Biomarkers in chronic graft-versus-host disease: quo vadis?

Biomarkers are increasingly used for diagnosis and treatment of transplant-related complications including the first biomarker-driven interventional trials of acute graft-versus-host disease (GvHD). In contrast, the development of biomarkers of chronic GvHD (cGvHD) has lagged behind due to a broader variety of manifestations, overlap with acute GvHD, a greater variation in time to onset and maximum severity, and lack of sufficient patient numbers within prospective trials. An international workshop organized by a North-American and European consortium was held in Marseille in March 2017 with the goal to discuss strategies for future biomarker development to guide cGvHD therapy. As a result of this meeting, two areas were prioritized: the development of prognostic biomarkers for subsequent onset of moderate/severe cGvHD, and in parallel, the development of qualified clinical-grade assays for biomarker quantification. The most promising prognostic serum biomarkers are CXCL9, ST2, matrix metalloproteinase-3, osteopontin, CXCL10, CXCL11, and CD163. Urine-proteomics and cellular subsets (CD4+ T-cell subsets, NK cell subsets, and CD19+CD21low B cells) represent additional potential prognostic biomarkers of cGvHD. A joint effort is required to verify the results of numerous exploratory trials before any of the potential candidates is ready for validation and subsequent clinical application.

Assessment of imatinib as first-line treatment of chronic myeloid leukemia: 10-year survival results of the randomized CML study IV and impact of non-CML determinants.

Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400 mg/day (n=400) could be optimized by doubling the dose (n=420), adding interferon (IFN) (n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival was 80% and 10-year relative survival was 92%. Survival between IM400 mg and any experimental arm was not different. In a multivariate analysis, risk group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs other) influenced survival significantly, but not any form of treatment optimization. Patients reaching the molecular response milestones at 3, 6 and 12 months had a significant survival advantage. For responders, monotherapy with IM400 mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease, more life-time can currently be gained by carefully addressing non-CML determinants of survival.

Altered immune reconstitution of B and T cells precedes the onset of clinical symptoms of chronic graft-versus-host disease and is influenced by the type of onset.

We analyzed lymphocyte subpopulations and cytokines 3 months after allogeneic hematopoietic stem cell transplantation aiming to identify predictive cellular and serum markers for chronic graft-versus-host disease (cGVHD). Samples of 49 patients (pts) (no cGVHD (n = 14), subsequent quiescent onset (n = 16), de novo onset of cGVHD (n = 19)) were analyzed in the absence of active GVHD by flow cytometry and enzyme-linked immunosorbent assay. All mean absolute cell counts are presented as cells per microliter; relative cell counts are presented as percentage of lymphocytes. Pts with subsequent de novo cGVHD had significantly higher relative and absolute counts of CD4+ T cells including higher absolute counts of CD4+ memory T cells (22.36%; 206.55/µl; 136/µl, respectively) compared to pts with subsequent quiescent onset of cGVHD (12.41%; 83.42/µl; 54.3/µl) and pts without cGVHD (10.55%) with regard to relative counts of CD4+ T cells. Similarly, significantly more relative and absolute regulatory T cell numbers (CD4+FOXP3+) were detected in pts with de novo onset of cGVHD (3.08% and 24.63/µl) compared to those in pts without (1.25% and 9.06/µl) or with quiescent onset of cGVHD (1.15% and 6.91/µl). Finally, relative B cell counts, including naïve and memory B cells, were also significantly decreased in pts developing quiescent cGVHD (0.85, 0.73, 0.12% resp.) when compared to pts with de novo onset (5.61, 5.24, 0.38%). The results demonstrate that alterations in immune reconstitution are already present before onset of clinical symptoms and differ between de novo and quiescent onset of disease.

Long-term outcome of patients with newly diagnosed chronic myeloid leukemia: a randomized comparison of stem cell transplantation with drug treatment.

Tyrosine kinase inhibitors represent today's treatment of choice in chronic myeloid leukemia (CML). Allogeneic hematopoietic stem cell transplantation (HSCT) is regarded as salvage therapy. This prospective randomized CML-study IIIA recruited 669 patients with newly diagnosed CML between July 1997 and January 2004 from 143 centers. Of these, 427 patients were considered eligible for HSCT and were randomized by availability of a matched family donor between primary HSCT (group A; N=166 patients) and best available drug treatment (group B; N=261). Primary end point was long-term survival. Survival probabilities were not different between groups A and B (10-year survival: 0.76 (95% confidence interval (CI): 0.69-0.82) vs 0.69 (95% CI: 0.61-0.76)), but influenced by disease and transplant risk. Patients with a low transplant risk showed superior survival compared with patients with high- (P<0.001) and non-high-risk disease (P=0.047) in group B; after entering blast crisis, survival was not different with or without HSCT. Significantly more patients in group A were in molecular remission (56% vs 39%; P=0.005) and free of drug treatment (56% vs 6%; P<0.001). Differences in symptoms and Karnofsky score were not significant. In the era of tyrosine kinase inhibitors, HSCT remains a valid option when both disease and transplant risk are considered.

Extracorporeal photopheresis in 62 patients with acute and chronic GVHD: results of treatment with the COBE Spectra System.

In this retrospective analysis, 30 patients with acute GVHD (aGVHD) and 32 patients with chronic GVHD (cGVHD) treated with extracorporeal photopheresis (ECP) performed by the COBE Spectra System were evaluated. After 3 months of ECP treatment, a CR and PR were observed in 9 (30%) and 6 (20%) patients with aGVHD and in 2 (6%) and 12 (38%) patients with cGVHD. In 16 (53%) patients with aGVHD and 9 (28%) with cGVHD ECP treatment was already stopped after 3 months. One (3%) patient with aGVHD and 7 (22%) patients with cGVHD received new additional immunosuppressive therapy started during the first 3 months of ECP treatment and were classified as 'nonresponder' with regard to ECP. Of these patients a PR was achieved in one patient with aGVHD and in three patients with cGVHD. Steroids could be tapered by 50 in 83% of patients with aGVHD and in 29% of patients with cGVHD after 3 months of ECP treatment. Patients with aGVHD achieving a CR or PR showed a significant improved OS after allo-SCT (P=0.019). ECP is associated with significant response rates and successful reduction of steroids in patients with GVHD.

Vaccination against pandemic H1N1 (2009) in patients after allogeneic hematopoietic stem cell transplantation: a retrospective analysis.

PURPOSE: Limited data are available on immunologic responses to primary pandemic H1N1 (2009) vaccination in recipients of allogeneic hematopoietic stem cell transplantation (HSCT) recipients. In 2009 serologic responses to either pandemic H1N1 (2009) vaccine (n = 36) or pandemic H1N1 (2009) infection (n = 2) were studied in 38 HSCT recipients. METHODS: Responses were measured with a standard hemagglutination-inhibition assay. Fourteen patients had active chronic graft-versus-host disease (cGvHD) at the time of vaccination/infection and seven patients had cGvHD in remission; 11 patients had no immunosuppressive therapy, and 27 patients were on immunosuppressive therapy. Nineteen patients (53%) responded to pandemic H1N1 (2009) vaccination. Two patients had pandemic H1N1 (2009) infection without prior vaccination, and one patient had severe pandemic H1N1 (2009) infection with acute respiratory distress syndrome despite prior single vaccination. RESULTS: Non-responders to pandemic H1N1 (2009) vaccination more often had cGvHD (65 vs. 53%) and received second- or third-line therapy (53 vs. 11%), while responders mostly had first-line therapy for cGvHD. While vaccine responders had no or single agent immunosuppressive therapy, non-responders frequently received moderate or intense immunosuppressive therapy. All vaccine recipients previously treated with rituximab were non-responders. CONCLUSIONS: In summary, the overall response to pandemic H1N1 (2009) vaccination in HSCT recipients was modest. Patients receiving combined immunosuppressive therapy for steroid-refractory cGvHD barely responded to pandemic H1N1 (2009) vaccination.

Recipient NOD2/CARD15 status affects cellular infiltrates in human intestinal graft-versus-host disease.

Nucleotide-binding oligomerization domain 2/caspase recruitment domain 15 (NOD2/CARD15) polymorphisms have been identified as risk factors of both Crohn's disease and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. However, the role of these receptors of innate immunity in the pathophysiology of gastrointestinal GVHD is still poorly defined. Immunohistological features of intestinal GVHD were analysed in gastrointestinal biopsies from 58 patients obtained at the time of first onset of intestinal symptoms. The observed changes were correlated with concomitant risk factors and the presence of polymorphisms within the pathogen recognition receptor gene NOD2/CARD15. Intestinal GVHD was associated with a stage-dependent decrease in CD4 T cell infiltrates and an increase in CD8 T cells in the lamina propria; CD8 infiltrates correlated with extent of apoptosis and consecutive epithelial proliferation. The presence of NOD2/CARD15 variants in the recipient was associated with a significant loss of CD4 T cells: in a semiquantitative analysis, the median CD4 score for patients with wild-type NOD2/CARD15 was 1.1 (range 3), but only 0.4 (range 2) for patients with variants (P = 0.002). This observation was independent from severity of GVHD in multivariate analyses and could not be explained by the loss of forkhead box P3(+) T cells. Our results suggest a loss of protective CD4 T cells in intestinal GVHD which is enhanced further by the presence of NOD2/CARD15 variants. Our study might help to identify more selective therapeutic strategies in the future.

Pharmaceutical and cellular strategies in prophylaxis and treatment of graft-versus-host disease.

Acute and chronic GVHD after allogeneic hematopoetic stem cell transplantation are still associated with significant morbidity and mortality. For prophylaxis of acute GVHD calcineurin inhibitors in combination with an antimetabolite (MTX or MMF) are administered, and these therapies are based on controlled studies. New prophylaxis strategies include mTOR-inhibitors in combination with tacrolimus but require confirmation by controlled trials. First-line treatment of acute GVHD consists mainly of steroids with doses ranging from 1 mg/kg/day prednisone to 3 mg/kg/day methylprednisolone. Second-line treatment of acute GVHD after failure of steroids is less well defined due to the lack of controlled studies. Treatment options are the use of cytotoxic antibodies (ATG, campath), cytokine blocking agents (etanercept, daclizumab), immunomodulating modalities (photopheresis), and antimetabolites (pentostatin, MMF). Recently, cellular approaches were developed, such as the adoptive transfer of mesenchymal stem cells. Nevertheless steroid-resistant acute GVHD is still a main challenge in alloHSCT and associated with high mortality. First-line treatment of chronic GVHD is also based on steroids with 1 mg/kg/day prednisolone or prednisone, which are often combined with calcineurin inhibitors. There is no consensus on second-line treatment of chronic GVHD and most therapies are solely based on phase II trials. Treatment options are the use of immunomodulating modalities (photopheresis, mTOR-inhibitors) and antimetabolites (MMF, MTX, pentostatin). Recent reports showed an efficacy of rituximab in selected patients. Other treatment options are low dose total nodal irradiation or the use of antibodies like ATG. Moreover, successful topical treatment of manifestations of chronic GVHD manifestations has been reported consisting of topical steroids like budesonide, topical calcineurin inhibitors, or PUVA.

Assessing the potential role of photopheresis in hematopoietic stem cell transplant.

The First International Symposium on Photopheresis in Hematopoietic Stem Cell Transplantation was held in Vienna, Austria with an educational grant from Therakos Inc. from 25 May to 27 May 2005. Three general issues were addressed: (1) pathophysiology of graft-versus-host disease (GvHD), (2) induction of immune tolerance and the immunology of phototherapy and (3) current standard treatment and prevention strategies of acute and chronic GvHD and the use of extracorporeal photopheresis (ECP). The objectives of the meeting were to open a dialogue among leading researchers in photobiology, immunology, and hematopoietic stem cell transplantation; foster discussions and suggestions for future studies of the mechanism of action of ECP in acute and chronic GvHD; and promote collaboration between basic scientists and clinicians. As can be seen from the summaries of the individual presentations, important advances have been made in our understanding of GvHD, including the use of photoimmunology interventions and the development of robust model systems. It is our expectation that data from photoimmunology studies can be used to generate hypotheses in animal models that can further define the mechanism of action of ECP and help translate the findings to clinical trials of ECP for the prophylaxis and treatment of both chronic and acute GvHD.

Effect of the consumption of tomato paste on plasma prostate-specific antigen levels in patients with benign prostate hyperplasia.

The consumption of tomatoes and tomato products has been associated with a reduced risk of prostate cancer. We observed a decrease of 10.77% in prostate-specific antigen (PSA) levels in patients with benign prostate hyperplasia who were submitted to daily ingestion of tomato paste. This was an experimental rather than a controlled study with a sample of 43 men ranging in age from 45 to 75 years, all with histological diagnoses of benign prostate hyperplasia and plasma PSA levels of 4-10 ng/mL. All patients received 50 g of tomato paste once a day for 10 consecutive weeks and PSA levels were analyzed before, during and after the consumption of tomato paste. ANOVA for repeated measures was used to compare PSA levels before, during and after the consumption of tomato paste. The mean +/- SD PSA level was 6.51 +/- 1.48 ng/mL at baseline and 5.81 +/- 1.58 ng/mL (P = 0.005) after 10 weeks. Acceptance was good in 88.3, regular in 9.3, and poor in 2.3% of the patients. Dietary ingestion of 50 g of tomato paste per day for 10 weeks significantly reduced mean plasma PSA levels in patients with benign prostate hyperplasia, probably as a result of the high amount of lycopene in tomato paste. This was not a prostate cancer prevention study, but showed some action of tomato paste in prostate biology. The development of prostate cancer is typically accompanied by an increase in plasma PSA levels, thus any intervention that affects plasma PSA levels can suggest an impact in the progression of disease.

Effect of the consumption of tomato paste on plasma prostate-specific antigen levels in patients with benign prostate hyperplasia

The consumption of tomatoes and tomato products has been associated with a reduced risk of prostate cancer. We observed a decrease of 10.77 percent in prostate-specific antigen (PSA) levels in patients with benign prostate hyperplasia who were submitted to daily ingestion of tomato paste. This was an experimental rather than a controlled study with a sample of 43 men ranging in age from 45 to 75 years, all with histological diagnoses of benign prostate hyperplasia and plasma PSA levels of 4-10 ng/mL. All patients received 50 g of tomato paste once a day for 10 consecutive weeks and PSA levels were analyzed before, during and after the consumption of tomato paste. ANOVA for repeated measures was used to compare PSA levels before, during and after the consumption of tomato paste. The mean ± SD PSA level was 6.51 ± 1.48 ng/mL at baseline and 5.81 ± 1.58 ng/mL (P = 0.005) after 10 weeks. Acceptance was good in 88.3, regular in 9.3, and poor in 2.3 percent of the patients. Dietary ingestion of 50 g of tomato paste per day for 10 weeks significantly reduced mean plasma PSA levels in patients with benign prostate hyperplasia, probably as a result of the high amount of lycopene in tomato paste. This was not a prostate cancer prevention study, but showed some action of tomato paste in prostate biology. The development of prostate cancer is typically accompanied by an increase in plasma PSA levels, thus any intervention that affects plasma PSA levels can suggest an impact in the progression of disease.

CD4+CD25+ regulatory T cells in hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (SCT) is a well-established treatment modality for malignant and nonmalignant hematologic diseases. High-dose radio- and/or chemotherapy eradicate the hematopoietic system of the patient and induce sufficient immunosuppression to enable donor stem cell engraftment. The replacement of the recipient's immune system with that of the donor significantly contributes to the success of this treatment, since donor immune cells facilitate stem cell engraftment, provide protection from infections, and eliminate residual malignant or nonmalignant host hematopoiesis, thereby protecting from disease relapse in patients transplanted for leukemia or lymphoma (graft-versus-leukemia effect, GVL). Mediators of these beneficial effects are mature T cells within the stem cell graft. However, donor T cells can also attack host tissues and induce a life-threatening syndrome called graft-versus-host disease (GVHD). The challenge of allogeneic SCT is to find a balance between beneficial and harmful T cell effects, which at present is only insufficiently achieved by the use of immunosuppressive drugs. In the future, it might be possible to replace or support such medications by using the intrinsic regulatory capacity of the transplanted immune system, as represented by T cell subpopulations with suppressive activity, such as CD4+ CD25+ regulatory T (Treg) cells. In various mouse model systems, these cells have been shown to suppress GVHD while preserving the GVL effect. As the characterization of their human counterparts is rapidly progressing, their application in allogeneic SCT might soon be explored in clinical trials.

Advancing animal models of neoplasia through in vivo bioluminescence imaging.

Malignant disease is the final manifestation of complex molecular and cellular events leading to uncontrolled cellular proliferation and eventually tissue destruction and metastases. While the in vitro examination of cultured tumour cells permits the molecular dissection of early pathways in tumorigenesis on cellular and subcellular levels, only interrogation of these processes within the complexity of organ systems of the living animal can reveal the full range of pathophysiological changes that occur in neoplastic disease. Such analyses require technologies that facilitate the study of biological processes in vivo, and several approaches have been developed over the last few years. These strategies, in the nascent field of in vivo molecular and cellular imaging, combine molecular biology with imaging modalities as a means to real-time acquisition of functional information about disease processes in living systems. In this review, we will summarise recent developments in in vivo bioluminescence imaging (BLI) and discuss the potential of this imaging strategy for the future of cancer research.

Bioluminescence imaging of lymphocyte trafficking in vivo.

Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a complete understanding of immune function, as well as cell-cell and cell-tissue interactions in immune development and in response to insult. Although the location of cell populations in various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular and cellular changes in living animals have advanced to a point where it is possible to reveal the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune responses and the spatial distribution of lymphocytes within the body.

Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8(+) T lymphocytes. The ACTG Advanced Flow Cytometry Focus Group.

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174-179, 2000.

Noninvasive assessment of tumor cell proliferation in animal models.

Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, and noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, and proliferation of these cells in irradiated severe combined immunodeficiency (SCID) mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1x10(3) cells in the peritoneal cavity, 1x10(4) cells at subcutaneous sites and 1x10(6) circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1x10(6) cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, and this method will accelerate development of novel therapeutic strategies.

A simplified method of CD34+ cell determination for peripheral blood progenitor cell transplantation and correlation with clinical engraftment.

Enumeration of CD34+ cells by flow cytometry is the recognized standard for quantitating progenitor cells for peripheral blood progenitor cell (PBPC) transplantation. Although many clinical studies have confirmed that the time to neutrophil and platelet engraftment is inversely proportional to the number of CD34+ cells infused, the minimum number of CD34+ cells necessary to acheive rapid engraftment has not been satisfactorily determined. The lack of a standardized method for quantitation of CD34+ cells by flow cytometry (FCM) is often cited as the reason for this ambiguity. This report describes an FCM method for CD34+ cell determination that is simple, highly reproducible, comparatively inexpensive, and validated by excellent correlation with clinical engraftment. Pheresis samples are stained and fixed within 4 hours of collection. Two hundred fifty thousand events are acquired as list mode data using a forward scatter threshold. The discrete CD34+ population is enumerated using a CD34-phycoerythrin FL2 vs. side scatter plot and Paint-A-Gate Pro software. The method was validated by excellent statistical correlation with clinical engraftment. Using this method, we determined the number of CD34+ progenitor cells necessary to achieve rapid engraftment to be 2 x 10(6)/kg.

Defective granzyme B gene expression and lytic response in T lymphocytes infiltrating human renal cell carcinoma.

Granzyme B is a protein thought to play a pivotal role in the cytolytic functions of T cells. In view of this, the inducibility of this gene in freshly isolated T cells (T-TILs) infiltrating human renal cell carcinoma (RCC) in vitro was examined by using the reverse transcriptase-polymerase chain reaction (RT-PCR). A reduction in granzyme B messenger RNA (mRNA) expression in stimulated T-TILs from five of nine patients with RCC compared with autologous peripheral blood T cells was noted. The reduced expression was observed after multiple stimuli including anti-CD3 antibody, interleukin-2 (IL-2), and phytohemagglutinin (PHA). Because CD8+ T cells represent the predominant cytotoxic population, the ability of this cell population to express granzyme B mRNA after stimulation also was examined. When compared with CD8+ peripheral blood lymphocytes (T-PBLs) from patients with RCC and normal donors, the induction of granzyme B mRNA was reduced in CD8+ T-TILs. CD8+ T-TILs also had lower non-major histocompatibility complex (MHC)-restricted cytotoxic activity than did CD8+ T-PBLs against both Daudi cells and allogeneic RCC cell lines. These results show that in a subset of patients with RCC, depressed lytic activity of CD8+ TILs compared with CD8+ PBLs is present. Reduced granzyme B mRNA expression also was noted in selected patients.