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.

Functional hyposplenism after hematopoietic stem cell transplantation.

Functional hyposplenism is associated with chronic GvHD (cGvHD) following hematopoietic stem cell transplantation (HSCT). We hypothesized that hyposplenism measured by pitted red cell counts in cGvHD was transient and related to the severity of disease. We performed a serial, retrospective review of 36 pediatric post-HSCT patients' pit counts at BC Children's Hospital from 2005 to 2013 and compared those counts with the clinical course of patients with late acute GvHD (aGvHD)/cGvHD. Of the 36 patients, 22 had late aGvHD/cGvHD based on National Institutes of Health consensus criteria. Fourteen of 22 GvHD patients had an abnormal pitted red cell count. Ten of 14 abnormal patients had late acute or overlap GvHD syndrome, primarily gastrointestinal disease. A second cohort was prospectively evaluated in a multicenter adult HSCT biomarker trial. We identified 3 out of 10 control patients who had an abnormal pitted red cell count, 3 out of 10 with classic cGvHD and 5 out of 9 patients with overlap syndrome were abnormal. In both the retrospective and prospective studies, hyposplenism was present in patients without late aGvHD/cGvHD suggesting current guidelines regarding antibiotic prophylaxis against encapsulated bacteria after HSCT need to be re-addressed and abnormal pit counts could be used to guide prophylaxis in all HSCT patients.

Risk factors and timing of relapse after allogeneic transplantation in pediatric ALL: for whom and when should interventions be tested?

We previously showed that minimal residual disease (MRD) detection pre-hematopoietic cell transplant (HCT) and acute GvHD (aGvHD) independently predicted risk of relapse in pediatric ALL. In this study we further define risk by assessing timing of relapse and the effects of leukemia risk category and post-HCT MRD. By multivariate analysis, pre-HCT MRD <0.1% and aGvHD by day +55 were associated with decreased relapse and improved event-free survival (EFS). Intermediate leukemia risk status predicted decreased relapse, and improved EFS and overall survival (OS). Patients with pre-HCT MRD ⩾0.1% who did not develop aGvHD compared with those with MRD <0.1% who did develop aGvHD had much worse survival (2 years EFS 18% vs 71%; P=0.001, 2 years OS 46 vs 74%; P=0.04). Patients with pre-HCT MRD <0.1% who did not experience aGvHD had higher rates of relapse than those who did develop aGvHD (40% vs 13%; P= 0.008). Post-HCT MRD led to a substantial increase in relapse risk (HR=4.5, P<0.01). Patients at high risk of relapse can be defined after transplant using leukemia risk category, presence of MRD pre or post HCT, and occurrence of aGvHD. An optimal window to initiate intervention to prevent relapse occurs between day +55 and +200 after HCT.

New frontiers in pediatric Allo-SCT: novel approaches for children and adolescents with ALL.

Although most children with ALL can be cured by chemotherapy approaches, allogeneic hematopoietic cell transplant (HCT) therapy offers a better chance of cure to selected high-risk patients in first remission and most children who relapse. Although transplant-related mortality has decreased significantly in the past decade, relapse remains high after HCT for ALL; developing strategies to decrease relapse and improve survival are vital. Recent studies have shown that relapse risk can be accurately defined using measurements of minimal residual disease (MRD) both pre- and post-HCT and by knowing whether patients get GVHD in the first 2 months after transplant. With these risk definitions in hand, investigators are now applying novel agents and immunotherapeutic methods in attempt to lower MRD before transplant and modulate the GVL effect after transplant. With powerful new immunological approaches coming on line, the transplant process itself will likely expand to include pre and/or post-HCT interventions aimed at reducing relapse.

Long-term follow-up of imatinib in pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia: Children's Oncology Group study AALL0031.

We previously reported preliminary findings that post induction imatinib mesylate (340 mg/m(2)/day), in combination with intensive chemotherapy, resulted in outcomes similar to blood and marrow transplant (BMT) for pediatric patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We now report 5-year outcomes of imatinib plus intensive chemotherapy in 91 children (1-21 years) with and without allogeneic BMT (N=91). We explore the impacts of additional chromosomal abnormalities and minimal residual disease (MRD) by flow cytometry on outcomes. The 5-year disease-free survival was similar for Cohort 5 patients, treated with chemotherapy plus imatinib (70%±12%, n=28), sibling donor BMT patients (65%±11%, n=21) and unrelated donor BMT patients (59±15%; P=0.60, n=13). Patients with additional cytogenetic abnormalities had worse outcomes (P=0.05). End induction (pre-imatinib) MRD was not prognostic for Cohort 5 or allogeneic BMT patients, although limited by small numbers. The re-induction rate following relapse was similar to other higher-risk ALL groups. Longer-term follow-up confirms our initial observation of substantially good outcomes for children and adolescents with Ph+ ALL treated with imatinib plus intensive chemotherapy with no advantage for allogeneic BMT.

Anti-CD13 Abs in children with extensive chronic GVHD and their relation to soluble CD13 after allogeneic blood and marrow transplantation from a Children's Oncology Groups Study, ASCT0031.

Our group previously demonstrated a strong association between elevated plasma soluble CD13 enzyme activity and newly diagnosed extensive chronic GVHD (cGVHD) in children. As cytotoxic anti-CD13 Abs have been documented after blood and marrow transplant (BMT) in association with CMV infection and cGVHD, we hypothesized that soluble CD13 contributes to cGVHD pathogenesis by induction of CD13 reactive Abs and that anti-CD13 Abs could be additional biomarkers for newly diagnosed pediatric extensive cGVHD. Using prospectively collected plasma samples from pediatric allogeneic BMT (allo-BMT) subjects with cGVHD and controls without cGVHD enrolled in a large multi-institution Children's Oncology Group cGVHD therapeutic trial, we evaluated whether soluble CD13 correlates with induction of anti-CD13 Abs. We found that CD13 reactive Abs are present in a proportion of patients after allo-BMT, but did not seem to correlate with the presence of soluble CD13. Anti-CD13 Abs also did not meet our criteria as a diagnostic biomarker for cGVHD. These data do not confirm that induction of CD13 reactive Abs is a mechanism for cGVHD in children nor are part of the pathogenesis of cGVHD associated with elevated soluble CD13. The exact role of CD13 in cGVHD remains to be determined.

Intracranial calcification after cord blood neonatal transplantation for krabbe disease.

Infantile-onset Krabbe disease results from a deficiency of the lysosomal enzyme galactocerebrosidase and leads to death from profound central and peripheral demyelination. Neonatal hematopoietic cell transplantation may result in near-normal cognitive development and partial rescue of gross motor development. The long-term course of the disorder for treated patients seems to involve slowly progressive neurological impairment. We describe the detailed 3-year outcomes of this experimental procedure using umbilical cord blood in a prenatally-diagnosed newborn with Krabbe disease. Substantial perivascular calcifications and atrophy of the white matter developed in the first year post-transplantation. Despite persistent neuroradiological and electrophysiological evidence of leukodystrophy, at age 3 years she has had only mildly impaired non-motor development and moderately impaired motor skills. The cause of these severe white matter changes may have been due to ongoing Krabbe disease or to effects of the chemotherapy regimen or to an interaction of these factors. Extended long-term follow-up of children neonatally transplanted for Krabbe disease is needed before the full utility and limitations of neonatal transplantation can be determined.

High concordance from independent studies by the Children's Cancer Group (CCG) and Pediatric Oncology Group (POG) associating favorable prognosis with combined trisomies 4, 10, and 17 in children with NCI Standard-Risk B-precursor Acute Lymphoblastic Leukemia: a Children's Oncology Group (COG) initiative.

Chromosome aberrations have a major role in pediatric acute lymphoblastic leukemia (ALL) risk assignment. The Children's Cancer Group (CCG) and the Pediatric Oncology Group (POG) independently assessed the significance of trisomy for chromosomes 4, 10, and 17 in National Cancer Institute (NCI) Standard- and High-Risk ALL. Data from 1582 (CCG) and 3902 (POG) patients were analyzed. Eight-year event-free survivals (EFS) of 91% (CCG) and 89% (POG) (P < 0.001) were achieved in patients assigned to NCI Standard Risk whose leukemic cells had simultaneous trisomies 4, 10, and 17. Both groups showed the degree of favorable prognostic importance increased with the actual number of favorable trisomies. POG analyses also demonstrated hyperdiploidy (> or =53 chromosomes) was less of an independently significant prognostic factor in the absence of these key trisomies. This finding supported conclusions from previous CCG and POG studies that specific trisomies are more important than chromosome number in predicting outcome in pediatric B-precursor ALL. In NCI Higher Risk patients, the number of favorable trisomies was not prognostically significant, but showed the same trend. Moreover, specific trisomies 4, 10, and 17 remain associated with favorable prognosis in Standard-Risk B-precursor ALL, even in the context of very different treatment approaches between the groups.

The International Prognostic Scoring System (IPSS) for childhood myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukemia (JMML).

The International Prognostic Scoring System (IPSS) for myelodysplastic syndrome (MDS) is based upon weighted data on bone marrow (BM) blast percentage, cytopenia, and cytogenetics, separating patients into four prognostic groups. We analyzed the value of the IPSS in 142 children with de novo MDS and 166 children with juvenile myelomonocytic leukemia (JMML) enrolled in retro- and prospective studies of the European Working Group on childhood MDS (EWOG-MDS). Survivals in MDS and JMML were analyzed separately. Among the criteria considered by the IPSS score, only BM blasts <5% and platelets >100 x 10(9)/l were significantly associated with a superior survival in MDS. In JMML, better survival was associated with platelets >40 x 10(9)/l, but not with any other IPSS factors including cytogenetics. In conclusion, the IPSS is of limited value in both pediatric MDS and JMML. The results reflect the differences between myelodysplastic and myeloproliferative diseases in children and adults.

Expression of the adaptor protein BLNK/SLP-65 in childhood acute lymphoblastic leukemia.

Deficient expression of BLNK, an adaptor molecule crucial for normal B-cell development, is associated with increased pro-B/pre-B-cell expansion in mice. It has been proposed that BLNK deficiency is a primary cause of B-lineage acute lymphoblastic leukemia (ALL). We studied BLNK expression in the leukemic cells from 352 patients with childhood ALL (309 B-lineage; 43 T-lineage). By HG_U95Av2 Affymetrix GeneChip analysis, BLNK was expressed in 275 of 284 (96.8%) B-lineage ALL samples but in only one of 43 (2.3%) T-lineage ALL samples. Of 118 B-lineage ALL samples analyzed with the HG_U133A GeneChip, 117 (99.2%) expressed BLNK. All 30 primary B-lineage ALL samples studied by RT-PCR expressed BLNK transcripts; all 19 samples studied by Western blotting or flow cytometry expressed BLNK protein. Levels of BLNK in B-lineage ALL were as high as those of their normal counterparts; they were not related with genetic subgroups or differentiation stage. These results indicate that BLNK deficiency is a rare occurrence in childhood B-lineage ALL and is unlikely to be a common leukemogenic event as previously proposed.

Evaluation for synergistic suppression of T cell responses to minor histocompatibility antigens by chloroquine in combination with tacrolimus and a rapamycin derivative, SDZ-RAD.

The 4-aminoquinolines, chloroquine and hydroxychloroquine, can suppress chronic graft-versus-host disease (GVHD) following blood and marrow transplantation (BMT) in mice and humans, respectively. We hypothesized that chloroquine in combination with tacrolimus and the rapamycin derivative SDZ-RAD can synergistically suppress T cell responses and antigen-presenting cell (APC) function in vitro. We used the APC-dependent C57BL/6 anti-BALB.B T cell response and APC-independent anti-CD3epsilon antibody-induced response to evaluate the role of synergism between chloroquine and tacrolimus or SDZ-RAD on each component of a T cell response to minor histocompatibility antigens. We found that chloroquine with tacrolimus had a greater synergistic suppression of APC-dependent compared to the APC-independent T cell responses, with a combination index (CIx) for 50% inhibition by mean effect analysis of 0.16 and 0.50, respectively (a lower number indicates greater suppression). By contrast, chloroquine with SDZ-RAD had a similar CIx between the two responsed 0.50 vs0.45) suggesting only T cell suppression. Synergy between chloroquine and SDZ-RAD involved a direct effect on T cell cytokine production, whereas synergism between chloroquine and tacrolimus was due to an effect on both T cells and APCs. We conclude that the renal-sparing 4-aminoquinolines may be used syneristically with immunosuppressive drugs currently used for BMT.

Differential killing of pre-B acute lymphoblastic leukaemia cells by activated NK cells and the NK-92 ci cell line.

The use of NK cells in adoptive therapy for malignant disease is an area of great potential. Currently the only NK cell line in clinical trials is NK-92, an activated NK cell line with a broad range of cytotoxicity against malignant cells. The activity of NK-92 against pre-B acute lymphoblastic leukaemias, however, is highly variable. In this study we compare the cytotoxic mechanisms and signalling pathways utilized by NK-92 ci and IL-2 activated NK cells to mediate killing of pre-B acute lymphoblastic leukaemia cell lines. Deficiencies in TNF family mediated apoptosis, phosphoinositide-3 kinase dependent and phosphoinositide-3 kinase independent killing limit the efficiency of NK-92 ci against pre-B acute lymphoblastic leukaemia cells. Importantly, treatment of the poorly killed leukaemia cells with TNF-alpha augmented both phosphoinositide-dependent and -independent cytolysis.

Effect of gastrointestinal inflammation and age on the pharmacokinetics of oral microemulsion cyclosporin A in the first month after bone marrow transplantation.

Cyclosporin A (CsA) absorption is highly variable in BMT patients. Neoral, a new microemulsion formulation of CsA, permits increased absorption with less variable pharmacokinetic parameters in non-BMT patients. We evaluated the pharmacokinetics of CsA after BMT in patients received microemulsion CsA. Two oral doses of 3mg/kg were given 48 h apart between 14 and 28 days after allogeneic BMT in 20 adults, and one dose in seven children, while subjects were receiving a continuous i.v. infusion of CsA. Whole blood samples were taken throughout the dosing interval to calculate the incremental CsA exposure using maximum concentration (Cmax), time to Cmax (tmax), concentration at 12 h after the dose (C12), the area under the concentration-time curve (AUC), and to establish inter- and intra-patient pharmacokinetic variability. Drug exposure was substantially lower in children than adults, with an AUC of 861+/-805 vs 2629+/-1487 micromg x h/l (P = 0.001), respectively, and absorption was delayed and diminished in both groups by comparison with solid organ recipients. Intra-patient variability in adults for AUC was high at 0.59+/-0.34, while inter-patient variability, measured as the coefficient of variation (c.v.), was 0.55 for the first and 0.54 for the second dose. In adults, gastrointestinal (GI) inflammation due to either mucositis or GVHD resulted in a higher AUC of 3077+/-1551 microg x h/l compared to 1795+/-973 microg x h/l (P = 0.02), and a similar trend was observed in children. AUC seemed little affected by the CsA formulation (liquid or capsule), or co-administration with liquids or food. Trough (12 h) CsA levels correlated poorly with incremental AUC. Sparse sample modeling of the AUC using two-point predictors taken at 2.5 and 5 h after dosing accurately approximated AUC in adults (r2 = 0.94), while 1.5 and 5 h was superior in children (r2 = 0.98). These data suggest that 12 h postdose trough measurements of CsA may not be the most appropriate way to evaluate CsA blood concentrations in order to establish therapeutic efficacy in BMT patients. Based on this study, the dose of microemulsion CsA should be adjusted based on recipient age, and the presence of GI inflammation secondary to mucositis or GVHD. These data would suggest that sparse sampling at time points earlier than the trough more accurately reflects the AUC and may correlate more closely with therapeutic efficacy early post-BMT.

Age is the major determinant of recurrence in pediatric differentiated thyroid carcinoma.

BACKGROUND: A relationship between young age and increased risk of recurrence of pediatric differentiated thyroid carcinoma has been suggested; however, no attempts have been made to assess the prognostic factors or efficacy of treatment in very young children with this malignancy. The objectives of this study were to evaluate the association of age with outcome in pediatric differentiated thyroid carcinoma and to compare the clinical, pathologic, prognostic, and treatment variables between younger and older children with this disease. PROCEDURE: A retrospective review of all patients presenting to the British Columbia's Children's Hospital or British Columbia Cancer Agency <17 years of age at diagnosis with differentiated thyroid carcinoma between January, 1955, and December, 1996, was completed. RESULTS: Thirty-eight patients were identified, 12 of whom were 10 years. An association between young age and extrathyroidal tumor invasion was identified (P = 0.016); however, the latter factor did not independently predict outcome. There was a trend for suppressive doses of thyroid hormone to improve outcome, particularly with increasing age at diagnosis, but this was not statistically significant. CONCLUSIONS: Age is the major determinant of recurrence in pediatric differentiated thyroid carcinoma. The results suggest different tumor biology in young children requiring novel approaches to therapy to decrease recurrence rates.

Effect of nucleated marrow cell dose on relapse and survival in identical twin bone marrow transplants for leukemia.

The impact of cell dose (number of nucleated donor cells per kilogram recipient weight) on transplantation outcome is controversial and may differ for allogeneic and identical twin (syngeneic) bone marrow transplants. We studied the association between cell dose and outcome in 100 unmanipulated identical twin bone marrow transplantations for leukemia, reported to the International Bone Marrow Transplant Registry between 1985 and 1994, using Cox proportional hazards regression for multivariate analyses. Cell doses ranged from 0.3 to 7.4 x 10(8) nucleated cells/kg (median, 3.0 x 10(8)cells/kg). Median follow-up was 75 months. Five-year cumulative incidences of transplant-related mortality with high (more than 3 x 10(8) cells/kg) versus low (less than or equal to 3 x 10(8) cells/kg) cell doses were 2% (95% confidence interval [CI], 0% to 8%) versus 10% (95% CI, 4% to 20%), respectively. Five-year probabilities of leukemia-free survival were 53% (95% CI, 39% to 67%) and 37% (95% CI, 23% to 52%), respectively. In multivariate analysis, among patients surviving in remission at least 9 months after transplantation, those receiving high cell doses were at significantly lower risk for treatment failure (relapse or death) than those receiving low cell doses (RR, 0.27; 95% CI, 0.12 to 0.6; P =.001). Lower treatment failure resulted from fewer relapses in the high cell dose group (RR for relapse, 0.28; 95% CI, 1.2 to 0.66; P =.003). These findings suggest that outcomes after syngeneic bone marrow transplantation could be improved by transplanting more than 3 x 10(8) nucleated cells per kilogram. The benefit of high cell dose on relapse may represent a delayed graft-versus-leukemia effect.

A population-based study of childhood myelodysplastic syndrome in British Columbia, Canada.

Myelodysplastic syndrome (MDS) is considered to be very rare in children. However, the only two published population-based studies reported widely divergent incidence figures. To further explore the epidemiology of childhood MDS and to evaluate the accuracy of cancer registry and treatment trial data, we conducted a population-based study of children aged 0-14 years in British Columbia (BC), Canada, between 1982 and 1996. MDS was diagnosed in 31 cases corresponding to an annual incidence of 3.2 per million children or 6% of all leukaemias, compared with an incidence of 6.0/million for acute myeloid leukaemia (AML), and of 0.5/million for chronic myeloid leukaemia. There was a non-significant (P = 0.19) trend toward an increase in MDS incidence with time, the increase was partly explained by an increasing number of patients with Down syndrome. Associated abnormalities were found in 48% of the MDS cases with Down syndrome as the most common (seven cases). Only one third of the MDS cases were correctly registered in the Cancer Registry and less than half of the eligible MDS patients were enrolled on a cooperative group study. Data on MDS from treatment-based studies and cancer registries were inaccurate and seemed to significantly underestimate the incidence of MDS in children.