Isolation and Characterization of a Spontaneous Lymphocytic Leukemia (L-76) in the Strain 2 Guinea Pig1

A partial characterization of a transplantable guinea pig leukemia (L-76) that arose spontaneously in a young female strain 2 animal is described. The leukemia appears to be histologically, pathologically, and karyologically similar to the L.C lymphoblastic leukemia, which also arose spontaneously 24 years earlier. The presence of C3 receptor sites on the surface of the leukemia indicates it to be of B-cell origin. Although the L-76 leukemia originated in a strain 2 animal, it also produces a rapidly progressing, lymphoblastic leukemia in strain 13, Hartley, and strain 2 x strain 13 F, hybrid adult guinea pigs. An inoculation of 2 x 10' blast cells obtained from peripheral blood induced a stem cell leukemia in the strain 2 host within 12 days, with white blood cell counts of 2 to 3 x 10s cells/cu mm at expiration. Titration data indicated that as few as 10 cells injected s.c. were capable of transmitting a systemic disease, which reached a terminal phase by 40 days. Pathological examination indicated involvement of the entire hematopoietic system, with almost all organs infil trated by massive concentrations of neoplastic lymphoblasts. Treatment of the leukemia with Cytoxan (cyclophosphamide), 100 mg/kg, provided a remission period that lasted 4 to 5 weeks with eventual relapse and death due to the leukemia. Treatment of leukemic guinea pigs with 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitro sourea (MECCNU) alone or in combination with cyclophosphamide resulted in long-term survivors apparently "cured" of leukemia. INTRODUCTION The L2C leukemia was isolated in the 1950's by Congdon and Lorenz (2) as 1 of a series of spontaneous and radiationinduced leukemias observed in the NIH guinea pig colony over a period of years. This leukemia, which by this time has evolved into a group of sublines based on subtle genetic and immunological differences, is the only trans plantable guinea pig leukemia currently available (8). Al though the LjC leukemia has been of great value in a variety of immunological studies, the lack of additional recently isolated leukemias has hampered efforts in areas such as the tumor virology of the guinea pig and has limited gener alizations on the etiology of leukemia in the guinea pig. In this report we describe a spontaneous lymphoblastic leukemia recently isolated in a strain 2 guinea pig (desig nated L-76 leukemia). Results on its transplantability, his tology, pathology, karyology, and other biological charac teristics will be presented in addition to comparing these properties to those of the UC-En leukemia. MATERIALS AND METHODS Animals. Inbred strain 2-N guinea pigs were obtained from the Frederick Cancer Research Center Animal Farm, Frederick, Md., while strain 13, random-bred Hartley, and strain 2 x strain 13 F, hybrid guinea pigs were obtained from herds maintained at the NIH Animal Farm, Bethesda, Md. Animals were housed in stainless-steel cages and fed Wayne guinea pig chow and water ad libitum and kale twice weekly. All animals were age matched and weighed 300 to 400 g when experiments were initiated. Leukemias. The history and biological properties of the L2C (EN subline) leukemia have been previously described (4, 6, 7, 8, 10, 13, 18). The L,C-EN subtype was kindly sup plied by Dr. I. Green (NIH, Bethesda, Md.). The L-76 leuke mia arose spontaneously in 1976 in a 5.5-month-old female strain 2 guinea pig. At necrospy the disease involved the en tire hematopoietic system with generalized enlargement of the spleen and lymph nodes, typical of an acute leukemia. A peripheral WBC was greater than 200,000/cu mm. Blood and tissue specimens were collected and shipped to us on ice by Dr. R. Hong from the Frederick Cancer Research Center Animal Farm. Upon arrival the blood and a spleen homogenate were immediately injected into normal strain 2 guinea pigs. These recipient guinea pigs eventually died of leukemia 3 to 4 weeks later. The L-76 leukemia is maintained as a transplantable cell line in strain 2 guinea pigs and is passaged serially every other week as a s.c. injection of 2 x 106 viable leukemic blast cells obtained from peripheral blood. Material for in vivo transplantation was obtained from animals displaying WBC greater than 100,000/cu mm. Peripheral blood was collected via a cardiac puncture into heparinized syringes (5 units/ml) and diluted 1:3 with 0.01 M PBS,3 pH 7.2. Separation of blast cells was performed by centrifugation on a Ficoll-Hypaque gradient (1). After removal from the gradient, the cells were washed 3 times in cold PBS and either utilized the same day or preserved by storage in liquid nitrogen. Viability was generally greater than 95% for fresh cells and 85% for thawed cells. The material used for all in vivo and in vitro studies was obtained from leukemic 1 This work was supported by Contract N01-CP-53566 within the Virus Cancer Program of the National Cancer Institute. 2 To whom requests for reprints should be addressed. Received January 30, 1978; accepted July 5, 1978. 3 The abbreviations used are: i.d.. intradermal; CFA, complete Freund's adjuvant; CY, cyclophosphamide; MECCNU, 1-(2-chloroethyl)-3-((rarjs-4methylcyclohexyl)-1-nitrosourea; E, erythrocyte; EAC, erythrocyte antibody complement; RDDP. RNA-dependent DNA-polymerase; MST. median sur vival time; PBS, phosphate-buffered saline (0.01 M) containing 50 ^g genta-