Symposium on Pediatric Oncology
Diagnostic Methods in Pediatric Oncology Blaise E. Favara, M.D.*
Proper management of the child with neoplasia requires application of the knowledge and experience of an expert pediatric oncologic team. The "community standard of practice" today should be that of the nearest or most convenient pediatric cancer center. As soon as neoplasia is considered in the differential diagnosis of a child's illness, the primary physician should consult a pediatric oncologist. That consultation should result initially in a thoughtful plan concerning the best means of making the diagnosis with least risk of morbidity and mortality to the young patient. Effective therapy can be carried out only if a correct specific diagnosis is made. If a decision is made to transfer the patient to a center for diagnostic studies, the referring physician can anticipate that modern diagnostic methods will be available and used if necessary. Should the decision be to proceed with studies locally, outside of a center, it is imperative that primary physician, local pathologist, consulting pediatric oncologist, and pediatric pathologist work closely to assure that the most valuable specimen is obtained for diagnosis, and that appropriate studies are done. Certainly, it is unwise to expect every pathology laboratory to provide the sophisticated methodology available in the cancer center, but if "pre-biopsy planning" as described above is carried out, the level of diagnostic expertise need not be compromised. Too often biopsy material obtained at significant risk to the patient is inadequate or improperly processed. The habit of thoughtless formalin fixation of all tissues must be broken. The community standard of practice of diagnostic pathology should also be that of the regional pediatric cancer center when neoplasia is being considered in a child. The tissue or pathologic diagnosis should be the expression of an expert opinion based on clinical information and careful study of tissue to which appropriate methods have been applied. The game of "looking at the slide cold" in order to avoid bias, is often entertaining, but making a diagnosis of a malignant disorder with all of its implications is serious business. *Pathologist and Director of Laboratories, The Children's Hospital, Denver, Colorado Supported in part by National Cancer Institute grant CA-12247-01Al.
Pediatric Clinics of North America-Vol. 23, No.1, February 1976
55
56
BLAISE
E.
FAVARA
Before describing specific diagnostic techniques and their application, I would like to comment on the concept of descriptive pathology and clinical correlation beyond conventional diagnosis. The benefits of wedding clinical investigation to good patient care have never been demonstrated more beautifully than in the field ofpediatric oncology. The evolution of complex treatment regimens for specific diseases has brought hope where there was none, as described elsewhere in this symposium. Disease-specific therapy, of course, demands specific correct diagnosis, but response to therapy correctly applied varies. If strong correlations were to be established between special characteri~tics of a tumor and its behavior, therapy could be further customized. It is thus desirable to characterize malignant tissue and cells as thoroughly as capabilities permit and to carefully correlate those characteristics with response to therapy. This step beyond diagnosis lies in the province ofthe research wing of the cancer center's diagnostic unit, but since diagnostic material is the substrate for such work, a joint service-research approach is essential.
ANATOMIC PATHOLOGIC TECHNIQUES IN DIAGNOSIS Although there are important clinical laboratory methods of value in the study of children with suspected malignant neoplasia only anatomic pathological techniques will be considered here. Anatomic methods as described here are those techniques which are applied to the study of malignant tissue. For the purpose of this review, these methods are categorized as conventional morphological, cytochemical, enzyme-cytoche'TIical, ultrastructural, and functional. Conventional Morphologic Methods It is imperative that gross specimens be examined thoroughly and that samples be wisely taken for sections. Multiple samples from large specimens are required for representation of the tumor histology. Buffered 10 per cent formalin is a useful all-purpose fixative; however, Zenker's fixative is better for hematopoietic and lymphoid tissues. Tissue should be 2 to 3 mm thick or less and should be placed in fixative as soon as possible and fixed for no less that 4 hours before processing. Osseous or calcified material should be fixed adequately in formalin and decalcified by a suitable method carefully controlled in order to prevent soft tissue distortion. Paraffin block sections 6 microns in thickness are generally adequate, but lymphoid and hematopoietic tissues require sectioning at 2 to 4 microns for proper interpretation. Well prepared sections stained with hematoxylin and eosin, when studied by an individual experienced in pediatric oncologic pathology, are sufficient for arriving at a correct specific diagnosis in most instances. When frozen sections are required to assist the surgeon in judging adequacy of the operative resection or in assessing adequacy of a diagnostic biopsy, interpretation should be rendered in view of all available clinical infornation and in a conservative manner.
DIAGNOSTIC METHODS IN PEDIATRIC ONCOLOGY
57
Smears of bone marTOW aspirated material should be prepared by a skilled technologist in the operating area. Use of an automatic staining device,* commonly used for blood smears, has been found to yield excellent Wright stains in a reproducible fashion. Biological fluids, such as cerebrospinal fluid, pleural or ascitic fluid, or fluid content of a cyst, are best prepared by use of the cytocentrifuge. Cells can be concentrated and presented as a monolayer with proper use of this instrument. The air-dried cell preparation can then be stained in the same way that bone marTOW or blood smears are handled. Cytochemical Methods Hundreds of cytochemical stains have been employed to characterize the histopathology of tumors. The repertory of such stains in a sophisticated laboratory may be very large, but few are used with any frequency in a diagnostic unit. The following stains are indispensable in the study of pediatric oncologic material: periodic acid-Schiff (PAS) stain for carbohydrate; Masson trichrome stain for connective tissue; phosphotungstic acid-hematoxylin (PTAH) stain for neuroglial fibers, fibrin, muscle striations, and blephroplasts; Sudan IV stain for lipid; and methyl green pyronin (MGP) for RNA and DNA. Forethought is required for proper use of cytochemical methods since fixation and processing affect the results. For example, tissues to be stained for water-soluble carbohydrate must be fixed in an anhydrous alcohol and processed in an anhydrous fashion. Formalin-fixed tissues yield poor results with methyl green pyronin stain, but Zenker's fixative gives excellent results. Enzyme-Cytochemical Methods The application of enzymatic techniques to the study of biopsy material has reached its peak in the field of metabolic disorders, however, certain cytologic methods are quite useful in diagnostic pediatric oncology. The use ofa battery of enzyme-cytochemical studies including alphanaphthol acetate esterase, chloracetate esterase, and peroxidase is essential for precise classification of the leukemias and lymphomas. These tests can be performed on fresh, air-dried monolayers of cells such as those obtained from lymph node imprints, smears of aspirated bone marTOW, peripheral blood smears, and cytocentrifuge preparations of biological fluids. Solid tumors, dissociated by mechanical means and smeared or cytocentrifuged, can also be studied by such techniques. Materials so prepared can be transported to the appropriate laboratory for these special studies within 10 days of procurement of the specimen without compromise of results. Table 1 shows characteristic staining qualities of hematopoietic cells.
'Ames Hema-Tek, Ames Company, Elkhart, Indiana.
I.•
58
BLAISE
E.
FAVARA
Table 1. Cytochemical and Enzyme-Cytochemical Methods in the Study of Malignant Tissue in Children CYTOCHEMICAL AND
CELL TYPE
ENZYME-CYTOCHEMICAL
(MONOCYTIC OR
STAIN
Alphanaphthol acetate esterase Chloroacetate esterase Periodic acid-Schiff Peroxidase Sudan IV
LYMPHOCYTIC
HISTIOCYTIC)
GRANULOCYTIC
+
+ granular
+ ± diffuse ±
±
+ diffuse + +
Ultrastructural Methods Transmission electron microscopy (TEM) and, more recently, scanning electron microscopy (SEM) have been used to characterize tumor and malignant cells. Our experience with TEM over the past seven years in the study of pediatric tumors has provided us with a useful tool for diagnosing the difficult case, for confirming diagnoses made by light microscopy, and for further characterizing tumors. The value of SEM in diagnostic oncology seems to be limited, but our experience ofless than one year with this technique is too brief for further comment. The failure of SEM to differentiate lymphocyte subclasses in a reliable way is a disappointment. For good quality electron microscopy it is essential that tissues be placed in proper fixative immediately. We fix the specimen in the operating room immediately upon removal by placing a 1 x 1 mm cube of tissue in cold 2.5 per cent glutaraldehyde. Material so obtained and fixed can be sent to a laboratory with electron microscopy facilities. Buffering and additional processing are carried out later on batches of specimens. In unusual situations where ultrastructural studies might be of value, but tissue was not fixed in glutaraldehyde initially, an effort should be made to process tissues fixed in formalin. Results of such studies are variable in terms of degree of tissue preservation, but may be rewarding nonetheless.
Functional Methods Tissue culture and identification of lymphoreticular cell surface markers have become important tools in tumor characterization. Both methods require prompt special handling of specimens. Although the diagnostic value of studying living tumor cells in culture media is limited, such material is important to the investigator, and recent developments in immunotherapy will no doubt result in greater demands for such techniques. Our own practice is to attend the biopsy procedure and promptly place a portion of the specimen in culture media using sterile technique. Solid tumor tissue is dissociated. Cell suspensions prepared from dissociated solid tumor, bone marrow, or peripheral blood, after appropriate processing, are also frozen under controlled conditions in liquid nitrogen and kept for subsequent
«
59
DIAGNOSTIC METHODS IN PEDIATRIC ONCOLOGY
investigation. Some of the material is processed for tissue culture which is periodically observed for morphological patterns and developed as a cell line in long-term culture for investigative purposes. Tissue patterns and cell characteristics observed in this system can at time be useful in making a diagnosis. Biopsy material can be placed in appropriate "holding media" and sent to a center for additional processing if directions for handling are followed carefully. The exciting recent developments in the field of immunology and hematopoietic-reticuloendothelial pathology have led to functional classifications of malignant lymphomas 4 and leukemias. 3 The relationship of malignant hematopoietic-reticuloendothelial cells to the immune system forms the basis of such classifications. Lymphocytes are cells with different morphologic and functional characteristics. The morphology of lymphocytes changes as they carry out various functions and evolve from resting to active state. Lymphocytes may be small cells with indented or round nucleus, small with cleaved nucleus, large with cleaved nucleus, large without cleaved nucleus, etc. They may be immunoblasts before becoming a plasma cell. What morphologists once called reticulum cells or histiocytes in Hodgkin's disease and other lymphomas, and in normal and reactive lymphoid tissues actually are in most instances lymphocytes. The Reed Sternberg cell, so important in hematopoietic pathology, is a lymphocyte in the immunoblast stage. Lymphocytes, the malignant cells in most leukemias and lymphomas, therefore have many faces. In addition, lymphocytes with identical morphology by light and electron microscopy differ in their origin and function. Thymic derived lymphocytes (T cells) are related to cell-mediated immunity, whereas those lymphocytes (B cells) which are precursors of plasma cells and involved in humoral immunity are derived from a site which is equivalent to the bursa of Fabricius in the fowl, perhaps the fetal liver and/or bone marrow in the human. Detection of cell surface markers on lymphocytes is currently the means of identifying T and B cells. Those cells without markers are designated null (N) cells. Although there are a number of techniques for detection of the various markers (Table 2) a relatively simple technique is illustrated in Figure l. Using one test, T cells, B cells, null cells, and monocytes can be quantitated in cell suspensions of bone marrow, peripheral blood, cellular Table 2. Cell Surface Markers on Lymphoreticular Cells MONOCYTES B CELLS
Membrane bound Ig Anti T cell serum Sheep RBC rosettes
HISTIOCYTES
+
+
E
EAC (IgM)* EA CIgG)
T CELLS
+ +
*Chicken erythrocytes may be used.
+ +
60
BLAISE
E.
FAVARA
Mononuclear cell suspension
+ Latex granules-phagocytosis by monocytes _ _ _ _ _ _ _ _ _ _
+ / Chicken red blood cells coated with antibody in presence of weak com- ---+ EAC plement
Monocytes with latex markers
/ rosettes~
+
~
B lymphocytes
Sheep red blood cells (untreated) ------+~ E rosettes No reaction
T lymphocytes N lymphocytes
Figure 1. Combined method for surface markers. Note that red blood cells of chickens are elliptical and nucleated making a striking distinction from the red blood cells of sheep.
biological fluids, and dissociated tumor properly processed. We find that cell suspensions that are promptly put into sterile acid, citrate, dextrose (ACD) (available in most blood banks) can be studied with satisfactory results up to 24 hours later. Leukemias or lymphomas are therefore subclassed as to the type of lymphocyte present. For example, we might designate a malignant lymphoma as being malignant lymphoma-immunoblastic B cell type, or acute lymphogenous leukemia-null cell type. These brief descriptions of technique are intended to provide the reader with some idea of what methods are available and useful for pediatric oncologic diagnosis and what each entails in terms of specimen processing. They can be carried out on material obtained outside the laboratory performing such tests, provided that preparatory methods are followed carefully, appropriate fixative or cell support media are on hand, and a system for rapid transportation to the laboratory is available.
Application of Methodology Knowledge of the information that can be derived from these special studies and how such information is used in diagnosis and clinicopathological correlation is vital. Leukemias and malignant lymphomas are the most common malignant disorders affecting children. Although there is no standard classification of the leukemias, the application of cytochemical techniques is necessary for even the most basic differentiation ofleukemic subtypes. It is absolutely essential that lymphogenous and nonlymphogenous types of leukemia be differentiated since the prognosis and therapy differ greatly. This can be done in most instances with careful study of Wright's stained and PAS stained smears of aspirated bone marrow, however the application of a profile of special studies is more rewarding. 2 We routinely study the leuke'nic cell population by cytochemical and enzy'ne cytochemical 'TIethods (Table 1), by transmission and scanning electron
DIAGNOSTIC METHODS IN PEDIATRIC ONCOLOGY
61
microscopy, and additionally the lymphogenous leukemias are characterized by cell surface markers. The results of these studies permit classification of the leukemias as follows: Acute lymphogenous leukemias Lymphoblastic Multiphasic lymphogenous (microlymphoblastic, prolymphocytic, subacute) T, B, N cell subtypes Nonlymphogenous leukemias Myelogenous Acute myeloblastic Multiphasic myelogenous Progranulocytic Myelomonocytic Erythroleukemia Chronic granulocytic Monocytic Acute monoblastic Acute monocytic Histiocytic? Acute undifferentiated leukemia
When a similar approach is taken to the study of malignant lymphomas, a functional classification as proposed by Lukes and Collins can be used to classify these disorders in children. 4 Since follicular lymphomas, Sezary's syndrome, mycosis fungoides, and myelomas are extremely rare in children the remaining categories are indeed few: T cell lymphomas Convoluted lymphocytic Immunoblastic sarcoma B cell lymphomas Plasmacytoid lymphocytic Immunoblastic sarcoma Burkitt's Undefined lymphocytic (null cell) Histiocytic Unclassified
Among the solid tumors of children those affecting the central nervous system are the most common. Precise categorization of brain tumors is greatly aided by cytochemical stains, transmission electron microscopy, and observation of patterns of growth in tissue culture. Gliomas of ependymal origin are readily distinguished from those of astrocytic origin by ultrastructural characteristics which may not be readily apparent using light microscopy. The PTAH stain is quite useful for identifying astrocytic processes in a tumor. In the study of other solid tumors we find transmission electron microscopy to be of great value in those with poorly differentiated histologic features. Such tumors are relatively common in children. In some instances a tu mor is identified ultrastructurally by recognition of cell organelles which are specific for the cell type comprising the tumor. An example of this specificity may be seen in rhabdomyosarcoma. Organization of actin and myosin filaments and Z bands in the cytoplasm of some sufficiently differentiated cells clearly indicates a striated muscle source of the neoplasm (Figure 2, A).
62
BLAISE
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FAVARA
Figure 2. A, Rhabdomyosarcoma with cytoplasmic actin and myosin filaments organized in some areas into Z bands (arrow). (x 28,600.) B, Neuroblastoma. Cell processes contain secretory granules (thick arrow) and microtubules (thin arrow). (x 38,000.)
DIAGNOSTIC METHODS IN PEDIATRIC ONCOLOGY
63
Figure 2 (Continued). C, Carcinoma of pancreas. Acinar cell type identified by presence of zymogenic granules (arrow )incytoplasm. (x 8,000. )D, Langerhans' granules designate this histiocyte as a Langerhans' cell in a child with cutaneous and osseous disease. (x 86,800.)
64
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E.
FAVARA
Neuroblastoma can usually be shown to have neurosecretory granules and microtubules in the cytoplasn ofthose cells differentiated to that degree (Figure 2,B). Such granules are present in the cytoplasm of the characteristic cells ofthe APUD system 4 in general, neuroblastoma being the most common tumor ofthat system in children. Neuroblastoma cells in tissue culture develop characteristic neuroprocesses within 48 hours after innoculation and can be readily identified in this way. In some instances cell organelles are common to a variety of cells and one must therefore rely on their arrangement and their association with other organelles in determining histogenesis of a given neoplasm. Examples of such tumors are primary hepatic tumors, nephroblastoma, fibrosarcoma, and tumors arising from ovary and testis. We find the electron microscope to be quite useful in the study of pancreatic tumors since the cytoplasmic granules of endocrine (APUDrelated) and acinar cells ofthe pancreas are quite characteristic. A poorly differentiated carcinoma ofthe pancreas shown by electron microscopy to be of acinar cell origin is depicted in Figure 2,C. Here the zymogenic granules are specific for the histogenetic cell line. Recently, a number of patients referred to our center had been diagnosed as having carcinomas metastatic to the lymph nodes; however, on further study, the patients proved to have immunoblastic sarcomas. The methyl green pyronin stain for cytoplasmic RNA and the electron microscope have been quite helpful in identifying lymphoid cells and thus correctly diagnosing the process as a lymphomatous one. Appropriate therapy can then be given and an extensive search for a primary tumor outside the lymphoreticular system is avoided. The reticuloses (histiocytoses) occurring in children present particularly troublesome problems in diagnosis, and no doubt represent one of the more confusing areas of pediatric pathology.5 It is not clear as to whether these processes are reactive, aberrant immune responses or indeed neoplastic in type. The identification of Langerhans' granules (Figure 2, D) in cells involved in these processes seems to be a marker of what is called the histiocytosis X spectrum. This spectrum encompasses "Letterer-Siewe, Hand-Schiiller-Christian, and eosinophilic granuloma syndromes." This organelle, the Langerhans' granule (detectable only by electron microscopy), seemingly characterizes a special type ofhistiocyte and may indicate benign behavior of the pathologic process. Those reticuloses in which Langerhans' granules are absent tend in general to behave in a more aggressive and possibly lethal fashion. Application of the multiple techniques described in this presentation will no doubt help to further understanding of this extremely confusing group of pathologic processes.
SUMMARY Diagnosing malignant disorders in pediatric patients demands a good working relationship between informed clinician and informed pathologist and the application of sophisticated methodology in order to arrive at
DIAGNOSTIC METHODS IN PEDIATRIC ONCOLOGY
65
correct specific diagnoses and thus appropriate patient management programs. Since neoplastic and related disorders are not common in children, every occurrence of these disorders should be viewed as an investigational opportunity in order to increase our understanding of pathogenesis and ultimately to reduce the morbidity and mortality in children.
REFERENCES l. Baylin, S. B.: Ectopic production of hormones and other proteins by tumors. Hosp.
Pract., 10: 117-126, 1975. 2. Haegert, D. G., Stuart, J., and Smith, J. L.: Acute lymphoblastic leukaemia; A heterogeneous disease. Brit. Med. J., 1 :312-314, 1974. 3. Hayhoe, F. G. J., and Cawley, J. C.: Acute leukemia: Cellular morphology, cytochemistry and fine structure. Clin. Haematol, 1 :49-94, 1972. 4. Lukes, R. J., and Collins, R. D.: A functional classification of malignant lymphomas. In Reticuloendothelial System-International Academy of Pathology Monographs. Baltimore, Williams and Wilkins Co., 1975, pp. 213-242. 5. Newton, W. A., Jr., and Hamoudi, A. B.: Histiocytosis: A histologic classification and clinical correlation. In Perspectives in Pediatric Pathology, Vol. l. Chicago, Year Book Medical Publishers, Inc., 1973, pp. 251-283. The Children's Hospital 1056 East Nineteenth Avenue Denver, Colorado 80218
.. .,;
Diagnostic Methods in Pediatric Oncology Blaise E. Favara, M.D.*
Proper management of the child with neoplasia requires application of the knowledge and experience of an expert pediatric oncologic team. The "community standard of practice" today should be that of the nearest or most convenient pediatric cancer center. As soon as neoplasia is considered in the differential diagnosis of a child's illness, the primary physician should consult a pediatric oncologist. That consultation should result initially in a thoughtful plan concerning the best means of making the diagnosis with least risk of morbidity and mortality to the young patient. Effective therapy can be carried out only if a correct specific diagnosis is made. If a decision is made to transfer the patient to a center for diagnostic studies, the referring physician can anticipate that modern diagnostic methods will be available and used if necessary. Should the decision be to proceed with studies locally, outside of a center, it is imperative that primary physician, local pathologist, consulting pediatric oncologist, and pediatric pathologist work closely to assure that the most valuable specimen is obtained for diagnosis, and that appropriate studies are done. Certainly, it is unwise to expect every pathology laboratory to provide the sophisticated methodology available in the cancer center, but if "pre-biopsy planning" as described above is carried out, the level of diagnostic expertise need not be compromised. Too often biopsy material obtained at significant risk to the patient is inadequate or improperly processed. The habit of thoughtless formalin fixation of all tissues must be broken. The community standard of practice of diagnostic pathology should also be that of the regional pediatric cancer center when neoplasia is being considered in a child. The tissue or pathologic diagnosis should be the expression of an expert opinion based on clinical information and careful study of tissue to which appropriate methods have been applied. The game of "looking at the slide cold" in order to avoid bias, is often entertaining, but making a diagnosis of a malignant disorder with all of its implications is serious business. *Pathologist and Director of Laboratories, The Children's Hospital, Denver, Colorado Supported in part by National Cancer Institute grant CA-12247-01Al.
Pediatric Clinics of North America-Vol. 23, No.1, February 1976
55
56
BLAISE
E.
FAVARA
Before describing specific diagnostic techniques and their application, I would like to comment on the concept of descriptive pathology and clinical correlation beyond conventional diagnosis. The benefits of wedding clinical investigation to good patient care have never been demonstrated more beautifully than in the field ofpediatric oncology. The evolution of complex treatment regimens for specific diseases has brought hope where there was none, as described elsewhere in this symposium. Disease-specific therapy, of course, demands specific correct diagnosis, but response to therapy correctly applied varies. If strong correlations were to be established between special characteri~tics of a tumor and its behavior, therapy could be further customized. It is thus desirable to characterize malignant tissue and cells as thoroughly as capabilities permit and to carefully correlate those characteristics with response to therapy. This step beyond diagnosis lies in the province ofthe research wing of the cancer center's diagnostic unit, but since diagnostic material is the substrate for such work, a joint service-research approach is essential.
ANATOMIC PATHOLOGIC TECHNIQUES IN DIAGNOSIS Although there are important clinical laboratory methods of value in the study of children with suspected malignant neoplasia only anatomic pathological techniques will be considered here. Anatomic methods as described here are those techniques which are applied to the study of malignant tissue. For the purpose of this review, these methods are categorized as conventional morphological, cytochemical, enzyme-cytoche'TIical, ultrastructural, and functional. Conventional Morphologic Methods It is imperative that gross specimens be examined thoroughly and that samples be wisely taken for sections. Multiple samples from large specimens are required for representation of the tumor histology. Buffered 10 per cent formalin is a useful all-purpose fixative; however, Zenker's fixative is better for hematopoietic and lymphoid tissues. Tissue should be 2 to 3 mm thick or less and should be placed in fixative as soon as possible and fixed for no less that 4 hours before processing. Osseous or calcified material should be fixed adequately in formalin and decalcified by a suitable method carefully controlled in order to prevent soft tissue distortion. Paraffin block sections 6 microns in thickness are generally adequate, but lymphoid and hematopoietic tissues require sectioning at 2 to 4 microns for proper interpretation. Well prepared sections stained with hematoxylin and eosin, when studied by an individual experienced in pediatric oncologic pathology, are sufficient for arriving at a correct specific diagnosis in most instances. When frozen sections are required to assist the surgeon in judging adequacy of the operative resection or in assessing adequacy of a diagnostic biopsy, interpretation should be rendered in view of all available clinical infornation and in a conservative manner.
DIAGNOSTIC METHODS IN PEDIATRIC ONCOLOGY
57
Smears of bone marTOW aspirated material should be prepared by a skilled technologist in the operating area. Use of an automatic staining device,* commonly used for blood smears, has been found to yield excellent Wright stains in a reproducible fashion. Biological fluids, such as cerebrospinal fluid, pleural or ascitic fluid, or fluid content of a cyst, are best prepared by use of the cytocentrifuge. Cells can be concentrated and presented as a monolayer with proper use of this instrument. The air-dried cell preparation can then be stained in the same way that bone marTOW or blood smears are handled. Cytochemical Methods Hundreds of cytochemical stains have been employed to characterize the histopathology of tumors. The repertory of such stains in a sophisticated laboratory may be very large, but few are used with any frequency in a diagnostic unit. The following stains are indispensable in the study of pediatric oncologic material: periodic acid-Schiff (PAS) stain for carbohydrate; Masson trichrome stain for connective tissue; phosphotungstic acid-hematoxylin (PTAH) stain for neuroglial fibers, fibrin, muscle striations, and blephroplasts; Sudan IV stain for lipid; and methyl green pyronin (MGP) for RNA and DNA. Forethought is required for proper use of cytochemical methods since fixation and processing affect the results. For example, tissues to be stained for water-soluble carbohydrate must be fixed in an anhydrous alcohol and processed in an anhydrous fashion. Formalin-fixed tissues yield poor results with methyl green pyronin stain, but Zenker's fixative gives excellent results. Enzyme-Cytochemical Methods The application of enzymatic techniques to the study of biopsy material has reached its peak in the field of metabolic disorders, however, certain cytologic methods are quite useful in diagnostic pediatric oncology. The use ofa battery of enzyme-cytochemical studies including alphanaphthol acetate esterase, chloracetate esterase, and peroxidase is essential for precise classification of the leukemias and lymphomas. These tests can be performed on fresh, air-dried monolayers of cells such as those obtained from lymph node imprints, smears of aspirated bone marTOW, peripheral blood smears, and cytocentrifuge preparations of biological fluids. Solid tumors, dissociated by mechanical means and smeared or cytocentrifuged, can also be studied by such techniques. Materials so prepared can be transported to the appropriate laboratory for these special studies within 10 days of procurement of the specimen without compromise of results. Table 1 shows characteristic staining qualities of hematopoietic cells.
'Ames Hema-Tek, Ames Company, Elkhart, Indiana.
I.•
58
BLAISE
E.
FAVARA
Table 1. Cytochemical and Enzyme-Cytochemical Methods in the Study of Malignant Tissue in Children CYTOCHEMICAL AND
CELL TYPE
ENZYME-CYTOCHEMICAL
(MONOCYTIC OR
STAIN
Alphanaphthol acetate esterase Chloroacetate esterase Periodic acid-Schiff Peroxidase Sudan IV
LYMPHOCYTIC
HISTIOCYTIC)
GRANULOCYTIC
+
+ granular
+ ± diffuse ±
±
+ diffuse + +
Ultrastructural Methods Transmission electron microscopy (TEM) and, more recently, scanning electron microscopy (SEM) have been used to characterize tumor and malignant cells. Our experience with TEM over the past seven years in the study of pediatric tumors has provided us with a useful tool for diagnosing the difficult case, for confirming diagnoses made by light microscopy, and for further characterizing tumors. The value of SEM in diagnostic oncology seems to be limited, but our experience ofless than one year with this technique is too brief for further comment. The failure of SEM to differentiate lymphocyte subclasses in a reliable way is a disappointment. For good quality electron microscopy it is essential that tissues be placed in proper fixative immediately. We fix the specimen in the operating room immediately upon removal by placing a 1 x 1 mm cube of tissue in cold 2.5 per cent glutaraldehyde. Material so obtained and fixed can be sent to a laboratory with electron microscopy facilities. Buffering and additional processing are carried out later on batches of specimens. In unusual situations where ultrastructural studies might be of value, but tissue was not fixed in glutaraldehyde initially, an effort should be made to process tissues fixed in formalin. Results of such studies are variable in terms of degree of tissue preservation, but may be rewarding nonetheless.
Functional Methods Tissue culture and identification of lymphoreticular cell surface markers have become important tools in tumor characterization. Both methods require prompt special handling of specimens. Although the diagnostic value of studying living tumor cells in culture media is limited, such material is important to the investigator, and recent developments in immunotherapy will no doubt result in greater demands for such techniques. Our own practice is to attend the biopsy procedure and promptly place a portion of the specimen in culture media using sterile technique. Solid tumor tissue is dissociated. Cell suspensions prepared from dissociated solid tumor, bone marrow, or peripheral blood, after appropriate processing, are also frozen under controlled conditions in liquid nitrogen and kept for subsequent
«
59
DIAGNOSTIC METHODS IN PEDIATRIC ONCOLOGY
investigation. Some of the material is processed for tissue culture which is periodically observed for morphological patterns and developed as a cell line in long-term culture for investigative purposes. Tissue patterns and cell characteristics observed in this system can at time be useful in making a diagnosis. Biopsy material can be placed in appropriate "holding media" and sent to a center for additional processing if directions for handling are followed carefully. The exciting recent developments in the field of immunology and hematopoietic-reticuloendothelial pathology have led to functional classifications of malignant lymphomas 4 and leukemias. 3 The relationship of malignant hematopoietic-reticuloendothelial cells to the immune system forms the basis of such classifications. Lymphocytes are cells with different morphologic and functional characteristics. The morphology of lymphocytes changes as they carry out various functions and evolve from resting to active state. Lymphocytes may be small cells with indented or round nucleus, small with cleaved nucleus, large with cleaved nucleus, large without cleaved nucleus, etc. They may be immunoblasts before becoming a plasma cell. What morphologists once called reticulum cells or histiocytes in Hodgkin's disease and other lymphomas, and in normal and reactive lymphoid tissues actually are in most instances lymphocytes. The Reed Sternberg cell, so important in hematopoietic pathology, is a lymphocyte in the immunoblast stage. Lymphocytes, the malignant cells in most leukemias and lymphomas, therefore have many faces. In addition, lymphocytes with identical morphology by light and electron microscopy differ in their origin and function. Thymic derived lymphocytes (T cells) are related to cell-mediated immunity, whereas those lymphocytes (B cells) which are precursors of plasma cells and involved in humoral immunity are derived from a site which is equivalent to the bursa of Fabricius in the fowl, perhaps the fetal liver and/or bone marrow in the human. Detection of cell surface markers on lymphocytes is currently the means of identifying T and B cells. Those cells without markers are designated null (N) cells. Although there are a number of techniques for detection of the various markers (Table 2) a relatively simple technique is illustrated in Figure l. Using one test, T cells, B cells, null cells, and monocytes can be quantitated in cell suspensions of bone marrow, peripheral blood, cellular Table 2. Cell Surface Markers on Lymphoreticular Cells MONOCYTES B CELLS
Membrane bound Ig Anti T cell serum Sheep RBC rosettes
HISTIOCYTES
+
+
E
EAC (IgM)* EA CIgG)
T CELLS
+ +
*Chicken erythrocytes may be used.
+ +
60
BLAISE
E.
FAVARA
Mononuclear cell suspension
+ Latex granules-phagocytosis by monocytes _ _ _ _ _ _ _ _ _ _
+ / Chicken red blood cells coated with antibody in presence of weak com- ---+ EAC plement
Monocytes with latex markers
/ rosettes~
+
~
B lymphocytes
Sheep red blood cells (untreated) ------+~ E rosettes No reaction
T lymphocytes N lymphocytes
Figure 1. Combined method for surface markers. Note that red blood cells of chickens are elliptical and nucleated making a striking distinction from the red blood cells of sheep.
biological fluids, and dissociated tumor properly processed. We find that cell suspensions that are promptly put into sterile acid, citrate, dextrose (ACD) (available in most blood banks) can be studied with satisfactory results up to 24 hours later. Leukemias or lymphomas are therefore subclassed as to the type of lymphocyte present. For example, we might designate a malignant lymphoma as being malignant lymphoma-immunoblastic B cell type, or acute lymphogenous leukemia-null cell type. These brief descriptions of technique are intended to provide the reader with some idea of what methods are available and useful for pediatric oncologic diagnosis and what each entails in terms of specimen processing. They can be carried out on material obtained outside the laboratory performing such tests, provided that preparatory methods are followed carefully, appropriate fixative or cell support media are on hand, and a system for rapid transportation to the laboratory is available.
Application of Methodology Knowledge of the information that can be derived from these special studies and how such information is used in diagnosis and clinicopathological correlation is vital. Leukemias and malignant lymphomas are the most common malignant disorders affecting children. Although there is no standard classification of the leukemias, the application of cytochemical techniques is necessary for even the most basic differentiation ofleukemic subtypes. It is absolutely essential that lymphogenous and nonlymphogenous types of leukemia be differentiated since the prognosis and therapy differ greatly. This can be done in most instances with careful study of Wright's stained and PAS stained smears of aspirated bone marrow, however the application of a profile of special studies is more rewarding. 2 We routinely study the leuke'nic cell population by cytochemical and enzy'ne cytochemical 'TIethods (Table 1), by transmission and scanning electron
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microscopy, and additionally the lymphogenous leukemias are characterized by cell surface markers. The results of these studies permit classification of the leukemias as follows: Acute lymphogenous leukemias Lymphoblastic Multiphasic lymphogenous (microlymphoblastic, prolymphocytic, subacute) T, B, N cell subtypes Nonlymphogenous leukemias Myelogenous Acute myeloblastic Multiphasic myelogenous Progranulocytic Myelomonocytic Erythroleukemia Chronic granulocytic Monocytic Acute monoblastic Acute monocytic Histiocytic? Acute undifferentiated leukemia
When a similar approach is taken to the study of malignant lymphomas, a functional classification as proposed by Lukes and Collins can be used to classify these disorders in children. 4 Since follicular lymphomas, Sezary's syndrome, mycosis fungoides, and myelomas are extremely rare in children the remaining categories are indeed few: T cell lymphomas Convoluted lymphocytic Immunoblastic sarcoma B cell lymphomas Plasmacytoid lymphocytic Immunoblastic sarcoma Burkitt's Undefined lymphocytic (null cell) Histiocytic Unclassified
Among the solid tumors of children those affecting the central nervous system are the most common. Precise categorization of brain tumors is greatly aided by cytochemical stains, transmission electron microscopy, and observation of patterns of growth in tissue culture. Gliomas of ependymal origin are readily distinguished from those of astrocytic origin by ultrastructural characteristics which may not be readily apparent using light microscopy. The PTAH stain is quite useful for identifying astrocytic processes in a tumor. In the study of other solid tumors we find transmission electron microscopy to be of great value in those with poorly differentiated histologic features. Such tumors are relatively common in children. In some instances a tu mor is identified ultrastructurally by recognition of cell organelles which are specific for the cell type comprising the tumor. An example of this specificity may be seen in rhabdomyosarcoma. Organization of actin and myosin filaments and Z bands in the cytoplasm of some sufficiently differentiated cells clearly indicates a striated muscle source of the neoplasm (Figure 2, A).
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Figure 2. A, Rhabdomyosarcoma with cytoplasmic actin and myosin filaments organized in some areas into Z bands (arrow). (x 28,600.) B, Neuroblastoma. Cell processes contain secretory granules (thick arrow) and microtubules (thin arrow). (x 38,000.)
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Figure 2 (Continued). C, Carcinoma of pancreas. Acinar cell type identified by presence of zymogenic granules (arrow )incytoplasm. (x 8,000. )D, Langerhans' granules designate this histiocyte as a Langerhans' cell in a child with cutaneous and osseous disease. (x 86,800.)
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Neuroblastoma can usually be shown to have neurosecretory granules and microtubules in the cytoplasn ofthose cells differentiated to that degree (Figure 2,B). Such granules are present in the cytoplasm of the characteristic cells ofthe APUD system 4 in general, neuroblastoma being the most common tumor ofthat system in children. Neuroblastoma cells in tissue culture develop characteristic neuroprocesses within 48 hours after innoculation and can be readily identified in this way. In some instances cell organelles are common to a variety of cells and one must therefore rely on their arrangement and their association with other organelles in determining histogenesis of a given neoplasm. Examples of such tumors are primary hepatic tumors, nephroblastoma, fibrosarcoma, and tumors arising from ovary and testis. We find the electron microscope to be quite useful in the study of pancreatic tumors since the cytoplasmic granules of endocrine (APUDrelated) and acinar cells ofthe pancreas are quite characteristic. A poorly differentiated carcinoma ofthe pancreas shown by electron microscopy to be of acinar cell origin is depicted in Figure 2,C. Here the zymogenic granules are specific for the histogenetic cell line. Recently, a number of patients referred to our center had been diagnosed as having carcinomas metastatic to the lymph nodes; however, on further study, the patients proved to have immunoblastic sarcomas. The methyl green pyronin stain for cytoplasmic RNA and the electron microscope have been quite helpful in identifying lymphoid cells and thus correctly diagnosing the process as a lymphomatous one. Appropriate therapy can then be given and an extensive search for a primary tumor outside the lymphoreticular system is avoided. The reticuloses (histiocytoses) occurring in children present particularly troublesome problems in diagnosis, and no doubt represent one of the more confusing areas of pediatric pathology.5 It is not clear as to whether these processes are reactive, aberrant immune responses or indeed neoplastic in type. The identification of Langerhans' granules (Figure 2, D) in cells involved in these processes seems to be a marker of what is called the histiocytosis X spectrum. This spectrum encompasses "Letterer-Siewe, Hand-Schiiller-Christian, and eosinophilic granuloma syndromes." This organelle, the Langerhans' granule (detectable only by electron microscopy), seemingly characterizes a special type ofhistiocyte and may indicate benign behavior of the pathologic process. Those reticuloses in which Langerhans' granules are absent tend in general to behave in a more aggressive and possibly lethal fashion. Application of the multiple techniques described in this presentation will no doubt help to further understanding of this extremely confusing group of pathologic processes.
SUMMARY Diagnosing malignant disorders in pediatric patients demands a good working relationship between informed clinician and informed pathologist and the application of sophisticated methodology in order to arrive at
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correct specific diagnoses and thus appropriate patient management programs. Since neoplastic and related disorders are not common in children, every occurrence of these disorders should be viewed as an investigational opportunity in order to increase our understanding of pathogenesis and ultimately to reduce the morbidity and mortality in children.
REFERENCES l. Baylin, S. B.: Ectopic production of hormones and other proteins by tumors. Hosp.
Pract., 10: 117-126, 1975. 2. Haegert, D. G., Stuart, J., and Smith, J. L.: Acute lymphoblastic leukaemia; A heterogeneous disease. Brit. Med. J., 1 :312-314, 1974. 3. Hayhoe, F. G. J., and Cawley, J. C.: Acute leukemia: Cellular morphology, cytochemistry and fine structure. Clin. Haematol, 1 :49-94, 1972. 4. Lukes, R. J., and Collins, R. D.: A functional classification of malignant lymphomas. In Reticuloendothelial System-International Academy of Pathology Monographs. Baltimore, Williams and Wilkins Co., 1975, pp. 213-242. 5. Newton, W. A., Jr., and Hamoudi, A. B.: Histiocytosis: A histologic classification and clinical correlation. In Perspectives in Pediatric Pathology, Vol. l. Chicago, Year Book Medical Publishers, Inc., 1973, pp. 251-283. The Children's Hospital 1056 East Nineteenth Avenue Denver, Colorado 80218
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