F. Gavosto

Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colony-stimulating factor.

The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 micrograms/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32 +/- 7 to 79 +/- 16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43 +/- 20 to 82 +/- 11%) and day 7 CFU-GM (from 41 +/- 14 to 56 +/- 20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26 +/- 14 to 41 +/- 6%, and that of erythroblasts increased from 25 +/- 12 to 30 +/- 12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents.

Unusual malignant tumours in 49 patients with HIV infection

Between December 1986 and December 1988, the Italian Cooperative Group on AIDS-Related Tumours documented 49 HIV-related tumours other than malignant lymphomas (ML) and Kaposis sarcomas (KS), predominantly among HIV-infected intravenous drug abusers (IVDA). Of 12 germinal testicular tumours collected, six were seminomas, two of which were pure embryonal and the other four embryonal mixed. Cervical carcinoma was observed in nine IVDAs (intraepithelial in eight and advanced, with rapid progression, in one). Lung cancer associated with HIV infection was reported in eight patients, of whom four had an adenocarcinoma, two a small cell carcinoma, one an epidermoid carcinoma and one a mesothelioma. All patients with non-small-cell-lung cancer (SCLC) were at stage III, while those with SCLC and mesothelioma had limited disease. Five out of eight presented with limited disease at onset. The median age was low; lung cancer occurred predominantly in young adults, of whom all but one were smokers. Three patients could not be treated; four died while on treatment because of progression of the neoplasia and one died of an overdose. Acute lymphoblastic leukaemia (ALL) was diagnosed in five patients. The immunophenotype was always Burkitt-like (L3), and acute myeloblastic leukaemia (M2) was diagnosed in one. Of the central nervous system (CNS) tumours, two cases of glioblastoma and one of medulloblastoma were described. Two cases of young adults with multiple myeloma and two cases of colorectal carcinoma were also reported. One case of chronic lymphocytic leukaemia, one anorectal carcinoma, one oral carcinoma, one pancreatic carcinoma, one thymoma, one kidney carcinoma, one malignant melanoma and thyroid carcinoma were also found. Testis carcinoma occurred mainly in patients in an early phase of HIV infection, without adversely affecting full-dose chemotherapy or radiotherapy. In situ cervical carcinoma treated with conization would suggest papanicolaou shear test screening in young IVDA. Lung carcinoma occurred in a young age group with rapid progression and resulted in death within 2 months. Intensive chemotherapy for ALL was not adversely affected by HIV infection and two complete remissions were achieved (11 and 15 months duration). This retrospective study shows that while oral and anorectal tumours were very rarely observed, a wide spectrum of other HIV-related solid tumours and leukaemias were found in this IVDA-based series. The incidence of such tumours is probably underestimated because they are not diagnostic of AIDS. The required therapeutic approaches may not necessarily be influenced by HIV infection, in contrast with the observed pattern for treatment of KS and ML in HIV-infected subjects.

Treatment of acute myeloid leukaemia patients with recombinant interleukin 2: a pilot study

Summary. Twelve patients with acute myeloid leukaemia (AML) with evidence of resistant disease were treated with recombinant interleukin 2 (rIL2) given intravenously by continuous infusion. No objective response to rIL2 alone was documented in the seven patients with advanced disease (20–90%, resistant blasts in the marrow), except for a partial response to rIL2 plus chemotherapy in one. Of the five patients with limited disease (8–15% marrow blasts), three obtained a complete disappearance of the blasts following two to four 5 d courses of rIL2 alone. One patient persists in fourth complete remission (CR) 30 months later, another obtained a third CR for 4 months, and the last remained in third CR for 9 months before relapsing. This latter patient achieved a fourth CR with low‐dose cytarabine. The remissions have been maintained with low‐dose monthly courses of rIL2 given on an out‐patient basis. Two AML did not respond to rIL2 alone; one, however, obtained a fourth CR with chemotherapy and rIL2. Administration of rIL2 was accompanied by organomegaly and leucocytosis, with a frequent lymphocytosis and increase in eosinophils and large granular lymphocytes, both in the blood and in the marrow. Side effects, though often severe, were controllable using a daily dose escalating protocol and never required intensive care treatment. The results of this pilot study indicate that treatment of AML patients with rIL2 is feasible and may result in the disappearance of chemotherapy‐resistant blasts in patients with limited but detectable disease. Further controlled trials in AML in CR appear warranted.

Idiopathic Myelofibrosis: a Possible Role for Immune-Complexes in the Pathogenesis of Bone Marrow Fibrosis

Summary. Sixteen patients with idiopathic myelofibrosis (IM) have been investigated with respect to the possibility that immune mechanisms may be of importance in the pathogenesis of bone marrow fibrosis. The following points appear relevant: (1) immune‐complexes (IC) are detectable with different techniques in a high percentage of patients with IM. Their presence is associated with evidence of bone‐marrow histological markers of immune activity. (2) IgG is the main Ig class in the composition of IM IC. The results obtained favour the hypothesis that autoimmune mechanisms are involved in IM patients.

Interleukin 2 does not promote the in vitro and in vivo proliferation and growth of human acute leukaemia cells of myeloid and lymphoid origin

The effect of recombinant interleukin 2 (IL2) on the in vitro and in vivo proliferation and growth of human acute leukaemia cells of both myeloid and lymphoid origin was investigated. In none of the 25 primary samples tested could a continuously in vitro growing cell line be obtained by adding IL2 to the culture medium. Although IL2 induced a proliferative signal in three of the 31 acute leukaemias analysed, the overall 3H‐thymidine uptake of the neoplastic cells was significantly reduced (P<0.05) in the presence of IL2. The unlikelihood of an important proliferative signal triggered by IL2 was confirmed in a semisolid clonogenic assay, which failed to document an increased colony growth in the 26 samples studied. Furthermore, using a colorimetric assay as a test for cell proliferation and survival, in seven of the 11 fresh acute leukaemia samples tested a 22–40% reduction in viability was observed in the presence of IL2, while in the remaining four, IL2 was ineffective. In order to investigate the effect of IL2 in an in vivo setting, an experimental model in heavily immunosuppressed nu/nu mice was established. In no case did IL2 promote the in vivo proliferation and growth of human myeloid and lymphoid acute leukaemia cells injected in the mice. On the contrary, with seven of the eight leukaemic cell lines which gave rise spontaneously to leukaemic masses, this could be prevented when the mice received locally 300 U of IL2 three times daily for 90 d. IL2 also blocked the growth in vivo of three fresh acute leukaemia samples (two myeloid and one lymphoid). Co‐culture experiments using leukaemic cell lines and increasing numbers of normal lymphocytes suggest that the inhibitory effect of IL2 is probably exerted via an indirect mechanism. These findings, coupled to the well‐documented ability of IL2 to generate lymphokine activated killer cells cytolytic against leukaemic blasts, further point to the potential role of immunotherapy with IL2 in the management of patients with haematological malignancies.

Induction and Persistence of Complete Remission in a Resistant Acute Myeloid Leukemia Patient after Treatment with Recombinant Interleukin-2

A complete and persistent clinico-hematologic remission was obtained in an M4 acute myeloid leukemia patient after treatment with recombinant interleukin 2 (rIL2) alone. After two autologous bone-marrow transplantations and in the third relapse with 10% persistent blasts in the marrow, the patient was treated with two intensive courses of rIL2 given by continuous infusion over a period of 13 days. rIL2 administration was accompanied by significant side effects and followed by notable hematological, clinical and immunological modifications. Complete remission was achieved after these two courses and has been maintained with monthly low-dose cycles of rIL2 given on an out-patient basis. Eighteen months after starting treatment with rIL2 the patient is well and in persistent remission.

Multimarker phenotypic characterization of adult and childhood acute lymphoblastic leukaemia: An Italian multicentre study

Summary. A multicentre phenotypic study was carried out in Italy combining conventional immunological techniques with monoclonal antibody (MoAb) analysis in 190 cases of adult and childhood acute lymphoblastic leukaemia (ALL), in an attempt to define better the lineage relationship of the neoplastic cells. Of the 140 children evaluated, 79·3% expressed the common ALL (cALL) antigen (all analyses performed by MoAb), 11·4% were T‐ALL and 9·3% were non‐T, non‐B, non‐common (‘null’) ALL. The proportion of adult cALL cases was slightly lower (64% of the 50 cases studied) than that of childhood ALL, whilst the incidence of T‐ALL was significantly higher in adults than in children (26%v. 11·4%, P <0·05). Because of the high proportion of cALL cases, the incidence of ‘null’ ALL in adult patients was similar (10%) to that of children, and lower than previously reported. The recognition of early pre‐T‐ALL cases (T1+, RFT2+, T10+, T6‐, T11‐, E‐) contributed to the overall low proportion of ‘null’ ALL; prior to the use of MoAb, such cases would probably have been classified as undifferentiated acute leukaemia or ‘null’ ALL. The search for B‐cell‐related markers showed that the incidence of pre‐B‐ALL cases (cytoplasmic immunoglobulin positive cases) was similar in adults and in children (25·6% and 32%, respectively). Furthermore, the great majority of cases studied expressed the BA‐1 antigen (92·8% of adults and 79% of children), whilst the BA‐2 antigen was found in 53% of cases (tested only in children), confirming a hierarchy in the expression of B‐cell related markers in CALL: BA‐1, BA‐2, Cylg. Several of the ‘null’ cases also expressed the BA‐1 antigen on a variable proportion of cells, pointing to a possible B‐cell origin of the blasts.

Late DNA replication pattern in human haemopoietic cells: A comparative investigation using a high resolution quantitative autoradiography

Abstract A quantitative autoradiographic technique was used to compare DNA duplication during the final phase of the synthesis period (about 30 min) in chromosomes of lymphocytes, erythroblasts and human bone marow cells. The duration of the G2 period in the three tissues was determined and a calculation was made in order to establish at which point before the beginning of this period each pair of chromosomes had completed synthesis. It was observed that the length of the single G2 period is very much the same in all three tissues. It was therefore possible to conclude that, in 19 chromosome pairs, synthesis will terminate in the last 10 min of the S period and in the other 5 pairs in the last 5 min. The synthetic activity rate of each pair of chromosomes, in the three types of haemopoietic cells, did not present significant differences either in the overall period or in the time intervals into which this period was subdivided (using time analysis based on a cumulative distribution method). Detection of late-labelling zones at the subchromosomic level, using a new technique, made it possible to obtain characteristic patterns for many chromosomes; but we were unable to detect qualitative or quantitative differences between the cells of different tissues.

Retinoic acid enhances the growth of only one subpopulation of granulomonocyte precursors

The effect of various concentrations of retinoic acid on the growth of committed granulomonopoietic precursors (CFU-GM) was studied on semisolid agar cultures. Retinoic acid increased the growth of day 14 CFU-GM but had no significant effect on day 7 CFU-GM. Since the latter are believed to represent the progeny of day 14 CFU-GM it is suggested that the effect of retinoic acid is confined to the early stages of granulomonocytic differentiation.

The problem of anaemia in the acute leukaemias: Kinetic study☆

Abstract In vivo pulse-labelling with 3 H-thymidine was employed to obtain red cell kinetics data in 3 untreated cases of acute myeloblastic leukaemia (AML). The K out of the proliferating into the non-proliferating compartment was below the sum of the individual compartment birth rates in all 3 cases: this sign of ineffective red cell erythropoiesis was equally evident whether an S phase of 13 or 16 hr was assumed. Grain distribution values provided evidence in favour of a divisionless maturation of erythroblasts. A fall in the number of stem cells available for erythroblastic differentiation and ineffective erythropoiesis are held to be the principal mechanisms responsible for anaemia in AML.