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Keywords:

  • LYPD3;
  • NSCLC;
  • biomarker;
  • AAH;
  • metaplasia

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The protein C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor, is a potential new biomarker in non-small cell lung cancer, with high levels of expression recently shown to correlate to poor survival of adenocarcinoma patients. In this study, C4.4A immunoreactivity in precursor lesions of lung squamous cell carcinoma and adenocarcinoma was investigated by stainings with a specific anti-C4.4A antibody. In the transformation from normal bronchial epithelium to squamous cell carcinoma, C4.4A was weakly expressed in basal cell hyperplasia but dramatically increased in squamous metaplasia. This was confined to the cell membrane and sustained in dysplasia, carcinoma in situ, and the invasive carcinoma. The induction of C4.4A already at the stage of hyperplasia could indicate that it is a marker of very early squamous differentiation, which aligns well with our earlier finding that C4.4A expression levels do not provide prognostic information on the survival of squamous cell carcinoma patients. In the progression from normal alveolar epithelium to peripheral adenocarcinoma, we observed an unexpected, distinct cytoplasmic staining for C4.4A in a fraction of atypical adenomatous hyperplasias, while most bronchioloalveolar carcinomas were negative. Likewise, only a fraction of the invasive adenocarcinomas was positive for C4.4A. With a view to the prognostic impact of C4.4A in adenocarcinoma patients, this finding might suggest that C4.4A could be an early biomarker for a possibly more malignant subtype of this disease.

Despite intense research efforts, survival rates in non-small cell lung cancer (NSCLC) have not improved significantly over the last decades and remain at only ∼ 15% after 5 years.1, 2 The poor prognosis of these patients is mainly due to presentation at advanced, inoperable disease stages. There is, therefore, considerable interest in identifying markers that can detect NSCLC as early as possible, with an ultimate goal of treating the disease already at the premalignant stage.3

One such potential biomarker is the C4.4A protein,4, 5 which is a structural homologue to the urokinase-type plasminogen activator receptor.6 Expression of C4.4A in normal tissues is mostly found in suprabasal layers of squamous epithelium such as the esophagus and skin,7–9 and it is thus absent from healthy lung tissue. C4.4A has, however, been reported to be upregulated in lung tumor tissue,5, 10 and we have recently shown that C4.4A has a pronounced prognostic impact in NSCLC, primarily ascribed to a severely compromised overall survival of adenocarcinoma (AC) patients with high C4.4A expression.11

The purpose of the current study was to clarify when C4.4A is induced during development of pathological precursor lesions in lung epithelia, through a description of C4.4A expression in normal epithelium, premalignant lesions, and invasive carcinomas of the lung. We have studied the progression to squamous cell carcinoma (SCC) and AC, from normal bronchial and alveolar epithelium, respectively, through preneoplastic and neoplastic lesions defined by the WHO classification of lung tumors,12 and used tissue from benign lung diseases for comparison. The multistage pathogenesis preceding manifest pulmonary SCC is well-defined, comprising basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ (CIS), and invasive SCC.12–14 As for the progression to AC, the presumed precursor lesions are termed atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC).14, 15

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patient material

Surgically resected, archival lung tissue samples from a total of 78 patients were included in the study. The main histopathological diagnoses are listed in Supporting Information Table 1. For each patient, 1–4 representative formalin-fixed, paraffin-embedded tissue blocks were selected from the archives of the Departments of Pathology at Gentofte and Rigshospitalet (Copenhagen University) Hospitals (Denmark), yielding a total of 113 specimens, so as to include tumor tissue, premalignant alveolar and bronchial lesions, and reactive alveolar changes. Hematoxylin and eosin-stained slides were reviewed by two certified pathologists (ES-R, ODL), in order to verify the presence of the relevant histological changes in these samples. Serial sections were then processed for immunohistochemistry to reveal expression patterns of C4.4A. Conductance of the study was approved by the local ethical committee (Protocol H-D-2007-0133), and no patients had objected to the use of their tissue for research purposes, as confirmed by the Danish registry for use of tissue.

Immunohistochemistry

Immunohistochemical stainings were performed under standardized conditions using a rabbit polyclonal anti-C4.4A antibody as described previously.7 In brief, antigen retrieval was done with 10 μg/ml Proteinase K (15 min, 37°C), after which the sections were incubated with 1% (v/v) hydrogen peroxide for 15 min, and subsequently overnight at 4°C with 5 μg/ml anti-C4.4A antibody. Horseradish peroxidase-labeled anti-rabbit IgG (Envision, K4003, Dako, Glostrup, Denmark) was used for detection (100 μl/section) 45 min at room temperature, followed by development with NovaRED (Vector Laboratories, Burlingame, CA) for 10 min and counterstaining with Mayer's hematoxylin (Histolab Products Ab, Gothenburg, Sweden). Staining specificity was challenged by preabsorption of the antibody with a 10-fold molar excess of recombinant C4.4A protein7 before addition to the sections, parallel stainings with the monoclonal antibody 11H108 and a rabbit IgG of irrelevant specificity (X903, Dako), as well as omitting the primary antibody (Supporting Information Figs. 1a–1d).

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Figure 1. C4.4A reactivity in bronchial epithelium during the progression to squamous cell carcinoma. Bronchial epithelium was immunostained with 5 μg/ml polyclonal rabbit anti-C4.4A antibody and counterstained with Mayer's hematoxylin. The expression of C4.4A increased from basal cell hyperplasia (b), to incomplete (c, f) and complete (d, e) squamous metaplasia, while normal bronchial epithelium was devoid of C4.4A (a). Panels f and e are magnifications of c and d, respectively. Expression of C4.4A was sustained in squamous dysplasia (g), CIS (h), and invasive SCC (i). Scale bars: 25 μm (a–d, g); ∼ 6 μm (e, f); 50 μm (h); 100 μm (i). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Scoring of C4.4A staining

Semi-quantitative evaluation of C4.4A staining was performed independently by two pathologists (ES-R, ODL), with identical results for most specimens, and consensus being reached for the few discrepant cases. Each of the investigated lesions present in a given specimen was classified separately as negative (0), weakly/focally positive (1+), positive (2+) or strongly positive (3+) (Supporting Information Figs. 1e–1h and Supporting Information Table 1). Multiple examples of a given lesion from the same patient were scored with the mean of the individual specimens. In the case of multifocal AAH lesions, each focus was quantified separately (1–15 foci), where after a weighted score was calculated by multiplying the number of foci with the score for each intensity level (0-3+) and dividing by the total number of foci for each sample (Supporting Information Table 2). A final score for AAH was obtained by rounding off to the closest integer. For the majority of lung resections, tissue representative of more than one of the investigated lesions could be found in either the same or separate blocks.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

C4.4A expression in the progression to pulmonary SCC

Normal bronchial epithelium, as evaluated in 26 patients (Table 1), was negative for C4.4A (Fig. 1a), both in morphologically normal-appearing bronchi adjacent to tumor tissue in cancer patients, and in tissue from benign lung diseases. In 22 cases, a weak to moderate, mostly cytoplasmic staining, was found in basal cell hyperplasia of the pseudostratified bronchial epithelium (Fig. 1b). This reactivity for C4.4A was dramatically intensified upon transition to non-malignant squamous metaplasia (Figs. 1c–1f), which was observed in 17 patients covering all disease categories of the study. Complete transepithelial metaplasia was always accompanied by a strong staining for C4.4A in almost 100% of the cells (Figs. 1d–1e), and this was predominantly associated with the plasma membrane. In most cases, C4.4A expression reflected the extent of metaplasia, increasing in intensity and membrane association with increasing involvement of the epithelial layer (Fig. 1; compare panels c+f with d+e). In contrast to basal cell hyperplasia, bronchial goblet cell hyperplasia was always negative for C4.4A (Table 1; n = 20).

Table 1. C4.4A expression in reactive, premalignant, and malignant lung tissue
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The pattern of C4.4A reactivity in squamous dysplasia (n = 5) was more or less identical to that observed in complete metaplasia (Fig. 1g). The limited number of CIS available in this material (n = 3) showed a more heterogeneous expression as compared to metaplasia and dysplasia, varying in the intensity and frequency of positive cells, but C4.4A reactivity was usually confined to the premalignant cells (Fig. 1h). This pattern was sustained in invasive SCC (Fig. 1i; n = 6), where C4.4A-expressing cells primarily resided in keratinizing areas of high differentiation.

C4.4A expression in the progression to pulmonary AC

In general, as for bronchial epithelium, normal alveolar epithelium (n = 33) was devoid of C4.4A (Fig. 2a), with only few scattered weakly positive type II pneumocytes. The reactivity for C4.4A in premalignant AAH lesions (n = 30) was variable, sometimes even within foci in the same tissue block (Supporting Information Table 2). Final scores yielded 17% negative, 37% weakly positive, and 37% positive foci (Table 1). Ten percent were strongly positive with a notable, cytoplasmic reaction in the atypical alveolar cells (Figs. 2c–2d). To determine if the C4.4A pattern observed in these atypical type II pneumocytes was specific for AAH or if it was generally found in reactive alveolar epithelium, we included patients with usual interstitial pneumonia, cystic fibrosis, and emphysema, showing various degrees of reactive changes. Overall, C4.4A was absent in most of these diseases (18/22 negative cases; Fig. 2b). Four patients had nonetheless a few scattered and weakly positive pneumocytes, but none of these displayed as strong reactivity as that observed in AAH. Notably, the difference in C4.4A expression between reactive and AAH lesions was statistically significant, as assessed by a test for independence (exact p value < 0.0001). When present, C4.4A staining of atypical type II pneumocytes in AAH lesions exhibited a rather unusual subcellular compartmentalization without the distinct confinement to the cell membrane, but our validation protocol unambiguously substantiated the specificity of this expression pattern (Figs. 2c–2f).

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Figure 2. C4.4A expression in alveolar epithelium during the progression to adenocarcinoma. Alveolar epithelium was immunostained with 5 μg/ml polyclonal rabbit anti-C4.4A antibody and counterstained with Mayer's hematoxylin. Normal (a) and reactive (b) alveolar epithelium was devoid of C4.4A, as contrasted to a C4.4A-positive AAH lesion (c, d). Preabsorption of the polyclonal antibody with recombinant C4.4A protein (e, f) quenches the reactivity observed in panels c and d. Panels d and f are magnifications of c and e, respectively. The lower panel shows examples of a C4.4A-negative BAC (g) and a C4.4A-positive invasive AC (h). Scale bars: 50 μm (a, b, g, h); 100 μm (c, e); 25 μm (d, f). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The majority of BACs demonstrated either no (62%) or very little (31%) C4.4A (Fig. 2g; n = 26). As opposed to SCC, only 28% of the invasive ACs were positive for C4.4A, the remaining 72% being either negative or weakly positive (Table 1; n = 18). When an AC contained C4.4A, the pattern was often focal, but the staining was quite prominent in those cells that reacted (Fig. 2h).

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we investigated the localization of C4.4A in the progression from normal bronchial epithelium, to basal cell hyperplasia, squamous metaplasia, dysplasia, CIS, and ultimately invasive SCC, as well as from normal alveolar epithelium to invasive AC, through the stages of AAH and BAC. Normal expression of C4.4A in stratified squamous epithelia is stringently regulated, as typified by its confinement to the squamous side of squamo-columnar junctions.9 In line with this, the present study reveals a clear difference between invasive SCC and AC, with the former generally being positive and the latter negative for C4.4A. This observation conforms with a prognostic study in NSCLC, where high levels of C4.4A were found in 77% of SCCs and only 25% of ACs.11 A larger study on tumor tissue samples from a new patient cohort similarly divides these histological variants into two separate groups with regard to C4.4A expression (Jacobsen et al, manuscript in preparation). Stimulation with phorbol 12-myristate 13-acetate, which can commit cells to terminal squamous differentiation, furthermore leads to an upregulation of the protein in suprabasal keratinocytes of murine skin, while basal keratinocytes remain negative.7 Our investigation of transitions in pulmonary epithelia recapitulates this scenario, as illustrated by the absence of C4.4A in morphologically normal bronchial epithelium, being replaced by prominent membrane-associated C4.4A expression in squamous metaplasia (Fig. 1). Surprisingly, C4.4A was also detected in basal cell hyperplasia (Fig. 1b). One plausible explanation for this unexpected finding is that these C4.4A-positive cells already have entered the very earliest phases of the metaplastic conversion process that will replace the normal pseudostratified columnar epithelium of the bronchi with a stratified squamous epithelium. Hence, C4.4A may be a novel biomarker that is induced before this squamous differentiation is morphologically manifest. In Table 1, we have, therefore, termed these lesions “Basal cell hyperplasia/early metaplasia” to mirror their intermediate nature. This is also reflected in the gradual increase in the levels of C4.4A seen in the development from normal bronchial epithelium to squamous metaplasia (Table 1). Other reports have advocated that respiratory basal cells are indeed the progenitors of squamous metaplastic cells and presumably preneoplastic epithelium.12, 14, 16–18 In agreement with this assumption, we did not observe any C4.4A expression in hyperplasia of bronchial goblet cells (Table 1).

The early non-malignant transdifferentiation stage of the bronchial airways, in which C4.4A expression is induced, is most likely temporarily populated by those cells that upon neoplastic transformation eventually will make up the malignant SCC. Delineating the function of C4.4A, which possibly has a bearing on cell–cell interactions,7, 8, 19 might consequently increase our knowledge of the mechanisms engaged in early lung cancer pathogenesis. A corollary to the appearance of C4.4A already in basal cell hyperplasia/early metaplasia is that it would be too early a biomarker to reflect the subsequent malignant transformation per se, given that these lesions are believed to be reactive changes not linked to an increased risk of cancer progression.20–22 This is in excellent coherence with the lack of prognostic information from C4.4A on the survival of SCC patients.11

In sharp contrast, C4.4A expression in resected lung tumor tissue from AC, which has become the most common form of lung cancer,15 was shown to be a robust biomarker for poor survival of patients with this histological subtype.11 In the investigated cases of AAH, considered a precursor lesion for peripheral pulmonary AC, 17% were C4.4A-negative, and C4.4A could also occasionally be weakly expressed in reactive changes in benign lung diseases. This precludes the potential use of C4.4A as a general biomarker for early disease in the alveolar compartment. 10% of AAHs were, however, strongly positive (3+) by immunohistochemical staining for C4.4A (Figs. 2c–2d), even though AAH does not bear any morphological resemblance to squamous differentiation. AAHs do not necessarily progress to malignancy,23 but it has been proposed that they constitute secondary events arising independently of the primary tumor,24 which might indicate a risk of developing a second primary tumor. In this context, the positive fraction of AAH lesions is intriguing, given that also for the AC cases, only a subgroup displays C4.4A reactivity (28%). Some patients exhibited significant variation in the intensity of C4.4A expression in individual foci within the same tissue block (Supporting Information Table 2). The clinical consequences of this observation remain to be established, but there might exist subpopulations of AAH cells, accompanied by distinct levels of C4.4A, with different malignant potential in the progression to AC. A prospective study with longitudinal sampling, e.g. in a mouse lung cancer model,25 would be necessary to unveil whether a C4.4A-positive AAH could eventually develop into a C4.4A-positive AC. If there were a relationship between C4.4A-positivity in AAH and an ensuing AC, it is tempting to speculate that C4.4A could be used as an early marker of a supposedly more aggressive subtype of AC.

The present immunohistochemical study has revealed the appearance of C4.4A at very early stages of transformation in both central and peripheral airways. Future studies aiming at uncovering the biological function of C4.4A will clarify its potential as a biomarker in NSCLC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Mette Andersen, Öznur Turan, Jette Pedersen, Lotte Frederiksen, and John Post for excellent technical assistance, and Ib Jarle Christensen (The Finsen Laboratory) for statistical help.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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IJC_26305_sm_SuppFig1.tif18168KSupporting Information
IJC_26305_sm_SuppInfo.doc298KSupporting Information

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