Bone Marrow Reaction in Patients with Chronic GvHD - An Immunohistochemical Analysis of Microvessels and Lymphocytes.

Chronic GvHD (cGvHD) is associated with endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in skin biopsies as well as increase of von Willebrand factor in plasma (BC Biedermann, Lancet 2002). Moreover an increased quantity of CD45RO positive lymphocytes has been observed in bone marrow biopsies (bmb) in CML pts with acute GvHD (aGvHD) grade III and lV, who were transplanted from unrelated donors (J Thiele, Bone marrow transplantation 2001). We analysed factors of T-cell activation and microvessels in bmb of pts with cGvHD to evaluate the function of the bone marrow in cGvHD.

Methods: In an associated protocol of a prospective phase II study in 35 pts for the therapy of bone loss following HSCT with a bisphosphonate one bmb was taken prior of entry into the study and embedded in paraffin after decalcification. The interval between HSCT and time of biopsy was median 477 +/−998 days. For conventional semiquantitative evaluation of marrow and spongiosa a Giemsa staining was used. For immunological quantitative evaluation the following monoclonal antibodies were used with the APAAP method: CD34 and von Willebrand factor (VWF) for endothels of microvessels and CD45RO and CD8 for T-lymphocytes subsets. The pts were divided in 5 groups:

1. neither aGvHD nor cGvHD;

2. no cGvHD but aGvHD before entry;

3. cGvHD limited;

4. cGvHD extensive inactive;

5. cGvHD extensive active, needing immunosuppression.

Results: Conventionally analysed no pt had evidence of vasculitis. 1 pt of group 4 had one small immunoreactive aggregate of lymphocytes another pt of group 5 had a diffuse infiltration with lymphocytes. 32 pts had no or very small reduction of spongiosa and 3 had severe reduction of spongiosa < 15%. The significant immunological results were:

The number of CD34 as well as the number of vWF positive microvessels increased with the grade of involvement and activity of cGvHD (11,1 vs 25,3 respectively 8,7 vs 18,1). Additionally the content of CD8 positive lymphocytes increased with the grade and activity of disease. In contrast to reported skin biopsies, bone marrow capillaries were not destroyed by cytotoxic lymphocytes with increased activity. The bone marrow seems to show a different immunological reaction in contrast to pts with cGvHD of whom the target organs are skin, liver or gut. This was not observed on conventional biopsies. Further studies will be directed to the pattern of microvessels with regard to hematopoietic function and relapse.