were heated with cell conditioning solution (CC1, Ventana) according to the manufacturer’s instructions. Primary antibodies against CD4 (clone 1F6, dilution: 1:30, Novocastra, Newcastle Upon Tyne, UK), CD8 (clone C8/114B, dilution: 1:100, DAKO), perforin (clone 5B10, dilution: 1:20, Novocastra Laboratories LTD), granzyme (clone Gr B-7, dilution: 1:25, DAKO), FoxP3 (code ab10563, dilution: 1:400, Abcam) and MHC-I (2-microglobulin) (clone 2l, dilution: 1:1000, RDI Research Diagnostic Inc.) were applied adjusted to the Ventana Benchmark system after performing titrations. iVIEW-DAB was used as chromogen. For each section, sixteen high power fields (16 × 100 m2 ) were identified randomly. The amount of stained cells in the sixteen fields was averaged. Areas of normal lung, scar tissue, necrosis and clusters of anthracotic pigment were excluded from the analysis.
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3. Results 3.1. Patient’s history A 61 year-old Caucasian woman with end-stage UIP underwent bilateral, sequential lung transplantation through bilateral anterio-lateral thoracotomy in September 2008. The patient had a 20 pack-year history of smoking, suspended twenty years earlier, a history of tuberculosis lymphadenitis at the age of 26, treated by 6 months of tuberculostatic drugs and an early stage cervical cancer at the age of 41, cured by surgery. The donor lung was obtained from a 60 year-old Caucasian woman, a non-smoker, who was healthy except for type II diabetes mellitus. The arterial pO2 prior to explantation of the donor lung was on 100% oxygen up to 400 mmHg and preoperative imaging showed no abnormalities. The recipient’s clinical course after transplantation was without any complications. She was extubated one day after surgery and was transferred from the ICU to the regular ward on the second day after transplantation. She had a low-risk status with regard to CMV, EBV and Toxoplasma, as determined by standard procedures. Immunosuppression was started with cyclosporine, mycophenolat and steroids and she was discharged from the hospital 4 weeks after transplantation. Surveillance bronchoscopy before discharge showed unremarkable anastomoses and mild signs of acute rejection (ISHLT-classification A1, B0 [7]). There was no evidence of pathologic microorganisms in the bronchoalveolar lavage fluid. Surveillance bronchoscopy, transbronchial biopsy and lung function tests were carried out once per month for about 6 months and revealed unremarkable results. Signs of rejection (ISHLT A0, B0) had resolved, and no dysplasia or malignancy was seen, except for a generalised epithelial metaplasia and mild inflammatory reaction.
Fig. 1. PET-CT. Seven months after bilateral lung transplantation a PET-CT analysis was performed. (a,b) Two intrapulmonary nodules in the middle lobe (red arrows). (c) The large right-sided paracarinar lymph node with high FDG accumulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Post-transplantation bronchitis was diagnosed after 6.5 months and treated with antibiotics. One month later the patient still showed a slightly decreased lung function (FEV1) as compared to previous values. At that time the patient refused a bronchoscopic biopsy, but agreed to perform a CT scan to detect any indications of chronic rejection. The CT scan revealed a large right-sided paracarinar lymph node of 6 cm in diameter and two small intrapulmonary nodules in the middle lobe, all of which suggested a malignant process. The following PET-CT scan showed strong FDG positivity in the lymph node region with a SUV (standardized uptake value) max of 10.7 (Fig. 1). Histopathology revealed a large cell carcinoma (LC) with partial sarcomatoid differentiation, which was staged as cT4 based on re-evaluation of the CT scan. At that time the immunosuppression consisted of cyclosporine with a target CO /C2 level of 100/700 g/l, mycophenolat 2 × 1 g and prednisone 7.5 mg. We decided with the patient to start neoadjuvant chemotherapy with pemetrexed (500 mg/m2 ) combined with carboplatin (75 mg/m2 ), as a precaution due to the nephrotoxic effect of cisplatin and cyclosporine. Under chemotherapy immunosuppression was reduced to the cyclosporine target level of 100/600 g/l, mycophenolat to 2 × 250–500 mg and prednisone was increased to 10 mg. After 3 cycles the SUV in the PET-CT decreased to a maximum of 6.7 with a partial metabolic response and stable morphology. The patient was subjected to an extended right-sided pneumonectomy with partial resection of the vena cava superior. The primary postoperative FEV1 was 0.91 l. The postsurgical recovery was uneventful except for weight loss of 6 kg over 3 months and a reduced physical performance status (Karnofsky 80%). The pathological staining after the resection was ypT4 ypN2 (2/7), stage IIIA. A follow-up PET-CT after 4 months revealed multiple metastatic lesions in the bone and a new lesion in the superior lingua of the left lung. The patient died 7 months after initial diagnosis.
Fig. 2. SSP Genovision analysis of tumor DNA. HLA haplotype analysis of the tumor DNA revealed expression of HLA-DR11 (shown here), which was only expressed by the donor. The graft recipient expressed HLA-A2, -A29(19), -B7, -B44(12), -DR14(6), -DR7, whereas the donor expressed HLA-A1, -A23(9), -B8, -B49(21), -DR3, -DR11(5).
B8, -B49(21), -DR3, -DR11(5). HLA haplotype analysis of the tumor DNA revealed expression of HLA-DR11, which was only expressed by the donor (Figs. 2 and 3). 3.3. Laboratory parameters Laboratory findings included normal blood values for AST, ALT, gGT and bilirubin, the creatinine remained stable at a high level of 140 mol/l (Ref.: 44–80 mol/l) before and after transplantation. The haemoglobin stabilized to 10 g/dl (Ref.: 11.7–15.3 g/dl) with a persisting lymphopenia (<1.5 × 103 lymphocytes/l) after transplantation. 3.4. Peri- and intratumoral immune infiltration Immunohistochemical analysis of the tumor revealed a dense peri- and intratumoral infiltration by CD8+ and CD4+ T cells (Fig. 4a and b). The tumor expressed MHC class I (Fig. 4c), which is a prerequisite for recognition by CD8+ T cells. The CD8+ T cells, however, did not express the effector molecules perforin and granzyme B (Figs. 4d, e and 5) and thus appeared deficient of T lymphocyte effector function. FoxP3+ T cells were present in the tumor tissue (Fig. 4f).
3.2. HLA haplotype analysis 4. Discussion The tumor was typed by HLA haplotype analysis (SSP, GenoVision, Oslo, Norway) and was identical with the organ donor. The graft recipient expressed HLA-A2, -A29(19), -B7, -B44(12), -DR14(6), -DR7, whereas the donor expressed HLA-A1, -A23(9), -
There is accumulating evidence for immunological editing and control of cancer in the non-immunocompromised host [11,12]. Immunosuppression in association with organ transplantation is
Fig. 3. Absolute lymphocyte count. Lymphocyte count was determined by standard methods over time.
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Fig. 4. Immunohistochemical analysis of the tumor. (a) Cytotoxic CD8+ T cells staining, 20X, (b) helper CD4+ T cells staining, 20X, (c) MHC-I staining, MHC-I is required for CD8+ T cell activation, 20X, (d) perforin staining, perforin is produced in CD8+ T cells upon activation, 20X, (e) granzyme B staining, granzyme B also is produced in CD8+ T cells upon activation, 20X, and (f) FoxP3 staining, FoxP3 is expressed in CD4+ regulatory T cells, 20X.
Fig. 5. Quantification of CD8-, perforin- and granzyme B- positive cells in the tumor. For each section, sixteen random high power fields (HPF, 16 × 100 m2 ) were evaluated and the average number ± SD of positive cells per HPF is shown. Areas of normal lung, scar tissue, necrosis and clusters of anthracotic pigment were excluded from the analysis.
required to protect the graft from rejection but concomitantly compromises immunological control mechanisms against infectious diseases and cancer [8,9]. This report describes a patient, who received a bilateral lung transplantation and developed NSCLC 7 months later. The tumor was found to be donor-derived as determined by HLA haplotype analysis. During chemotherapy and thereafter the immunosuppression was reduced first to reduce the risk of infection due to additional immunosuppression arising through chemotherapy and second to attempt a host versus tumor effect. In the PET-CT the patient had no objective morphological tumor response but a moderate reduction in FDG-uptake after 3 cycles of neoadjuvant chemotherapy. In the surgical specimen, despite a prominent immune infiltrate in the tumor, the latter was not rejected, likely to be explained by the detected lack of effector function of infiltrating CD8+ T cells as well as the presence of regulatory FoxP3+ T cells. In most cases, cancer in immunosuppressed organ transplant recipients arises from cells of recipient origin (most commonly skin cancer or lymphoma). Donor-derived cancers arising in the host have been described in the literature [5,13–15]. In such cases, it is suggested that before transplantation outgrowth of occult tumor cells was controlled by the donor’s uncompromised immune system and under immunosuppressive therapy minimal residual disease, early stage cancer or premalignant conditions may have an advantage for outgrowth in the recipient.
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Cancer chemotherapy, radiotherapy and surgery are standard treatment options for patients with NSCLC. The treatment of NSCLC in transplanted patients has not been investigated. Post-transplant lymphoproliferative disease (PTLD) is a common heterogeneous group of lymphoid neoplasms following solid organ transplantation. Standard treatment for early lesions of PTLD is reduction of immunosuppression. Recommendations for the treatment of donor-related melanoma also includes withdrawal of immunosuppression and explantation of the allograft. In cancer patients with life-sustaining organ transplantation, withdrawal of immunosuppression and graft removal is not feasible. Reduction of immunosuppression, urgent retransplantation and/or standard cancer treatment are the only possible salvage strategies. In clinical practice success with standard treatment, e.g. radiotherapy or chemotherapy, in immunosuppressed cancer patients is rare. In mouse models it has been shown that the efficacy of radiotherapy [16] and chemotherapy [17] depends on intact immune effector functions. This report supports that the lack of efficacy may be explained by the missing help from the immune system to effectively control cancer. Current protocols of nontargeted immunosuppression are clearly insufficient for prevention and treatment of cancer in vital organ transplanted patients. 4 classes of immunosuppressive agents are used in lung transplant patients: corticosteroids, calcineurin inhibitors, inhibitors of nucleotide biosynthesis and mTOR inhibitors. Interestingly, mTOR inhibitors have both immunosuppressive and anti-cancer effects [10]. They have shown promise in maintenance immunosuppressive regimens in lung transplantation but further studies are needed to identify the optimal way to use these agents. Conflict of interest The authors state no conflict of interest. Acknowledgment We thank Ms. Barbara Rüsi for technical assistance. This work was supported by Grants from the Cancer Research Institute, the Science Foundation for Oncology (SFO, Zurich), the Hanne
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