- Email: [email protected]
Pulmonary haptoglobin: a new marker for adenocarcinomas of the lung?
70
9.
10.
11. 12.
Pathology (2011), 43(1), January
CORRESPONDENCE
12 additional follicular dendritic cell tumors and 6 additional interdigitating dendritic cell tumors. Am J Surg Pathol 2004; 28: 988–98. Jaffe R, Pileri SA, Facchetti, et al. WHO classification of histiocytic and dendritic cell neoplasms. World Health Organisation Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press, 2008; 353–67. Chan JKC. Proliferative lesions of follicular dendritic cells: an overview, including a detailed account of follicular dendritic cell sarcoma, a neoplasm with many faces and uncommon etiologic associations. Adv Anat Pathol 1997; 4: 387–411. Perez-Ordonez B, Erlandson RA. Follicular dendritic cell tumor: report of 13 additional cases of a distinctive entity. Am J Surg Pathol 1996; 20: 944–55. Pileri SA, Grogan TM, Harris NL, et al. Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology 2002; 41: 1–29.
Table 1 Overview of primary antibodies and antigen retrieval procedures for immunohistochemistry Antibody
Clone
Producer
Haptoglobin TTF1
HG-36 SPT24
Abcam, UK DCS, Germany
SP-B
SPM 158
SP-A
PE-10
Zytomed Systems, Germany DCS, Germany
Napsin
KCG1.1
Zytomed Systems, Germany
Dilution
Antigen retrieval
1/100 1/400
2 min FastEnzyme 30 min citric acid buffer pH 6 30 min citric acid buffer pH 6 30 min Tris EDTA pH 9 30 min citric acid buffer pH 6
1/50 1/200 1/200
DOI: 10.1097/PAT.0b013e3283419e2a
Pulmonary haptoglobin: a new marker for adenocarcinomas of the lung? Sir, Lung cancer is the most common cancer worldwide with a 5-year survival of 60% for stage I and approximately 15% for more advanced stages. Early detection and identification of beginning cancer is a prerequisite for successful therapy.1 Therapeutically, there are long standing differences between the treatment regimens for different subtypes of lung cancer which are divided mainly into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Differentiation between SCLC and NSCLC is of great therapeutic relevance. With regard to this, appropriate immunohistochemical procedures have been developed.2 Novel chemo-therapeutic approaches have recently been developed for NSCLC, which is known as a largely chemo-resistant tumour. In the according clinical studies substantial differences between adenocarcinomas and squamous cell carcinomas or large cell carcinomas have been shown with regard to adequate therapeutic regimens.3,4 Therefore, sub-differentiation of NSCLC is currently becoming more of a focus and increasingly becoming a central element within therapeutic decisions. Increased haptoglobin (Hp) levels have been found in the serum of patients suffering from lung cancer and the possibility of Hp as a potential serum biomarker candidate has been discussed.5 Despite its well described occurrence in blood and liver, Hp has been previously discovered in human lung and lung cancer tissues, respectively.6 It has been shown that this acute phase protein does not originate solely from the liver, but is also expressed and synthesised in the human lung. Hp inhibits phytohaemagglutinin (PHA)-induced blastogenesis of lymphocytes, which has been suggested to protect tumours against immunological attack.7 Apffel and Peters8 proposed that carbohydrate-rich plasma proteins produced by the liver are localised on the surface of cancer cells, enabling them to escape the immunological attack of the host. These proteins have been identified as acute phase reactants and their increased expression in tissues and serum may enable neoplastic cells to evade immunological detection.9,10 In this study we attempted to analyse the specific
expression of pulmonary haptoglobin (pHp) in human lung cancer tissues and compare it to common markers of adenocarcinomas. Immunohistochemistry and heat-induced antigen retrieval were conducted as shown in Table 1. In short, 1 mm thick sections of formalin fixed, paraffin embedded tissues mounted on SuperFrost slides (Menzel Glas, Germany) were deparaffinised by incubation in xylene (2 10 min). Endogenous peroxidases were blocked by 10 min incubation in 3% H2O2. Primary antibodies were diluted in antibody diluent (Zytomed Systems, Germany) as described in Table 1. For detection, a one-step polymer system conjugated with horseradish peroxidases was applied according to the manufacturer’s instructions (ZytoChemPlus HRP One-Step Polymer anti-Mouse/Rabbit; Zytomed Systems, Germany). Colour reaction was performed using aminoethylcarbazole (permanent AEC Kit; Zytomed Systems) as a substrate. A total of 119 formalin fixed, paraffin embedded tumour tissues were immunohistochemically analysed for expression of pHp. Pulmonary haptoglobin was found to be expressed in 35 of 72 (48.6%) adenocarcinomas of the lung, which was set in relation to other markers like TTF-1 (79.1%), SP-A (51.3%), SP-B (48.6%) and napsin (84.1%) (Fig. 1). Expression of pHp in squamous cell carcinomas (n ¼ 47) was generally absent; only one case locally showed negligible signals (data not shown).
Fig. 1 Expression of haptoglobin and common markers for adenocarcinomas of the lung. Seventy-two tumour tissues were subjected to immunohistochemistry and treated as described; 35 of 72 were positive for Hp (48.6%), 57 for TTF-1 (79.1%), 37 for SP-A (51.3%) and 35 for Sp-B (48.6%). Expression of napsin could only be analysed in 53 of 63 samples (84.1%).
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
CORRESPONDENCE
In adenocarcinomas, pHp showed a comparable sensitivity to SP-A and SP-B, and a lower sensitivity when compared to TTF-1 and napsin as illustrated in Fig. 1. Interestingly, some cases have been observed which exclusively express either pHp and napsin (pHpþ/napsinþ, 4.7%) or pHp and TTF-1 (pHpþ/TTF-1þ, 3.1%). Cases which were positive for TTF-1 or napsin with either SP-A or SP-B positivity and expression of pHp accounted for 14%. In 15 cases, SP-A and SP-B (SP-A/SP-B/TTF-1þ/napsinþ, 26.9%) showed no coexpression with either TTF-1 or napsin (Fig. 2). SP-A and SP-B are commonly regarded as highly specific markers for adenocarcinomas with limited sensitivity compared to TTF-1; the same holds true for pHp. The specificity of the highly sensitive marker TTF-1 is limited since
71
it detects tumours of the thyroid and small cell lung cancer as well. Napsin is not only expressed in adenocarcinomas of the lung, but also in renal cell carcinomas and papillary thyroid carcinomas.11 Taken together, 7.8–14.0% of our investigated samples would have caused difficulties in diagnosis of adenocarcinomas if expression of pHp had not been addressed. With 1.3 million deaths per year, lung cancers are the most prominent cause of death from cancer.1 Among these, about 40% are adenocarcinomas12 which account for 520 000 deaths each year. Addressing pHp as an additional marker would have a serious impact on diagnostics and subsequent therapy. Based on this, from at least 40 560 (7.8%) up to 72 800 (14%) cases of uncertain diagnosis each year could be avoided if pHp was used as an additional marker.
Fig. 2 Expression profile of adenocarcinomas which were subjected to immunohistochemistry. Negative cases of expression for a marker are indicated with – and no colour. In positive cases of expression, each marker is coded with þ and a marker-specific colour.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
72
Pathology (2011), 43(1), January
CORRESPONDENCE
Fig. 3 Exemplary immunohistochemical detection of haptoglobin in four different adenocarcinomas of the lung. (A–D) Intense cytoplasmic and granular staining of tumour cells can be observed. Positive signals are displayed in red (permanent AEC).
In conclusion, our findings demonstrate constant expression of haptoglobin in adenocarcinomas of the lung (Fig. 3). Pulmonary haptoglobin has not only been shown to be simultaneously expressed with other common markers but also in cases where few others give positive results. These promising initial data should be verified in large scale studies. pHp may be proven to be a useful new marker for diagnostic differentiation of adenocarcinomas from squamous cell carcinomas. Acknowledgements: The authors thank Jasmin Tiebach and Maria Lammers for excellent technical assistance. M. Abdullah*§ S. Marwitz*§ D. Ka¨hler* H. Schultz* C. Kuglerz P. Zabel{ E. Vollmer* T. Goldmann* *Clinical and Experimental Pathology, and {Medical Clinic, Research Center Borstel, Borstel, and zHospital Großhansdorf, Department of Thoracic Surgery, Großhansdorf, Germany; §these authors contributed equally Contact S. Marwitz. E-mail: [email protected] 1. World Health Organization (WHO). Cancer. Fact Sheet 297. Geneva: WHO, 2006. 2. Kaufmann O, Georgi T, Dietel M. Utility of 123C3 monoclonal antibody against CD56 (NCAM) for the diagnosis of small cell carcinomas on paraffin sections. Hum Pathol 1997; 28: 1373–8.
3. Lee HY, Ahn MJ, Park YH, et al. Adenocarcinoma has an excellent outcome with pemetrexed treatment in Korean patients: a prospective, multicenter trial. Lung Cancer 2009; 66: 338–43. 4. Smit EF, Burgers SA, Biesma B, et al. Randomized phase II and pharmacogenetic study of pemetrexed compared with pemetrexed plus carboplatin in pretreated patients with advanced non-small-cell lung cancer. J Clin Oncol 2009; 27: 2038–45. 5. Bharti A, Ma PC, Maulik G, et al. Haptoglobin alpha-subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res 2004; 24: 1031–8. 6. Abdullah M, Schultz H, Ka¨hler D, et al. Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues. Pathol Res Pract 2009; 205: 639–47. 7. Samak R, Edelstein R, Israel L. Immunosuppressive effect of acute-phase reactant proteins in vitro and its relevance to cancer. Cancer Immunol Immunother 1982; 13: 38–43. 8. Apffel CA, Peters JH. Tumors and serum glycoproteins. The ‘symbodies’. Prog Exp Tumor Res 1969; 12: 1–54. 9. Heo SH, Lee SJ, Ryoo HM, et al. Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS. Proteomics 2007; 7: 4292–302. 10. Kossowska B, Ferens-Sieczkowska M, Gancarz R, et al. Fucosylation of serum glycoproteins in lung cancer patients. Clin Chem Lab Med 2005; 43: 361–9. 11. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol 2010; 41: 20–5. 12. Travis WD. Pathology of lung cancer. Clin Chest Med 2002; 23: 65–81.
DOI: 10.1097/PAT.0b013e3283419f2a
Determination of a molecular signature of acute T-cell-mediated renal allograft rejection using quantitative real-time RT-PCR of 45 genes on a low density array Sir, Histopathological assessment of core biopsies remains the gold standard for the diagnosis of acute rejection in the renal
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
9.
10.
11. 12.
Pathology (2011), 43(1), January
CORRESPONDENCE
12 additional follicular dendritic cell tumors and 6 additional interdigitating dendritic cell tumors. Am J Surg Pathol 2004; 28: 988–98. Jaffe R, Pileri SA, Facchetti, et al. WHO classification of histiocytic and dendritic cell neoplasms. World Health Organisation Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press, 2008; 353–67. Chan JKC. Proliferative lesions of follicular dendritic cells: an overview, including a detailed account of follicular dendritic cell sarcoma, a neoplasm with many faces and uncommon etiologic associations. Adv Anat Pathol 1997; 4: 387–411. Perez-Ordonez B, Erlandson RA. Follicular dendritic cell tumor: report of 13 additional cases of a distinctive entity. Am J Surg Pathol 1996; 20: 944–55. Pileri SA, Grogan TM, Harris NL, et al. Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology 2002; 41: 1–29.
Table 1 Overview of primary antibodies and antigen retrieval procedures for immunohistochemistry Antibody
Clone
Producer
Haptoglobin TTF1
HG-36 SPT24
Abcam, UK DCS, Germany
SP-B
SPM 158
SP-A
PE-10
Zytomed Systems, Germany DCS, Germany
Napsin
KCG1.1
Zytomed Systems, Germany
Dilution
Antigen retrieval
1/100 1/400
2 min FastEnzyme 30 min citric acid buffer pH 6 30 min citric acid buffer pH 6 30 min Tris EDTA pH 9 30 min citric acid buffer pH 6
1/50 1/200 1/200
DOI: 10.1097/PAT.0b013e3283419e2a
Pulmonary haptoglobin: a new marker for adenocarcinomas of the lung? Sir, Lung cancer is the most common cancer worldwide with a 5-year survival of 60% for stage I and approximately 15% for more advanced stages. Early detection and identification of beginning cancer is a prerequisite for successful therapy.1 Therapeutically, there are long standing differences between the treatment regimens for different subtypes of lung cancer which are divided mainly into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Differentiation between SCLC and NSCLC is of great therapeutic relevance. With regard to this, appropriate immunohistochemical procedures have been developed.2 Novel chemo-therapeutic approaches have recently been developed for NSCLC, which is known as a largely chemo-resistant tumour. In the according clinical studies substantial differences between adenocarcinomas and squamous cell carcinomas or large cell carcinomas have been shown with regard to adequate therapeutic regimens.3,4 Therefore, sub-differentiation of NSCLC is currently becoming more of a focus and increasingly becoming a central element within therapeutic decisions. Increased haptoglobin (Hp) levels have been found in the serum of patients suffering from lung cancer and the possibility of Hp as a potential serum biomarker candidate has been discussed.5 Despite its well described occurrence in blood and liver, Hp has been previously discovered in human lung and lung cancer tissues, respectively.6 It has been shown that this acute phase protein does not originate solely from the liver, but is also expressed and synthesised in the human lung. Hp inhibits phytohaemagglutinin (PHA)-induced blastogenesis of lymphocytes, which has been suggested to protect tumours against immunological attack.7 Apffel and Peters8 proposed that carbohydrate-rich plasma proteins produced by the liver are localised on the surface of cancer cells, enabling them to escape the immunological attack of the host. These proteins have been identified as acute phase reactants and their increased expression in tissues and serum may enable neoplastic cells to evade immunological detection.9,10 In this study we attempted to analyse the specific
expression of pulmonary haptoglobin (pHp) in human lung cancer tissues and compare it to common markers of adenocarcinomas. Immunohistochemistry and heat-induced antigen retrieval were conducted as shown in Table 1. In short, 1 mm thick sections of formalin fixed, paraffin embedded tissues mounted on SuperFrost slides (Menzel Glas, Germany) were deparaffinised by incubation in xylene (2 10 min). Endogenous peroxidases were blocked by 10 min incubation in 3% H2O2. Primary antibodies were diluted in antibody diluent (Zytomed Systems, Germany) as described in Table 1. For detection, a one-step polymer system conjugated with horseradish peroxidases was applied according to the manufacturer’s instructions (ZytoChemPlus HRP One-Step Polymer anti-Mouse/Rabbit; Zytomed Systems, Germany). Colour reaction was performed using aminoethylcarbazole (permanent AEC Kit; Zytomed Systems) as a substrate. A total of 119 formalin fixed, paraffin embedded tumour tissues were immunohistochemically analysed for expression of pHp. Pulmonary haptoglobin was found to be expressed in 35 of 72 (48.6%) adenocarcinomas of the lung, which was set in relation to other markers like TTF-1 (79.1%), SP-A (51.3%), SP-B (48.6%) and napsin (84.1%) (Fig. 1). Expression of pHp in squamous cell carcinomas (n ¼ 47) was generally absent; only one case locally showed negligible signals (data not shown).
Fig. 1 Expression of haptoglobin and common markers for adenocarcinomas of the lung. Seventy-two tumour tissues were subjected to immunohistochemistry and treated as described; 35 of 72 were positive for Hp (48.6%), 57 for TTF-1 (79.1%), 37 for SP-A (51.3%) and 35 for Sp-B (48.6%). Expression of napsin could only be analysed in 53 of 63 samples (84.1%).
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
CORRESPONDENCE
In adenocarcinomas, pHp showed a comparable sensitivity to SP-A and SP-B, and a lower sensitivity when compared to TTF-1 and napsin as illustrated in Fig. 1. Interestingly, some cases have been observed which exclusively express either pHp and napsin (pHpþ/napsinþ, 4.7%) or pHp and TTF-1 (pHpþ/TTF-1þ, 3.1%). Cases which were positive for TTF-1 or napsin with either SP-A or SP-B positivity and expression of pHp accounted for 14%. In 15 cases, SP-A and SP-B (SP-A/SP-B/TTF-1þ/napsinþ, 26.9%) showed no coexpression with either TTF-1 or napsin (Fig. 2). SP-A and SP-B are commonly regarded as highly specific markers for adenocarcinomas with limited sensitivity compared to TTF-1; the same holds true for pHp. The specificity of the highly sensitive marker TTF-1 is limited since
71
it detects tumours of the thyroid and small cell lung cancer as well. Napsin is not only expressed in adenocarcinomas of the lung, but also in renal cell carcinomas and papillary thyroid carcinomas.11 Taken together, 7.8–14.0% of our investigated samples would have caused difficulties in diagnosis of adenocarcinomas if expression of pHp had not been addressed. With 1.3 million deaths per year, lung cancers are the most prominent cause of death from cancer.1 Among these, about 40% are adenocarcinomas12 which account for 520 000 deaths each year. Addressing pHp as an additional marker would have a serious impact on diagnostics and subsequent therapy. Based on this, from at least 40 560 (7.8%) up to 72 800 (14%) cases of uncertain diagnosis each year could be avoided if pHp was used as an additional marker.
Fig. 2 Expression profile of adenocarcinomas which were subjected to immunohistochemistry. Negative cases of expression for a marker are indicated with – and no colour. In positive cases of expression, each marker is coded with þ and a marker-specific colour.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
72
Pathology (2011), 43(1), January
CORRESPONDENCE
Fig. 3 Exemplary immunohistochemical detection of haptoglobin in four different adenocarcinomas of the lung. (A–D) Intense cytoplasmic and granular staining of tumour cells can be observed. Positive signals are displayed in red (permanent AEC).
In conclusion, our findings demonstrate constant expression of haptoglobin in adenocarcinomas of the lung (Fig. 3). Pulmonary haptoglobin has not only been shown to be simultaneously expressed with other common markers but also in cases where few others give positive results. These promising initial data should be verified in large scale studies. pHp may be proven to be a useful new marker for diagnostic differentiation of adenocarcinomas from squamous cell carcinomas. Acknowledgements: The authors thank Jasmin Tiebach and Maria Lammers for excellent technical assistance. M. Abdullah*§ S. Marwitz*§ D. Ka¨hler* H. Schultz* C. Kuglerz P. Zabel{ E. Vollmer* T. Goldmann* *Clinical and Experimental Pathology, and {Medical Clinic, Research Center Borstel, Borstel, and zHospital Großhansdorf, Department of Thoracic Surgery, Großhansdorf, Germany; §these authors contributed equally Contact S. Marwitz. E-mail: [email protected] 1. World Health Organization (WHO). Cancer. Fact Sheet 297. Geneva: WHO, 2006. 2. Kaufmann O, Georgi T, Dietel M. Utility of 123C3 monoclonal antibody against CD56 (NCAM) for the diagnosis of small cell carcinomas on paraffin sections. Hum Pathol 1997; 28: 1373–8.
3. Lee HY, Ahn MJ, Park YH, et al. Adenocarcinoma has an excellent outcome with pemetrexed treatment in Korean patients: a prospective, multicenter trial. Lung Cancer 2009; 66: 338–43. 4. Smit EF, Burgers SA, Biesma B, et al. Randomized phase II and pharmacogenetic study of pemetrexed compared with pemetrexed plus carboplatin in pretreated patients with advanced non-small-cell lung cancer. J Clin Oncol 2009; 27: 2038–45. 5. Bharti A, Ma PC, Maulik G, et al. Haptoglobin alpha-subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res 2004; 24: 1031–8. 6. Abdullah M, Schultz H, Ka¨hler D, et al. Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues. Pathol Res Pract 2009; 205: 639–47. 7. Samak R, Edelstein R, Israel L. Immunosuppressive effect of acute-phase reactant proteins in vitro and its relevance to cancer. Cancer Immunol Immunother 1982; 13: 38–43. 8. Apffel CA, Peters JH. Tumors and serum glycoproteins. The ‘symbodies’. Prog Exp Tumor Res 1969; 12: 1–54. 9. Heo SH, Lee SJ, Ryoo HM, et al. Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS. Proteomics 2007; 7: 4292–302. 10. Kossowska B, Ferens-Sieczkowska M, Gancarz R, et al. Fucosylation of serum glycoproteins in lung cancer patients. Clin Chem Lab Med 2005; 43: 361–9. 11. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol 2010; 41: 20–5. 12. Travis WD. Pathology of lung cancer. Clin Chest Med 2002; 23: 65–81.
DOI: 10.1097/PAT.0b013e3283419f2a
Determination of a molecular signature of acute T-cell-mediated renal allograft rejection using quantitative real-time RT-PCR of 45 genes on a low density array Sir, Histopathological assessment of core biopsies remains the gold standard for the diagnosis of acute rejection in the renal
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.