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Introduction

Aldehyde dehydrogenase (ALDH) 1 is a predominant isoform of the ALDH family in mammals, which oxidizes retinol to retinoic acid at the early stage of stem cell differentiation (1). ALDH1 is expressed in various stem/progenitor cells, such as hematopoietic and neural stem cells (1–3). In addition to normal tissues, a small population of tumor cells expresses ALDH1 at a high level. The ALDH1-high population of tumor cells is resistant to antitumor agents and exhibits a tumorigenic potential, suggesting that ALDH1 is one of the markers for cancer-initiating cells (CICs) (1). The presence of a large number of ALDH1-high cells is indicative of poor prognosis in cancers of the breast (1,4), lung (5), pancreas (6), bladder (7) and prostate (8).

CICs were first identified in leukemia (9). Although lymphoma is the most common disease among hematologic malignancies, CICs have not yet been determined in lymphoma. In the present study, ALDH1 expression was examined in tissues involved by diffuse large B-cell lymphoma (DLBCL) to investigate the presence of CICs. Unexpectedly, no lymphoma cells expressed ALDH1. Instead, ALDH1 expression was detected in surrounding non-tumorigenic cells. Stromal cells consist of vasculature, fibroblasts and inflammatory cells, such as lymphocytes, macrophages and dendritic cells (DCs). It is well known that stromal cells affect the character of tumors (10). For example, the macrophage subpopulation named M1 produces interleukin (IL)-12 which promotes tumoricidal responses, whereas another subpopulation, named M2, produces IL-10 which aids in tumor progression (11). In the present study, ALDH1 expression in non-tumorigenic stromal cells of tissues involved by DLBCL was evaluated, and its clinical implications were examined.

Materials and methods

Clinical samples

Forty-six patients, who were diagnosed with DLBCL at Osaka University Hospital during the period from April 2006 to December 2009 and whose follow-up data could be accessed, were examined. Clinicopathological findings in these 46 patients are summarized in Table I. There were 19 men and 27 women with ages ranging from 29 to 78 years (median age, 63). Location of DLBCL was nodal in 25 cases and extranodal in 21 cases. Immunohistochemically, the large lymphoid cells were CD20+ and CD3−. According to the criteria proposed by Hans et al (12), DLBCL is categorized into germinal center-type B cell lymphoma (GCB) (CD10+ or CD10−/bcl-6+/MUM1−) and non-GCB (CD10−/bcl-6−/MUM1+) type. The subtype of the studied DLBCL cases was GCB in 11 cases and non-GCB in 35 cases. Although the number of females and non-GCB cases was higher than that of males and GCB cases, there was no collection bias. Histological specimens were fixed in 10% formalin and routinely processed for paraffin-embedding. Paraffin-embedded specimens were stored in a dark room at room temperature at the Department of Pathology of Osaka University Hospital, and sectioned (4 μm) at the time of staining and stained with H&E and immunoperoxidase procedure.

Table I. Characteristics of the 46 DLBCL patients.

Table I.

Characteristics of the 46 DLBCL patients.

	No. of patients
Gender
Male	19
Female	27
Location
Nodal	25
Extranodal	21
Subtype
GCB	11
Non-GCB	35
Stage
I	18
II	7
III	4
IV	17
Treatment
Chemo-radiotherapy	10
Chemotherapy alone	28
Radiotherapy alone	1
Chemotherapy after surgery	2
Transplantation	4
No treatment	1
Response to therapies
CR	36
PR/NC	8
PD	1

[i] CR, complete response; PR, partial response; NC, no change; PD, progressive disease.

Based on physical examination records, surgical notes and pathological assessments of the specimens, the Ann Arbor staging system was applied. Eighteen cases were at stage I, 7 at stage II, 4 at stage III and 17 at stage IV. Ten patients received a combination of radiotherapy and chemotherapy, 28 received chemotherapy only, 2 received chemotherapy after surgery, 4 transplantation (3 auto- and 1 allo-transplantation), 1 received radiation therapy only and 1 received no therapy. The clinical outcome was evaluated according to the Guidelines of the International Workshop to Standardize Response Criteria for non-Hodgkin Lymphoma (13). The characteristics of the patients, such as age, tumor location and stage, in the present study were similar to those in previous reports (14,15). The proportion of male DLBCL patients in general was ∼55%, indicating that the proportion of female patients was slightly high in the present study. The study was approved by the Ethics Review Board of the Graduate School of Medicine, Osaka University.

Immunohistochemistry

ALDH1 expression was immunohistochemically examined using the anti-ALDH1 antibody (BD Biosciences, Franklin Lakes, NJ, USA). The sections were incubated with anti-ALDH1 diluted at x100, and subsequently processed using the ChemMate EnVision kit (Dako A/S, Glostrup, Denmark). DAB (Dako) was used as a chromogen. As the negative control, staining was carried out in the absence of the primary antibody. For immunophenotyping of DLBCL, the following monoclonal antibodies were used: CD20, CD3, Bcl-6, CD10 and MUM1 (Dako; dilution at 1:400, 1:50, 1:50, 1:100 and 1:100, respectively). Stained sections were evaluated independently by two pathologists (S.F. and E.M.).

Double staining of ALDH1 with CD20, fascin and CD68

Double staining of ALDH1 with CD20 (a marker for DLBCL cells), fascin (a marker for DCs; Dako) and CD68 (a marker for macrophages; Dako) was carried out using the EnVision G/2 doublestain system (Dako) according to the manufacturer’s protocol. For double staining with CD20, sections were first incubated with the anti-CD20 antibody (1:100), colored with DAB and, subsequently, ALDH1 expression was detected with Permanent Red (Dako). For double staining with CD68 or fascin, sections were initially incubated with ALDH1, colored with DAB and, subsequently, antigen retrieval was carried out using a Pascal pressurized heating chamber (Dako). After incubation with the anti-CD68 (1:100) or anti-fascin (1:100) antibody, sections were colored with Permanent Red. Since red fluorescence is released from Permanent Red, the red-color signal was detected with a fluorescence microscope (Biozero, Keyence, Osaka, Japan).

Statistical analysis

The Chi-square test was used to analyze the correlation between ALDH1 expression and clinicopathological characteristics of the lymphoma cases. Kaplan-Meier methods were used to calculate the overall survival (OS) rate, and differences in survival curves were evaluated with the log-rank test. p-values of <0.05 were considered statistically significant.

Results

Immunohistochemical findings

The expression of ALDH1 was examined in 46 DLBCL cases. The signals for ALDH1 were detected in two types of cells: cells with ample cytoplasm and fine chromatin and those with reticular structure (Fig. 1A). To confirm the absence of ALDH1 signals in DLBCL cells, double staining of ALDH1 and CD20 was performed; CD20+ large cells (brown-colored cells) did not express ALDH1 (Fig. 1B and C).

Figure 1. ALDH1+ cells in DLBCL. (A) Two types of cells expressed ALDH1: cells with ample cytoplasm and fine chromatin (arrows) and cells with reticular morphology (arrowheads). (B) Double staining of ALDH1 (red) and CD20 (brown). ALDH1+ cells are CD20−. (C) Fluorescent image of B. The red-colored ALDH1+ cells are clearly shown.

Double staining of ALDH1 and CD68

Since ALDH1+ cells with ample cytoplasm and fine chromatin morphologically resembled macrophages, the double staining of ALDH1 and CD68, a marker for macrophages, was carried out. ALDH1+ cells with ample cytoplasm and fine chromatin were positive for CD68 (Fig. 2A and B). By contrast, ALDH1+ cells with reticular morphology were negative for CD68 (Fig. 2A and B).

Figure 2. Double staining of ALDH1 and CD68 or fascin. (A) ALDH1 expression is shown in brown and CD68 in red. ALDH1+ cells with ample cytoplasm and fine chromatin are positive for CD68 (arrows), whereas ALDH1+ cells with reticular morphology are negative for CD68 (arrowheads). (B) Fluorescent image of A. CD68+ cells are shown. (C) ALDH1 expression is shown in brown and fascin in red. ALDH1+ cells with reticular morphology are positive for fascin (arrowheads), whereas ALDH1+ cells with ample cytoplasm and fine chromatin are negative for fascin (arrows). (D) Fluorescent image of C.

Double staining of ALDH1 and fascin

Reticular morphology in ALDH1+ cells suggested that a portion of the DCs was ALDH1+. To confirm this, double staining of ALDH1 and fascin was performed. ALDH1+ DC-like cells were positive for fascin (Fig. 2C and D).

Clinical implication of ALDH1+ cells in DLBCL

Clinical significance of the presence of ALDH1+ cells in DLBCL was examined. Seventeen and 7 of 46 cases possessed ALDH1+ macrophages and ALDH1+ DCs, respectively. The presence of ALDH1+ macrophages did not show any correlation with gender, location, subtype, stage of diseases and response to therapies (Table II). By contrast, the presence of ALDH1+ DCs was correlated with location (p=0.038) and response to therapies (p=0.039), although no correlation was detected in gender, subtype and stage of disease (Table III). There was a statistically significant difference in OS between patients with and without ALDH1+ DCs (p=0.006), although no difference was detected between patients with and without ALDH1+ macrophages (Fig. 3).

Figure 3. Kaplan-Meier plots. Overall survival (OS) curves of cases with and without ALDH1+ macrophages or DCs. Cases with ALDH1+ DCs showed a less favorable OS.

Table II. Correlation between the presence of ALDH1+ macrophages and clinicopathological parameters.

Table II.

Correlation between the presence of ALDH1+ macrophages and clinicopathological parameters.

	ALDH1+ macrophages		p-value
	Absent	Present
Gender			0.544
Male	11	8
Female	18	9
Location			0.090
Nodal	13	12
Extranodal	16	5
Subtype			0.964
GCB	7	4
Non-GCB	22	13
Stage			0.717
I	10	8
II	5	2
III	2	2
IV	12	5
Response to therapies			0.752
CR	23	13
PR/NC	5	3
PD	1	0

Table III. Correlation between the presence of ALDH1+ DCs and clinicopathological parameters.

Table III.

Correlation between the presence of ALDH1+ DCs and clinicopathological parameters.

	ALDH1+ DCs		p-value
	Absent	Present
Gender			0.180
Male	14	5
Female	24	3
Location			0.038
Nodal	18	7
Extranodal	20	1
Subtype			0.938
GCB	9	2
Non-GCB	29	6
Stage			0.555
I	15	3
II	7	0
III	3	1
IV	13	4
Response to therapies			0.039
CR	32	4
PR/NC	6	2
PD	0	1

Discussion

ALDH1 is reported to be one of the markers for CICs (1,8,16). To search for the presence of CICs in DLBCL, the ALDH1+ cells were immunohistochemically evaluated in lymphoid tissues involved by DLCBL. Unexpectedly, there were no ALDH1+ cells noted among the DLBCL cells. Instead, two types of cells in the stromal tissues expressed ALDH1: one with ample cytoplasm and fine chromatin, and another with reticular morphology. The former was stained with anti-CD68, and the latter with anti-fascin, indicating that ALDH1+ cells were macrophages and DCs, respectively.

Recently, ALDH1 expression has been reported in murine DCs derived from the gut (17), lung (5) and dermis (18). In humans, there have been no reports on ALDH1 expression in DCs. To our knowledge, this is the first report on ALDH1 expression in human DCs. Murine gut and dermal ALDH1+ DCs induce regulatory T cells (18,19), and thus may inhibit antitumorigenic immunity. The present study revealed that DLBCL patients with ALDH1+ DCs were associated with an unfavorable prognosis compared to those without, suggesting that human ALDH1+ DCs may induce regulatory T cells and inhibit antitumorigenic immunity. Further studies on the function of human ALDH1+ DCs may clarify their precise role in antitumorigenic immunity.

Recently, Guilliams et al found expression of ALDH1 in macrophages in intestinal lamina propria, but did not report on their function (18). To our knowledge, this is the first report on ALDH1+ macrophages in humans, but failed to show the clinical implication. The role of ALDH1 in macrophage function may be limited, at least in lymphoid tissues involved by DLBCL.

In conclusion, ALDH1+ macrophages and ALDH1+ DCs were found in the stromal tissues of DLBCL. Although no clinical implications were found to be associated with the presence of ALDH1+ macrophages, cases with ALDH1+ DCs demonstrated a less favorable prognosis compared to those without.

Acknowledgements

The authors thank Ms. Megumi Sugano, Ms. Etsuko Maeno and Ms. Takako Sawamura for the technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References