The presence of carboxypeptidase-M in tumour cells signifies epidermal growth factor receptor expression in lung adenocarcinomas: the coexistence predicts a poor prognosis regardless of EGFR levels

Abstract

Purpose: Carboxypeptidase-M (CPM) is a membrane-bound peptidase that metabolizes peptides, and is present in pneumocytes. CPM hydrolyses the C-terminal arginine of epidermal growth factor (EGF) resulting in des-Arg53-EGF which binds to the EGF receptor (EGFR) with an equal or greater affinity than native EGF. Therefore, this study focused on the possible presence of CPM in human lung adenocarcinomas (ADC) and evaluated the relationship between CPM and EGFR by assessing the impact of expressions on patient clinical outcome.

Methods: This is a retrospective study of 110 patients who underwent resection of the primary tumour (92) or metastatic tissues (18) for treatment or diagnosis. Immunohistochemistry (IHC) for CPM and EGFR was made in serial sections using standard methods.

Results: This study demonstrates for the first time that 23.6% of ADCs express carboxypeptidase-M (26/110), mainly in membrane-bound forms. The amounts and the extent of CPM within tumours vary from low levels to obviously overexpressed forms. The immunohistochemical positivity (+) for CPM in ADCs negatively correlated with disease survival. In addition, 80% of CPM+ adenocarcinomas (21/26) showed a coexpression with EGFR suggesting a high prevalence for coexistence. The follow up data indicated a significantly shorter 5-year survival time for patients with CPM+-EGFR+ (double-positive) tumours compared to those harbouring neoplasias negative for both proteins (9.5 vs. 60.4% survivals, P < 0.001).

Conclusion: The fact that CPM+ ADCs often co-express with EGFR suggests a functional-regulatory link between these proteins which might have therapeutical consequences. The present novel data could lead to improved IHC tests in lung adenocarcinomas for EGFR expression.

Fig. 1

Moderately differentiated adenocarcinoma (a) showing CPM immunopositivity (arrows) (b). Bronchioloalveolar carcinoma (c) with CPM immunopositivity (d). Note that the staining pattern for CPM is mainly cell membrane-associated and apical (arrows). A case of poorly differentiated adenocarcinoma (e): 50% of tumour cells showed minimal or trace amounts of CPM expression [f (score 1, grade 50)]; 20% of the same tumour exhibited intense membrane positivity [g (score 4, grade 80)], and 30% moderate immunostaining for CPM, respectively [h (score 3, grade 90)]. Therefore, the final grade for CPM: 220. The immunostained sections are counterstained with methyl-green. Original magnification ×20

Fig. 2

a–c EGFR⁺ ADC with low levels of CPM in tumour cells (not shown), and many CPM⁺ tumour-associated macrophages. a H-E; b CPM⁺ macrophages (arrows); c EGFR positivity in tumour cells (arrows). d, e Moderately differentiated lung adenocarcinoma with CPM–EGFR (double) positivity. d H-E; e CPM positivity; f EGFR immunostainings in different areas (70% co-existence). Note that the chromogenic substrate for CPM is VIP (purple) and DAB for EGFR (brown). g–i Immunofluorescence of a lung adenocarcinoma metastasis in the brain using serial sections to demonstrate colocalization. g CPM immunofluorescence with Texasred, and h EGFR with FITC (green) respectively, exhibiting positivities on the same tumourous gland. i An overlayed picture of g and h (50% opacity) indicating high degree of coexpression. All photographs ×20

Fig. 3

Kaplan–Meier curves for patient survival, stage I–IV

Fig. 4

Kaplan–Meier curves for patient survival. Stage I–II and stage III–IV