Heterogeneity in primary and metastatic prostate cancer as defined by cell surface CD profile

Abstract

Cluster designation (CD) antigens are cell surface markers that can be used to identify constituent cell populations of an organ. We have previously determined the CD phenotype of normal prostate parenchymal cells and are now extending this analysis to prostate cancer. Since expression of CD antigens is associated with cellular differentiation, cancer cells may differ from their normal counterpart in their CD profile. Compared with luminal secretory cells, prostate adenocarcinoma cells are frequently negative for CD10 and CD13, express increased levels of the cell activation molecule CD24, and decreased levels of the apoptosis-associated multifunctional enzyme CD38. Expression of CD57, CD63, CD75s, CD107a, CD107b, CD164, and CD166 by cancer cells is similar to that of secretory cells. Prostate basal epithelial cells do not express the CD antigens characteristic of prostate secretory cells; and the basal cell CD markers, CD29, CD44, CD49b, CD49f, CD104, and nerve growth factor receptor (NGFR) are not expressed by cancer cells. The preferential expression of secretory cell-associated CD markers by prostate cancer cells suggests a closer lineage relationship between cancer cells and secretory cells than basal cells. Although the above cancer CD phenotype was the most frequently seen, some prostate cancers contained populations of CD10- and/or CD13-positive cells, and CD57-negative cells. Furthermore, the cancer phenotype of tumor metastasis is different. Despite its low frequency in primary tumors, CD10 is expressed by virtually all of the nodal metastases of prostate cancer. In addition, stromal fibromuscular cells associated with primary prostate cancer differ from stromal cells in benign prostate tissue by an increased level of expression of the cell activation molecule, CD90. In summary, our data show that the CD marker expression profile of prostate cancer cells most closely resembles that of secretory prostate epithelial cells and that some prostate cancers consist of heterogeneous cell populations as distinguished by CD-marker expression profiles.

Figure 1

CD phenotype of prostate cancer. Shown are the staining results of CD10, CD13, CD24, CD26, CD38, CD57, CD75s, CD104, and CD107b on serial sections of specimen 01–064D. In this specimen, the tumor glands in the lower right of the field are negative for CD10 (the photomicrographs are identified by the CD molecules), CD13 and CD104; positive for CD24, CD26, CD38, CD57, CD75s, and CD107b. The tumor glands are visualized by their intense staining for CD24 compared to the larger benign glands. In contrast, tumor glands are stained weaker for CD38 compared to benign glands. They are also more uniformly stained for CD57, however, normal glands elsewhere in this specimen were well stained by CD57. CD104 stains the basal cell layer of benign glands, which is absent in cancer glands. Magnification is ×100.

Figure 2

Flow analysis of cancer. For the purpose of comparison, the number of positive events to each CD specificity (x axis) is adjusted to the percentage of CD57⁺ cells in either NP (13%) or CP (57.6%) (y axis), and tabulated. The finding that the cancer of specimen 99–010 is predominantly CD57⁺ was shown by immunohistochemistry of frozen blocks coded as 99–010E and 99–010F, although the tissue sample analyzed by flow cytometry was not the same as that represented in the tissue blocks. Staining results for CD10, CD13, CD104 and CD107b are also shown. CD57, CD10, CD13, CD26, and CD107b are reactive to luminal cells; CD44, CD104, CD29, and CD55 are reactive to basal cells; CD29, CD55, and CD49a are reactive to stromal fibromuscular cells; CD71 is reactive to multiple cell types; while CD53 and CD69 are reactive to white blood cells. Note the decreased positivity for CD10 and CD13 in CP. The CD44 and CD104 results are indicative of an under-representation of basal cells.

Figure 3

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Figure 3

Tumor heterogeneity. A: CD10⁺/CD13⁺ tumor glands. Two specimens, 01–089D and 01–090D, are contrasted. In the top panels, the cancer glands (middle) of 01–089D are not stained for CD10 and CD13 (whereas surrounding benign glands are stained). In the bottom panels, some of the small cancer glands in 01–090D are stained for CD10, and all for CD13. The cancer glands are negative for CD104 (not shown). Magnification is ×100. B: Adjacent, differentially stained tumor cell populations of high Gleason grade. Serial sections of specimen 01–081D were stained with the antibodies identified in the individual micrographs. Two tumor CD phenotypes are evident. Both types are positive for CD75s and negative for CD104 (stained are basal cells of benign glands, endothelial cells of blood vessels, and nerve cells in this field). The two types are otherwise indistinguishable by histomorphology. The Gleason score given for this specimen is 4 + 4. Magnification is ×100. C: Adjacent differentially stained tumor glands of low Gleason grade. One type of tumor glands is scored as CD10⁻/CD13⁻/CD57⁻ (blue arrow) while the other is scored as CD10⁺/CD13⁺/CD57⁺ (red arrow) in specimen 01–076D. Both cancer gland types are CD24⁺/CD26⁺/CD38⁺/CD75s⁺/CD104⁻. The Gleason score given for this specimen is 3 + 3. Magnification is ×100.

Figure 4

CD phenotype of a non-glandular pattern tumor. The Gleason score given for specimen 98–366D is 4 + 5, and the tumor mass (arrowed in the CD10 panel, note benign glands are stained) are CD10⁻/CD13⁻/CD24⁺/CD26⁺/CD38⁻/CD57⁺/CD75s⁺/CD104⁻/CD107b⁺. Except for the complete absence of CD38 staining, the phenotype is like that of the specimen shown in Figure 1. Magnification is ×100.

Figure 5

CD reactivity of prostate tumors. Thirty-one tumor specimens listed in the first column were scored for the expression of the CD markers listed. The subsequent columns indicate the immunoreactivity of the cancer to the CD antibodies indicated. Percentages of cancer showing positive immunostaining are presented in quartile with each filled box representing an increment of 25%. Unfilled boxes indicate no staining. Darker and lighter shading of the boxes are used to indicate increased intensity of staining in cancer versus non-cancer, as for CD24, and decreased intensity of staining, as for CD38, respectively. 99–022H1 and H2 are two separate tumor foci of the same specimen. For comparison, the CD phenotypes (as determined by flow cytometry) of cancer cell lines LNCaP, PC3, DU145, and CL1 are shown at the bottom. The lighter shaded boxes for CD24 in DU145 and CL1, and for CD104 in LNCaP are used to represent a labeling percentage of less than 25%. Because cultured cells were detached by trypsin before analysis epitopes of molecules like GPI-anchored CD24 might be affected by the enzyme treatment.

Figure 6

CD10 expression and Gleason components. The specimens in the first column are listed in the order of increasing percentage of CD10-positive cancer cells. The prostate cancer cases are characterized by percentages of Gleason 3, 4, and 5 components and the percentages of CD10⁺ cells in each component. For example, the tumor of 01–182D (first entry) is composed of 70% Gleason pattern 3, of which 0% is CD10-positive; 25% Gleason pattern 4, of which 0% is CD10-positive; and 5% Gleason pattern 5, of which 0% is CD10-positive. The tumor of 01–135C (36th entry) is composed of 50% Gleason pattern 5, of which 5% is CD10-positive; 40% Gleason pattern 4, of which 5% is CD10-positive; 10% Gleason pattern 5, of which 0% is CD10-positive. Its total positive percentage is 50 × 5/100 + 40 × 5/100 = 4.5%. The two cases highlighted in bold (98–395D and 97–233F) are known disease recurrence. The tumor volume is in ml. Some cases, like 99–155 (22.75% CD10-positive cancer cells) and 98–348 (55%), support the contention that presence of CD10⁺ cancer cells indicate likelihood of disease progression (as indicated by node involvement). Others, like 99–068 and 02–007, were assigned N+ yet the specimens 99–068D and 02–007C contained no CD10-positive cancer cells. However, our sampling only analyzed a portion of the tumor for many of these cases (see text). na, not available.

Figure 7

Lymph node metastases. Prostate cancer cells in the lymph nodes were identified by CD107b (and CD75s staining; and lymphocytes by CD45 staining). CD10⁺ cancer cells are evident in the two specimens, 00–061A7 and 99–022G1, shown. Staining appears to be localized to the apical surface of gland-like luminal space. CD10⁻ cancer cells are also evident in 00–061A7 (upper left). Magnification is ×100 (left) and ×400 (right).

Figure 8

CD reactivity of lymph node metastases. Sixteen specimens from four (available) different surgical cases (99–022, 00–061, 01–003, and 02–032) are listed. As in Figure 5, percentages of cancer showing positive immunostaining are presented in quartile with each filled box representing an increment of 25%. Unfilled boxes indicate no staining. The lighter shaded boxes indicate equivocal staining results. Note that the node patterns of 99–022G1, G2, G3 are similar to that of some tumor cells in the corresponding prostate, 99–022H1.

Figure 9

CD90 reactivity of cancer-associated stroma. The stromal fibromuscular cells of the two cancer foci found in specimen 01–178F are stained more intensely for CD90 than stromal fibromuscular cells of non-cancer tissue. Shown also are the staining results for CD10, CD45, and CD107b. The finding that staining is not due to infiltrating lymphocytes that are CD90⁺ is indicated by the low number of CD45-positive cells in the tumor foci. Both tumor and benign glands are stained for CD107b, tumor cells are negative for CD10. Magnification is ×40 except top right, which is ×100.