John H. Frenster, Michael M. Papalian, Marilyn A. Masek and Jeffrey A. Frenster,
Dept. of Medicine, Stanford University School of Medicine, Stanford, CA 94305

Abstract:

Introduction:

Materials and Methods
Patient Sources:
Electron Microscopy and Probe Analysis:
Morphometric Analysis:
Statistical Analysis:

Results
Subpopulations of Neoplastic Cells:
Subpopulations of Lymphocytes:
Activity of Apposed Lymphocytes:

Discussion:

Support:

References:
Additional References:

Links:

Abstract:

Five hundred and two cells, selected at random within the pretreatment-involved lymph nodes of patients with the nodular sclerosis type of Hodgkin's Disease, were analyzed by electron microscopy for in-vivo subcellular acivity and intercellular interaction. Subpopulations of neoplastic cells and of lymphocytes were recognized by ultrastructural criteria. Neoplastic cells displayed definite sequences of cell maturation, whereas lymphocytes displayed definite sequences of cell activation. Active lymphocytes were most often found apposed to the more active of the neoplastic cells, and the nuclei of such apposed lymphocytes displayed a progressive preferential asymmetry of distribution of active DNA templates into that half of the lymphocyte nucleus closest to the neoplastic cell. Such close apposition of active polysomal lymphocytes to neoplastic cells may correlate with increased patient survival in Hodgkin's Disease. 

Introduction:

Previous light microscopy studies (1, 2) have revealed a favorable close correlation between the number of lymphocytes within the involved pretreatment lymph nodes of patients with Hodgkin's disease and the eventual survival of the patient. these same studies have also revealed an adverse effect of increased numbers of neoplastic cells or of normal macrophages on patient survival (1).

Because light microscopy is limited in its optical resolution of subpopulations of cells and in its resolution of subcellular structures within individual cells, we have utilized electron microscopy to develop high-resolution acridine orange ultrastructural probes (3, 4) and morphometric methods (5) that have revealed subpopulations of lymphocytes and of neoplastic cells within sampled intact lymph nodes of human subjects (6). We now report the use of these high-resolution methods to analyze the subcellular activities and interactions of these cells within the involved pretreatment lymph nodes of newly diagnosed patients with Hodgkin's disease. 

Materials and Methods

Patient Sources:

Lymph nodes were obtained after informed consent from 3 untreated patients with the diagnosis of the nodular sclerosis form of Hodgkin's disease by surgical excision biopsy at the time of the original pathologic staging of their disease as described previously (7).

Electron Microscopy and Probe Analysis:

Lymph node biopsy specimens from patients during pathologic staging of their disease were fixed in the operating room with 5% glutaraldehyde in medium 199 (Grand Island Biological Co., Grand Island, N.Y.) at pH 6.5 and 4^o C for 2 hours immediately after surgical excision as described previously (7). The 1 mm³ blocks of fixed tissue were treated with 10^-3 M acridine orange (K & K Laboratories, Plainview, N.Y.; twice recrystallized) in medium 199 at pH 7.2 in the dark at 4^o C for 96 hours. The samples were next washed three times in medium 199 and incubated in the dark at 37^o C for 30 minutes in low-calcium spinner-type Eagle's minimum essential medium (Grand Island Biological Co.) at pH 7.4 and 0.8 mM MgCl₂ containing 1.0 mg/ml pancreatic DNase I (Worthington Biochemical Corp., Freehold, N.J.; electrophoretically separated from any contaminating RNase activity) (3). The incubated samples were then prepared for electron microscopy (8) by being post-fixed for 1 hour in 1% OsO₄, dehydrated with graded concentrations of ethanol and propylene oxide (9), and embedded in Epon. Thin sections were cut at 0.1 um thickness with an LKB ultramicrotome, stained with 5% uranyl acetate for 30 minutes, and examined at 80 kV under high resolution in a Siemens 1A electron microscope, and electron micrographs at 10,000-100,000 magnification were obtained by a Durst photo-enlarger. Control samples omitting acridine orange or omitting DNase did not reveal any reaction product (3).

Thirty electron microscopic grid squares were selected at random, and a complete enumeration of all 502 nucleated cells sectioned on the grid squares was performed. High-resolution electron micrographs at magnifications of 10,000 -100,000 were utilized to localize, measure, and count within each cell (4) the electron-dense reaction products formed as a complex between acridine orange and osmic acid indicative of active DNA templates (4, 6).Individual cells in the distinct stages of normal or neoplastic cell differentiation or cell division were identified independently by one observer utilizing the previously published ultrastructural criteria for lymphocytes (6) and neoplastic cells (7) in Hodgkin's disease. For the purpose of statistical chi-square analysis (10), lymphocytes were analyzed for activity of the cytoplasmic type (6) as monosomal (type 1), transitional (type 2), or polysomal (type 3), whereas neoplastic cells were analyzed for morphology of the nuclear type (6, 7) as Hodgkin's cells (type 4), mononuclear Reed-Sternberg cells (type 5), binuclear Reed-Sternberg cells (type 6), or multinuclear Reed-Sternberg cells (type 7).

Each of the interphase neoplastic cells was analyzed for chromatin type on a scale of 1 for condensed heterochromatin to 4 for extended euchromatin as described previously (11). The number of nuclear bodies (12), nucleoli (11), and nuclear blebs (13) found in each sectioned neoplastic cell was then separately counted.

Morphometric Analysis:

To analyze the possible relationships between lymphocyte activity and apposition to neoplastic cells, we analyzed separately each of the normal lymphocytes found to be apposed to a neoplastic cell by high-resolution morphometry in two dimensions at one section level (5) and utilized separate electron micrographs of each apposed lymphocyte at a 10,000-20,000 magnification. The sector of the neoplastic cell membrane was measured with a Leitz map-reader micrometer (7), and a straight chord line was then constructed in the neoplastic cell image joining the two ends of the apposition membrane sector. The longest nuclear dimension of the apposing normal lymphocyte was determined as the nuclear axis of the lymphocyte (5), and a rectangle outlining the nucleus of the lymphocyte was constructed parallel to the nuclear axis, with the center of each nucleus being determined by the intersection of the diagonals of each nuclear rectangle (5). The nuclear shape of each apposing normal lymphocyte was then expressed as the ratio of the long to the short dimensions of the nuclear rectangle (5).

The activation vector was then measured for each apposing normal lymphocyte by a line constructed to join the midpoint of the constructed chord in the neoplastic cell to the center of the nucleus of the apposing normal lymphocyte. The distance along the activation vector axis from the neoplastic cell membrane to the center of the nucleus of the apposing lymphocyte was measured as the activation vector length, and the diameter of the nucleus of the lymphocyte projected by perpendiculars onto the extension of the activation vector axis was measured as the nuclear projection length. The apposition membrane sector length was expressed as the ratio of the measured map-reader length of the sector to the nuclear projection length, whereas the sector/vector ratio was determined by the ratio of these measured lengths for each apposed normal lymphocyte.

The proximal cytoplasmic length was measured as the ratio of the length along the activation vector axis from the neoplastic cell membrane to the membrane of the nucleus of the lymphocyte divided by the total length of the apposing lymphocyte as measured along the extension of the activation vector axis. Similarly, the distal cytoplasmic length was measured as the ratio of the length along the extension of the activation vector axis from the nuclear membrane of the lymphocyte to the distal cell membrane of the lymphocyte divided by the total length of the apposing lymphocyte as measured along the extension of the activation vector axis. We then determined the distal/proximal cytoplasm ratio for each normal apposing lymphocyte by dividing distal cytoplasmic length by the proximal cytoplasmic length for that cell.

A line was then drawn through the nuclear center of each apposed lymphocyte perpendicular to the activation vector axis of that lymphocyte, and the nucleus of the lymphocyte was divided into near and far halves in relation to the neoplastic cell (5). The number of DNA probes in each near and far half nucleus was then counted within each apposed normal lymphocyte and expressed as the near/far DNA probe ratio of that lymphocyte (5).

Statistical Analysis:

The resulting ultrastructural probe and morphometric data were analyzed for significance of trend either by Mann's test for trend significance (14) or by chi-square tests with one degree of freedom (10) and one-tailed significance levels (15).

Results:

A total of 502 sectioned nucleated cells was analyzed within intact lymph nodes (Table 1).
 
 
 
 

Table 1. In vivo activity of lymph node cells in Hodgkin's disease.

Cell type	Number  of Cells	Percent of Cells Positive for DNA Probes, (%)	Mean DNA Probe Count of Positive Cells, (Mean +/- S.D.)
Lymphocytes	435	97.9	21.65 +/- 11.03
" Mitotic	2	0	-
Neoplastic Cells	29	75.8	29.13 +/- 17.02
" Mitotic	2	100.	25.00 +/- 5.65
Macrophages	9	88.8	9.60 +/- 10.06
Fibroblasts	15	53.3	18.50 +/- 4.73
Plasma Cells	2	100.	20.50 +/- 3.54
Eosinophilic Granulocytes	1	0	-
Endothelial Cells	4	100.	17.00 +/- 4.55
Monocytes	3	100.	32.33 +/- 10.93

Of these cells, 437 (87%) were found to be various types of normal lymphocytes, with 79 (18%) of these lymphocytes being found in touching-contact apposition to neoplastic cells; however only 2 (0.4%) of all lymphocytes were found to be in cell division, and these normal metaphase cells were negative for DNA probes, as was found previously for normal metaphase human bone marrow cells (4). In contrast, a total of only 31 (6.1%) of all cells was found to be of various types of neoplastic cells (Table 1); however 2 (6.4%) of these were found in cell division, and these metaphase neoplastic cells were postive for DNA probes, a phenomena found to date only within neoplastic cells (16).

Of the 8 major population cell types found within the intact lymph nodes (Table 1), normal monocytes tended to be the most actively labeled by DNA probes (32.33 +/- 10.93), whereas normal macrophages tended to be the least actively labeled (9.60 +/- 10.06; P<0.05).

Subpopulations of Neoplastic Cells:

When the neoplastic cells within the lymph nodes were subclassified on the basis of nuclear ultrastructural criteria previously published *6, 7), we found that the number of DNA probes decreased significantly with maturation of the neoplastic cells, being highest for immature Hodgkin's cells and lowest for the most mature multinuclear Reed-Sternberg cells (Table 2).
 
 
 
 

Table 2. In vivo activity of neoplastic cells in Hodgkin's disease.

Cell activity	Hodgkin's cellsa	Reed-Sternberg cells: Mono-nuclear	Bi- nuclear	Multi- nuclear	Significance of trendb Chi-square	P
DNA Probes	33.13	22.80	17.50	2.67	15.24	<0.0005
Chromatin type	3.63	3.60	3.47	3.75	NS	NS
Nuclear bodies	0.13	1.20	1.63	2.33	18.10	<0.0005
Nucleoli	1.63	1.60	1.25	2.33	NS	NS
Nuclear blebs	0.50	1.00	1.75	6.33	8.80	<0.005
Apposition membrane sector length	2.03	1.57	1.48	1.29	7.74	<0.005
Apposed  lymphocyte type	2.20	1.94	2.08	1.75	NS	NS
Apposed lymphocyte DNA probes	19.07	28.16	26.92	21.03	NS	NS

^a Mean = No. or type per neoplastic cell.
^b NS = not significant.

This change in numbers of DNA probes parallels the decrease in numbers of DNA probes seen with increasing degrees of cell differentiation and maturation in normal human bone marrow cells (4) and suggests that neoplastic cells in vivo are undergoing some type of abortive differentiation or pseudo-differentiation (17). This suggestion is further supported by the increasing number of nuclear bodies and nuclear blebs found in the more mature neoplastic cells (Table 2). Both nuclear bodies (12) and nuclear blebs (13) were shown previously to be associated with increasing degrees of cell differentiation and maturation in other cell systems. By contrast, the euchromatin state of nuclear chromatin organization did not vary significantly with maturation of of the neoplastic cells (Table 2), which suggests that DNA template inactivation within neoplastic cells in vivo can occur concurrently with persistence of the extended state of euchromatin (18). Similarly, the number of nucleoli did not vary significantly with increasing maturation of the neoplastic cells in vivo (Table 2).

Regarding external interactions of the neoplastic cells with apposing normal lymphocytes, the length of the apposition membrane sector was found to decrease significantly with increasing maturation of the neoplastic cells (Table 2), but the cytoplasmic type of apposed lymphocytes or the DNA probe activity of the apposed lymphocytes did not vary significantly with increasing maturation of the neoplastic cells in vivo (Table 2).

Subpopulations of Lymphocytes:

When the lymphocytes within the lymph nodes were subclassified on the basis of the ultrastructural cytoplasmic criteria published previously (6), we found that the number of nuclear DNA probes increased significantly with increasing activity of the cytoplasm of the lymphocyte, being lowest for monosomal lymphocytes and highest for polysomal lymphocytes (Table 3), with 100% of polysomal lymphocytes displaying active DNA templates within the cell nucleus (Table 3).
 
 
 
 

Table 3. In vivo effect of apposition on lymphocytes in Hodgkin's disease.

Cell activity	Monosomal lymphocytes	Transitional lymphocytes	Polysomal lymphocytes
Total lymphocytes:
Pecent of total lymphocytes	49.2	30.0	20.8
DNA probes (mean/lymphocyte)	19.8	21.0	24.8
Percent cells with probes	96.6	98.6	100.
Mann's test for trend significance:	P < 0.005
Apposed lymphocytes:
Percent of total apposed type	12.7	31.9	40.0
Percent increase in DNA probes	27.7	17.5	5.3
Percent increase in cells with DNA probes	3.4	1.4	0.0
Mann' test for trend significance:	P < 0.005

Although the majority of total lymphocytes within the lymph node was of the monosomal type, only a small fraction (12.7%) of these monosomal lymphocytes was found to be apposed to neoplastic cells, whereas a significantly larger fraction (40.0%) of polysomal lymphocytes was found to be apposed to neoplastic cells in vivo (Table 3). This observation suggests that lymphocytes apposed to neoplastic cells within Hodgkin's disease lymph nodes in vivo are more active than lymphocytes not so apposed, and this suggestion is supported by the demonstrated increase in DNA probe counts and increase in percentage of lymphocytes displaying active DNA templates within each cytoplasmic subpopulation of lymphocytes when these lymphocytes are found apposed to neoplastic cells compared to the total lymphocyte population (Table 3). Tight in vivo apposition by lymphocytes against neoplastic cells in Hodgkin's disease lymph nodes has been shown previously to correlate with cytotoxic reactions within such apposed neoplastic cells (7).

Activity of Apposed Lymphocytes:

When the in vivo activity of lymphocytes apposed to neoplastic cells within lymph nodes were analyzed by high-resolution morphometric techniques, we found that polysomal lymphocytes tended to be apposed to neoplastic cells of lesser maturation and greater activity than was the case for monosomal lymphocytes (Table 4).
 
 
 
 

Table 4. In vivo activity of apposed lymphocytes in Hodgkin's disease.

Cell activity	Monosomal lymphocytesa	Transitional  lymphocytes	Polysomal lymphocytes	Significance of trendb,  Chi-square	P
Apposed neoplastic cell type	5.60	5.26	5.25	2.80	<0.05
Apposition membrane sector	1.23	1.56	2.10	3.00	<0.05
Activation vector length	0.58	0.66	0.77	2.82	<0.05
Sector/vector ratio	2.43	2.56	2.98	2.94	<0.05
Proximal cytoplasm length	0.110	0.155	0.163	2.89	<0.05
Distal cytoplasm length	0.092	0.128	0.171	3.10	<0.05
Distal/proximal cytoplasm ratio	0.99	1.42	1.51	3.09	<0.05
Nuclear shape	1.51	1.62	1.64	2.78	<0.05
DNA probes	27.40	23.98	26.06	NS	NS
Near/far DNA  probe ratio	1.37	1.51	1.56	2.87	<0.05

^a Mean No. or type per lymphocyte.
^b NS = not significant.

In addition, the length of the apposition membrane sector, the length of the activation vector, and the sector/vector ratio were all significantly greater for polysomal apposed lymphocytes than for monosomal apposed lymphocytes (Table 4). These data are consistent with the possibility that greater in vivo activity of the neoplastic cells in turn induces greater in vivo activity of the lymphocytes apposed to these active neoplastic cells, compared to lymphocytes apposed to less active neoplastic cells.

In addition, the length of both the proximal and the distal cytoplasm in the apposed lymphocytes was found to increase significantly from monosomal lymphocytes to polysomal lymphocytes, as did the ratio of distal/proximal cytoplasm lengths, which indicates a greater amount of cytoplasm within the active polysomal lymphocytes compared to that within the less active monosomal lymphocytes (Table 4). The shape of the lymphocyte nucleus was similarly found to be significantly more elongated within polysomal lymphocytes compared to monosomal lymphocytes (Table 4), with more than 85% of all apposed lymphocytes displaying angles of less than 30^o between their long nuclear axis and the chord of apposition within the neoplastic cell, which indicates a strong tendency on the part of the nucleus of the lymphocyte to align itself with the apposition membrane of the neoplastic cell.

Although the absolute DNA probe counts of apposed lymphocytes did not vary significantly between the monosomal and polysomal apposed lymphocytes (Table 4), all three cytoplasmic types of lymphocytes showed increases in DNA probe counts while in the apposed position compared to the total lymphocyte population (Table 3), and the degree of preferential asymmetric distribution of DNA probes to that half of the nucleus of the lymphocyte closest to the apposed neoplastic cell increased significantly from less active monosomal lymphocytes to more active polysomal lymphocytes (Table 4).

Discussion.

Previous light microscopy studies have revealed that increased numbers of lymphocytes in the involved pretreatment lymph nodes of patients with Hodgkin's disease correlate strongly with an increased survival for the patient (1, 2), whereas increased numbers of neoplastic cells or of normal macrophages correlate with a decreased survival for the patient (1, 2). In addition, earlier electron microscopy studies have revealed that tight apposition of normal lymphocytes to neoplastic cells within the involved pretreatment lymph nodes of patients with Hodgkin's disease correlates strongly with cytotoxic reactions within such apposed neoplastic cells (7), which suggests that tight apposition by lymphocytes plays a favorable role in opposing neoplastic cell activity in vivo (7). Similarly, preliminary electron microscopic studies have revealed that the unfavorable effect of normal macrophages on patient survival in Hodgkin's disease may be in part related to the observed active phagocytosis of normal lymphocytes by normal macrophages in the involved lymph nodes of patients with Hodgkin's disease (19).

The electron microscopic studies reported here reveal that active polysomal lymphocytes are most likely to be apposed to active immature neoplastic cells, with the parameters of lymphocyte activation (polysomal cytoplasm, apposition membrane sector length, activation vector length, and sector/vector ratio) all signifiacantly increased within such apposed polysomal lymphocytes compared to apposed monosomal lymphocytes.

Moreover, the degree of preferential asymmetric distribution of active DNA templates (5) within the nuclei of apposed lymphocytes is significantly greater for active polysomal lymphocytes than it is for less active monosomal lymphocytes, which suggests that an activation signal from the neoplastic cell has reached as far as the nucleus of the apposed lymphocyte. Since species of low-molecular-weight-immune-RNA are capable of initiating antitumor cellular immune reactions within T-lymphocyte populations (20), and since the majority of apposing lymphocytes in Hodgkin's disease are of the T-type (21), and since low-molecular-weight RNA can return to the cell nucleus (22), there directing the synthesis of new RNA species (23), the possibility that such species of derepressor -immune RNA (24) are mediating an activation signal to the nuclei of apposed lymphocytes in Hodgkin's disease is under active investigation.

Support:

Supported in part by U. S. Public Health Service Research Grants CA10174 and CA13524 from the National Cancer Institute, by Research Grant IC-45 from the American Cancer Society, and by a Research Scholar Award from the Leukemia Society to J.H.F.

References:

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2. Livesey AE, Sutherland FI, Brown RA, et al, "Cytological Basis of Histological Typing of Diffuse Hodgkin's Disease", J. Clin. Pathol. 31, 551-559 (1978).

3. Frenster JH, "Electron Microscopic Localization of Acridine Orange Binding to DNA within Human Leukemic Bone Marrow Cells", Cancer Res. 31, 1128-1133 (1971).

4. Nakatsu SL, Masek MA, Landrum SR, and Frenster JH, "Activity of DNA Templates during Cell Division and Cell Differentiation", Nature 248, 334-335 (1974).

5. Frenster JH, Nakatsu SL, and Papalian MM, "Analysis of Nuclear Structure and Function during Cell Differentiation within Human Marrow", Blood 50, (suppl. 1) 146-147 (1977).

6. Frenster JH, Nakatsu SL, and Masek MA, "Ultrastructural Probes of DNA Templates within Human Bone Marrow and Lymph Node Cells", Adv. Cell Molec. Biol. 3, 1-19 (1974).
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8. Frenster JH, Allfrey VG, and Mirsky AE, "Repressed and Active Chromatin Isolated from Interphase Lymphocytes", Proc. Natl. Acad. Sci. U.S. 50, 1026-1032 (1963).

9. Kumakiri M, and Hashimoto K, "Acridine Orange-DNA Complex in Actinic Keratosis", J. Natl. Cancer Inst. 59, 839-844 (1977).

10. Mantel N, "Chi-Square Tests with One Degree of Freedom: Extensions of the Mantel-Haenszel Procedure", J. Am. Statist. Assoc. 58, 690-700 (1963).

11. Frenster JH, "Ultrastructure and Function of Heterochromatin and Euchromatin", in "The Cell Nucleus", (Busch H, ed.) vol. 1, New York: Academic Press, 1974, pp. 565-580. (Electron Micrographs).

12. Clark JH, Hardin JW, Padykula HA, et al, "Role of Estrogen Receptor Binding and Transcriptional Activity in the Stimulation of Hyperestrogenism and Nuclear Bodies", Proc. Natl. Acad. Sci. U.S. 75, 2781-2784 (1978).

13. Ahearn MJ, Trujillo JM, Cork A, et al, "The Association of Nuclear Blebs with Aneuploidy in Human Acute Leukemia", Cancer Res. 34, 2887-2896 (1974).

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