Karen Grimmett

Incidence and nature of CD20-negative relapses following rituximab therapy in aggressive B-cell non-Hodgkin's lymphoma: a retrospective review.

Summary. Re‐treatment with rituximab for B‐cell non‐Hodgkins lymphoma (NHL) relapsing after previous rituximab therapy has recently been shown to be clinically efficacious. Although the mechanism of resistance to rituximab re‐treatment in non‐responding patients is unknown, it is possible that loss of CD20 expression in the relapsed NHL could be important in some patients. We examined the incidence and nature of CD20 negative relapses following rituximab therapy in aggressive B‐cell NHL treated at our institution. Of a total of 18 patients who received rituximab, 13 have relapsed, with 10 patients subsequently undergoing repeat tissue biopsy. Six of these 10 patients (60%) were shown to have lost CD20 expression by either immunohistochemistry and/or flow cytometry. Furthermore, three of the six patients who relapsed with CD20‐negative NHL also suffered relapses at unusual anatomical sites. We conclude that loss of CD20 expression in aggressive B‐cell NHL relapsing post‐rituximab therapy is common. As such, repeat tissue biopsy should be undertaken to document CD20 expression by both flow cytometry and immunohistochemistry prior to considering repeated courses of rituximab in relapsed aggressive lymphomas.

Clinical response after intradermal immature dendritic cell vaccination in metastatic melanoma is associated with immune response to particulate antigen

Abstract. Metastatic melanoma is poorly responsive to treatment, and immunotherapeutic approaches are potentially beneficial. Predictors of clinical response are needed to identify suitable patients. We sought factors associated with melanoma-specific clinical response following intradermal vaccination with autologous melanoma peptide and particulate hepatitis B antigen (HBsAg)-exposed immature monocyte-derived dendritic cells (MDDC). Nineteen patients with metastatic melanoma received a maximum of 8, 2-weekly vaccinations of DC, exposed to HBsAg in addition to autologous melanoma peptides. A further 3 patients received an otherwise identical vaccine that did not include HBsAg. Patients were assessed 1–2 monthly for safety, disease volume, and cellular responses to HBsAg and melanoma peptide. There was no significant toxicity. Of 19 patients receiving HBsAg-exposed DC, 9 primed or boosted a cellular response to HBsAg, and 10 showed no HBsAg response. HBsAg-specific responses were associated with in vitro T cell responses to melanoma peptides and to phytohemagglutinin (PHA). Zero out of 10 non-HBsAg-responding and 4/9 HBsAg-responding patients achieved objective melanoma-specific clinical responses or disease stabilization – 1 complete and 2 partial responses and 1 case of stable disease (P=0.018). Development of melanoma-specific cellular immunity and T cell responsiveness to mitogen were greater in the group of patients responding to HBsAg. Therefore stimulation of an immune response to nominal particulate antigen was necessary when presented by melanoma peptide-exposed immature DC, to achieve clinical responses in metastatic melanoma. Since general immune competence may be a determinant of treatment response, it should be assessed in future trials on DC immunotherapy.

Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders

Adoptive immunotherapy using autologous Epstein‐Barr virus (EBV)‐specific cytotoxic T‐lymphocytes (auto‐CTL) can regress posttransplant lymphoproliferative disorders (PTLD). Widespread applicability of auto‐CTL remains constrained. Generation is time‐consuming, and auto‐CTL cannot be established in patients treated with the B‐cell depleting antibody rituximab. By contrast, pregenerated allogeneic CTL (allo‐CTL) offers immediate accessibility. Allo‐CTL has previously shown efficacy in “early” polyclonal‐ PTLD. We treated three patients with aggressive, advanced monoclonal‐PTLD following solid‐organ transplantation. All were refractory to at least three prior therapies. Despite HLA disparity, there was negligible toxicity, with early in vivo antiviral efficacy and reconstitution of EBV peptide‐specific immunity. Two patients attained complete remission (CR). One remains in CR 17 months following therapy, coincident with persistence of donor‐derived tumor targeted EBV‐specific CTL; the other died of non‐PTLD related pathology. In the third patient, autopsy demonstrated homing of allo‐CTL at the tumor site. Larger prospective studies of EBV‐specific allo‐CTL in PTLD are warranted.

Loss of PKR activity in chronic lymphocytic leukemia

There are a number of observations that suggest the dsRNA‐activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full‐length PKR is present but not active in 21 of 28 patient samples from B‐cell chronic lymphocytic leukemia (B‐CLL). PKR from these patients was unable to auto‐activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58IPK. We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B‐CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL.

A ≥1 log rise in RQ-PCR transcript levels defines molecular relapse in core binding factor acute myeloid leukemia and predicts subsequent morphologic relapse

Core binding factor acute myeloid leukemia (CBF AML), with t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and the associated fusion gene transcripts AML1/ETO or CBFβ/MYH11, has a favourable clinical prognosis although significant numbers of patients still suffer relapse. We examined the prognostic utility of serial bone marrow minimal residual disease (MRD) monitoring by RQ-PCR in a cohort of patients with CBF AML with long term clinical follow-up. Twenty-nine patients were evaluated with a median follow of 34 months. Twelve relapses occurred at a median of 11 months (range 4 – 17) from diagnosis. RQ-PCR levels at diagnosis, post-induction chemotherapy and post-consolidation were not predictive of outcome. However, a ≥1 log10 rise at any stage in transcript level relative to the level from a remission bone marrow sample correlated with inferior leukemia free survival (LFS) and imminent morphologic relapse (hazard ratio 8.6). Relapses occurred a median of 60 days (range 45 – 272) after a log10 rise. A ≥1 log10 rise in transcript levels strongly predicts subsequent morphologic relapse in CBF AML and therefore defines molecular relapse. Our data support a simple RQ-PCR model for prediction of impending relapse which has the potential for widespread clinical applicability. Prospective identification of high risk patients will enable clinical trials to assess the efficacy of treatment initiated at molecular relapse.

Selective accumulation of virus-specific CD8+ T cells within the peripheral blood stem cell compartment.

To the editor: The absence of cellular immunity is central to the pathogenesis of herpesvirus-mediated diseases after allogeneic hemopoietic stem cell transplantation (HSCT).[1][1],[2][2] For both bone marrow (BM)– and granulocyte-colony stimulating factor–mobilized peripheral blood stem cells

Epstein-Barr virus T-cell immunity despite rituximab

Immunosuppression following solid organ transplantation results in impaired T‐cell immunity and risk of Epstein–Barr virus (EBV)‐positive post‐transplant lymphoproliferative disorders (PTLD). The B‐cell targeting antibody rituximab has efficacy in PTLD. As B cells are the principle reservoir for EBV, we investigated the effect of rituximab on the persistence of EBV‐specific CD8+ T‐cell immunity. To avoid the confounding factor of concurrent immunosuppression to prevent transplant rejection, immunity was analysed in non‐transplanted lymphoma patients (i.e. a non‐PTLD setting). Cytomegalovirus‐specific T‐cell immunity was assessed as an internal control. Our data demonstrated that circulating B cells were not critical for maintaining EBV‐specific T‐cell immunity.

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-γ

Aims: An important consideration in the design of a tumour vaccine is the ability of tumour‐specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B‐CLL might use an escape strategy, the current studies compared B‐CLL and normal B cell MHC class I expression. Methods: Flow cytometry, TAP allele PCR and MHC class I PCR were used. Results: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B‐CLL cells in response to IFN‐γ was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B‐CLL cells upregulated TAP protein expression in response to IFN‐γ. Responsiveness of B‐CLL MHC class I mRNA to IFN‐γ was not impaired. Conclusions: The data suggest that MHC class I molecules might be less stable at the cell surface in B‐CLL than normal B cells, as a result of the described release of β2m and β2m‐free class I heavy chains from the membrane. This relative MHC class I expression defect of B‐CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.

Prognostic utility of spontaneous erythroid colony formation and JAK2 mutational analysis for thrombotic events in essential thrombocythaemia

Background:  Thrombotic events in essential thrombocythaemia (ET) are difficult to predict with current risk stratification based on age and prior history of thrombosis.