British Journal oj Dermatology (1975) 92, 273.
Tumour angiogenic factor associated with subcutaneous lymphoma JOHN E.WOLF AND WINTHROPE R.HUBLER, JR. Department of Dermatology, Baylor College of Medicine, 1200 Moursund Ave., Houston, Texas 77025
Accepted for publication 15 August 1974
SUMMARY
Growth and survival of cutaneous neoplasms are dependent on the development of a nutrient-supply ing vascular lifeline. A subcutaneous lymphoma is described which mimicked a vascular neoplasm because of the extensive telangiectasia overlying the tumour. Fragments of the lymphoma inserted in direct apposition to the hamster cheek pouch membrane or separated from the membrane by a microporous filter both induced dramatic ncovascularization. Control materials failed to induce significant vascular changes. A ditfusablc Tumour Angiogenic Factor may play a vital role in tumour survival and portends a therapeutic potential. As neoplastic cells proliferate and expand into a distinct tumour mass, their nutritional needs soon exceed the limits of interstitial fluids. It is then essential for the growing tumour that an adequate blood supply should be established. Ide, Baker & Warren (1939) and Algire & Chalkley (1945) have suggested that tumours are capable of meeting this Darwinian challenge by indirect stimulation of neovascularization in the host's vascular plexus. Goodall, Sanders & Shubik (1965) and Grcenblatt & Shubik (1968) have subsequently demonstrated neovascularization with tumour implants in the hamster cheek pouch; Folkman et al. (1970) have isolated a diffusablc Tumour Angiogenic Factor (TAF). In this report we document, clinically and experimentally, the elaboration of a TAF by a malignant subcutaneous lymphoma. CASE REPORT
A 4-year~old Oriental-American girl presented with a 2-o x 30 cm subcutaneous mass in the centra forehead. The lesion had been noted 4 months earlier after a traumatic injury and had slowly increased in size; it was firm, moveable and non-tender. The most striking clinical feature was dramatic erythema and telangiectasia overlying the lesion. The appearance was that of a vascular tumour and the initial clinical differential diagnosis included haematoma, haemangioma and haemangio-cndothelioma. An excisional biopsy was performed because of the increasing size of the lesion and a lobulated, 273
John E.Wolfand Winthrope R.HuhlerJr.
274
non-encapsulated tumour was retnoved and subtnitted for routine histopathology. Sections ot tutnour stained with haetnatoxylin and eosin revealed densely-packed dermal masses of stnall deeply basophilic cells extending into the subcutis (Figs, i and 2). The histopathological diagnosis was 'malignant lymphoma, undifferentiated, non-Burkitt type.'
FIGURE I. Dermal nodules of basophilic cells extending into the subcutis diagnosed as subcutaneous lymphoma (H & E, x 44),
Investigations
Physical examination was completely normal with the exception of the above-mentioned frontoglabellar mass. The white blood cell count was i8,9OO;mm'\ with 40",, lymphocytes and 5",, monocytes. Other laboratory findings were entirely within normal litnittj including the following: chest X-ray, skull X-ray, complete radiological bone survey, intravenous pyclogram, bone marrow aspiration, haemoglobin, haematocrit, platelet count, blood urea nitrogen, fasting blood glucose, and liver function tests. Since the white cell count was elevated and this type of lymphoma is often considered pre-leukaemic, the patient received a course of radiation therapy to the tumour site combined with systemic chemotherapy (vincristine, methotrexate and prednisone). Six months after surgery the patient is in good health with no signs of local tumour recurrence or systemic illness. Demonstration of TAF Fragments of the surgically excised lymphoma were transported to the laboratory in refrigerated normal saline and immediately implanted into hamster check pouch chambers by the classical tech-
Tumour angiogenic factor with lymphoma
275
FIGURE 2. The dtep dermal nodules of the excised lumour were composed ol' deeply basophilic lymphocytes.
nique of Sanders & Shubik (1964), Grccnblatt and Shubik (1968) and Warren and Shubik (1966). The immunologically 'privileged' domain of the hamster cheek pouch allowed us to observe the eifect of tumour implants on small blood vessels in an anatomically and physiologically normal setting. In six hamsters, 20 mm tumour fragments were implanted in direct apposition to the vascular membrane of the cheek pouch. In an additional six hamsters, similar implants were separated from the cheek pouch vascular bed by 6 0 mm discs of millipore filter (Type TA, 0 45 |im pore size, 25 fim thick) in order to test for the presence of a difFusablc Tumour Angiogenic Factor. Implanted materials were observed for 7-14 days with a stereoscopic binocular microscope and with an American Optical microscope with long distance objectives. The vascular response of the cheek pouch membrane to tumour grafts was contrasted with that to normal hiaman dermis, keloid, naevocellular nevi and inert control materials (Millipore filter, dialysis membrane). Lymphoma grafts stimulated neovascularization in the hamster cheek pouch directly and across a microporous membrane (Millipore) barrier. The usual sequence of vascular events consisted of arterial tortuosity and vasodilation, followed by early sprouting of tiny capillary buds, overgrowth of implants and establishment of complex functioning vascular arcades (Fig. 3). Controls showed occasional tortuosity and dilatation without angiogenesis. DISCUSSION
Greenblatt & Shubik (1968), Goodall et al. (1965) and Warren & Shubik (1966) demonstrated a diffusablcj angiogenically active substance in the hamster cheek pouch with direct tumour implants and transfilter diffusion studies. Folkman et al. (1970) have subsequently isolated a Tumour Angiogenic Factor and characterized it as a heat-labile^ cryostable iipoprotcin containing 25",, RNA and 50%
276
John E.Wolfand Wimhrope R.HiiblerJr.
FIGURE 3. Fragments of the subcutaneous iymphoma elicited the formation of reactive nuovascular arcades in the hamster check pouch membrane across a Millipore filter (>: 100).
carbohydrate. The exact mechanism of action of TAF is not entirely clear, but Warren, Greenblatt & Kommineni (1972), Folkman et al. (1970) and Cavallo et al. {i^-ji) offered evidence that the factor is directly mitogenic for capillary endothelial cells. In this report we have demonstrated the production of TAF by a malignant subcutaneous Iymphoma in order to italicize the immediate and potential dcrmatological implications of this angiogenic factor. The tumour reported was initially thought to be a blood vessel neoplasm when presented at a university teaching conference, thus emphasizing the clinically vascular appearance of the lesion. Human skin tumours arc often associated with alteration of cutaneous vessels—for example the telangicctasia seen with basal cell cpithclioma, subcutaneous mcdulloblastoma and metastatic carcinoma (Primack, 1973). These diagnostically significant vascular changes may be due to capillary proliferation induced by a Tumour Angiogenic Factor. There is also a chance that TAF may one day assume a therapeutic potential. Tannock (1968) has demonstrated that the expansion of a tumour mass is directly limited by Its blood supply and Folkman ct al. (1970) have suggested that tumour growth might be arrested by interfering with the elaboration and/or function of TAF. If so, cutaneous neoplasms, exquisitely visible and eminently accessible, might provide an excellent proving ground for the theory. In this report, we have utilized laboratory techniques to confirm a clinically relevant concept with widespread implications for dermatology.
Titmottr angiogenic factor with lymphoma
277
REFERENCES ALGIRE, G.H. & CHALKLEY, H . W . (1945) Vasctilar reactions of normal and malignant tissues i'» vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic tr^n^'plani^. Journal of the National Cancer Institute, 6,73CAVALLO, T.jSADiijR., FOLKMAN, J.& COTRNA,R.S. (1972) Tumor Angiogenesis: Rapid induction of endothelial mitosis demonstrated by autoradiography. Journal of Cell Biology, 54, 408. FOLKMAN, J., MERLER, E., ABERNATHY, C . & WILLIAMS, G . (1970) Isolation of a tumor factor responsible for
angiogenesis. _7oi^?7(ti/ of Experimental Medicine, 133, 275. GOODALL, G.M., SANDERS, A.G. & SHUBIK, P. (1965) Studies of vascular patterns in living tumors with a transparent chamber inserted in hamster cheek pouch. 7'"""'"'^ of the National Cancer Institute, 35, 497. GREENBLATT, M . & SHUBIK, P. (.1968) Tumor Angiogenesis: Transfiltcr diffusion studies in the hamster by the transparent chamber technique. Journal of the National Cancer Institute, 41, 111. IDE, H.G., BAKER, N . H . & WARREN, S.L. (1939) Vascularization of the Brown-Pearce rabbit epithelioma transplant as seen in the transparent ear chamber. American Journal of Roe?itgetiology, 42, 891. PRIMACK, A. (1973) Telangiectasia overlying a malignant tumor. Archives of Dermatology, 108, 704. SANDERS, A.G. & SHUBIK, P. (1964) A transplant window for use in the Syrian hamster. Israel Journal of Experimental Medicine, II, 118. TANNOCK, I . F . (1968) The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumor. British Journal of Cancer, 22, 258. WARREN, B.A. & SHUBIK, P. (1966) The growth of the blood supply to melanoma transplants in the hamster cheek pouch. Laboratory Investigations, 15, 464. WARREN, B.A., GREENBLATT, M . & KOMMINENI, V . R . C . (1972) Tumor angiogenesis: Ultrastructure of endo-
thelial cells in mitosis. British Journal of Experimental Pathology, 53, 216.
Tumour angiogenic factor associated with subcutaneous lymphoma JOHN E.WOLF AND WINTHROPE R.HUBLER, JR. Department of Dermatology, Baylor College of Medicine, 1200 Moursund Ave., Houston, Texas 77025
Accepted for publication 15 August 1974
SUMMARY
Growth and survival of cutaneous neoplasms are dependent on the development of a nutrient-supply ing vascular lifeline. A subcutaneous lymphoma is described which mimicked a vascular neoplasm because of the extensive telangiectasia overlying the tumour. Fragments of the lymphoma inserted in direct apposition to the hamster cheek pouch membrane or separated from the membrane by a microporous filter both induced dramatic ncovascularization. Control materials failed to induce significant vascular changes. A ditfusablc Tumour Angiogenic Factor may play a vital role in tumour survival and portends a therapeutic potential. As neoplastic cells proliferate and expand into a distinct tumour mass, their nutritional needs soon exceed the limits of interstitial fluids. It is then essential for the growing tumour that an adequate blood supply should be established. Ide, Baker & Warren (1939) and Algire & Chalkley (1945) have suggested that tumours are capable of meeting this Darwinian challenge by indirect stimulation of neovascularization in the host's vascular plexus. Goodall, Sanders & Shubik (1965) and Grcenblatt & Shubik (1968) have subsequently demonstrated neovascularization with tumour implants in the hamster cheek pouch; Folkman et al. (1970) have isolated a diffusablc Tumour Angiogenic Factor (TAF). In this report we document, clinically and experimentally, the elaboration of a TAF by a malignant subcutaneous lymphoma. CASE REPORT
A 4-year~old Oriental-American girl presented with a 2-o x 30 cm subcutaneous mass in the centra forehead. The lesion had been noted 4 months earlier after a traumatic injury and had slowly increased in size; it was firm, moveable and non-tender. The most striking clinical feature was dramatic erythema and telangiectasia overlying the lesion. The appearance was that of a vascular tumour and the initial clinical differential diagnosis included haematoma, haemangioma and haemangio-cndothelioma. An excisional biopsy was performed because of the increasing size of the lesion and a lobulated, 273
John E.Wolfand Winthrope R.HuhlerJr.
274
non-encapsulated tumour was retnoved and subtnitted for routine histopathology. Sections ot tutnour stained with haetnatoxylin and eosin revealed densely-packed dermal masses of stnall deeply basophilic cells extending into the subcutis (Figs, i and 2). The histopathological diagnosis was 'malignant lymphoma, undifferentiated, non-Burkitt type.'
FIGURE I. Dermal nodules of basophilic cells extending into the subcutis diagnosed as subcutaneous lymphoma (H & E, x 44),
Investigations
Physical examination was completely normal with the exception of the above-mentioned frontoglabellar mass. The white blood cell count was i8,9OO;mm'\ with 40",, lymphocytes and 5",, monocytes. Other laboratory findings were entirely within normal litnittj including the following: chest X-ray, skull X-ray, complete radiological bone survey, intravenous pyclogram, bone marrow aspiration, haemoglobin, haematocrit, platelet count, blood urea nitrogen, fasting blood glucose, and liver function tests. Since the white cell count was elevated and this type of lymphoma is often considered pre-leukaemic, the patient received a course of radiation therapy to the tumour site combined with systemic chemotherapy (vincristine, methotrexate and prednisone). Six months after surgery the patient is in good health with no signs of local tumour recurrence or systemic illness. Demonstration of TAF Fragments of the surgically excised lymphoma were transported to the laboratory in refrigerated normal saline and immediately implanted into hamster check pouch chambers by the classical tech-
Tumour angiogenic factor with lymphoma
275
FIGURE 2. The dtep dermal nodules of the excised lumour were composed ol' deeply basophilic lymphocytes.
nique of Sanders & Shubik (1964), Grccnblatt and Shubik (1968) and Warren and Shubik (1966). The immunologically 'privileged' domain of the hamster cheek pouch allowed us to observe the eifect of tumour implants on small blood vessels in an anatomically and physiologically normal setting. In six hamsters, 20 mm tumour fragments were implanted in direct apposition to the vascular membrane of the cheek pouch. In an additional six hamsters, similar implants were separated from the cheek pouch vascular bed by 6 0 mm discs of millipore filter (Type TA, 0 45 |im pore size, 25 fim thick) in order to test for the presence of a difFusablc Tumour Angiogenic Factor. Implanted materials were observed for 7-14 days with a stereoscopic binocular microscope and with an American Optical microscope with long distance objectives. The vascular response of the cheek pouch membrane to tumour grafts was contrasted with that to normal hiaman dermis, keloid, naevocellular nevi and inert control materials (Millipore filter, dialysis membrane). Lymphoma grafts stimulated neovascularization in the hamster cheek pouch directly and across a microporous membrane (Millipore) barrier. The usual sequence of vascular events consisted of arterial tortuosity and vasodilation, followed by early sprouting of tiny capillary buds, overgrowth of implants and establishment of complex functioning vascular arcades (Fig. 3). Controls showed occasional tortuosity and dilatation without angiogenesis. DISCUSSION
Greenblatt & Shubik (1968), Goodall et al. (1965) and Warren & Shubik (1966) demonstrated a diffusablcj angiogenically active substance in the hamster cheek pouch with direct tumour implants and transfilter diffusion studies. Folkman et al. (1970) have subsequently isolated a Tumour Angiogenic Factor and characterized it as a heat-labile^ cryostable iipoprotcin containing 25",, RNA and 50%
276
John E.Wolfand Wimhrope R.HiiblerJr.
FIGURE 3. Fragments of the subcutaneous iymphoma elicited the formation of reactive nuovascular arcades in the hamster check pouch membrane across a Millipore filter (>: 100).
carbohydrate. The exact mechanism of action of TAF is not entirely clear, but Warren, Greenblatt & Kommineni (1972), Folkman et al. (1970) and Cavallo et al. {i^-ji) offered evidence that the factor is directly mitogenic for capillary endothelial cells. In this report we have demonstrated the production of TAF by a malignant subcutaneous Iymphoma in order to italicize the immediate and potential dcrmatological implications of this angiogenic factor. The tumour reported was initially thought to be a blood vessel neoplasm when presented at a university teaching conference, thus emphasizing the clinically vascular appearance of the lesion. Human skin tumours arc often associated with alteration of cutaneous vessels—for example the telangicctasia seen with basal cell cpithclioma, subcutaneous mcdulloblastoma and metastatic carcinoma (Primack, 1973). These diagnostically significant vascular changes may be due to capillary proliferation induced by a Tumour Angiogenic Factor. There is also a chance that TAF may one day assume a therapeutic potential. Tannock (1968) has demonstrated that the expansion of a tumour mass is directly limited by Its blood supply and Folkman ct al. (1970) have suggested that tumour growth might be arrested by interfering with the elaboration and/or function of TAF. If so, cutaneous neoplasms, exquisitely visible and eminently accessible, might provide an excellent proving ground for the theory. In this report, we have utilized laboratory techniques to confirm a clinically relevant concept with widespread implications for dermatology.
Titmottr angiogenic factor with lymphoma
277
REFERENCES ALGIRE, G.H. & CHALKLEY, H . W . (1945) Vasctilar reactions of normal and malignant tissues i'» vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic tr^n^'plani^. Journal of the National Cancer Institute, 6,73CAVALLO, T.jSADiijR., FOLKMAN, J.& COTRNA,R.S. (1972) Tumor Angiogenesis: Rapid induction of endothelial mitosis demonstrated by autoradiography. Journal of Cell Biology, 54, 408. FOLKMAN, J., MERLER, E., ABERNATHY, C . & WILLIAMS, G . (1970) Isolation of a tumor factor responsible for
angiogenesis. _7oi^?7(ti/ of Experimental Medicine, 133, 275. GOODALL, G.M., SANDERS, A.G. & SHUBIK, P. (1965) Studies of vascular patterns in living tumors with a transparent chamber inserted in hamster cheek pouch. 7'"""'"'^ of the National Cancer Institute, 35, 497. GREENBLATT, M . & SHUBIK, P. (.1968) Tumor Angiogenesis: Transfiltcr diffusion studies in the hamster by the transparent chamber technique. Journal of the National Cancer Institute, 41, 111. IDE, H.G., BAKER, N . H . & WARREN, S.L. (1939) Vascularization of the Brown-Pearce rabbit epithelioma transplant as seen in the transparent ear chamber. American Journal of Roe?itgetiology, 42, 891. PRIMACK, A. (1973) Telangiectasia overlying a malignant tumor. Archives of Dermatology, 108, 704. SANDERS, A.G. & SHUBIK, P. (1964) A transplant window for use in the Syrian hamster. Israel Journal of Experimental Medicine, II, 118. TANNOCK, I . F . (1968) The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumor. British Journal of Cancer, 22, 258. WARREN, B.A. & SHUBIK, P. (1966) The growth of the blood supply to melanoma transplants in the hamster cheek pouch. Laboratory Investigations, 15, 464. WARREN, B.A., GREENBLATT, M . & KOMMINENI, V . R . C . (1972) Tumor angiogenesis: Ultrastructure of endo-
thelial cells in mitosis. British Journal of Experimental Pathology, 53, 216.
