Seah H. Lim

Cancer‐testis antigens in haematological malignancies

Immunotherapy is an attractive therapeutic option for patients with haematological malignancies. Until recently, the progress in the development of tumour vaccines for haematological malignancies had been slow due to the lack of suitable targets. Cancer‐testis (CT) antigens are potentially suitable molecules for tumour vaccines of haematological malignancies because of their high immunogenicity in vivo and their relatively restricted normal tissue distribution. This review evaluates the properties and potential functions of CT antigens. We discuss the expression of CT antigens in patient with haematological malignancies and provide evidence in support of their immunogenicity in vivo in these patients. We also address the role of ‘epigenetic’ regulation of CT antigens in haematological malignancies and how hypomethylating agents could induce the expression of some of these antigens in tumour cells to overcome the problem of heterogeneity of expression of the antigen within individual tumour specimens. Data implicating the interaction of the promoter genes of some of these CT antigens with the MeCP2 protein also suggest the potential role of the histone deacetylase inhibitors in inducing antigen expression in tumour cells. Finally, we discuss the direction of future research in advancing the development of tumour vaccines for haematological malignancies.

B-cell depletion for 2 years after autologous stem cell transplant for NHL induces prolonged hypogammaglobulinemia beyond the rituximab maintenance period

Although autologous stem cell transplant (ASCT) may benefit patients with relapsed or high-risk nonHodgkin lymphoma (NHL), many patients still relapse and die of their disease. Most relapses occur within three years after transplant. In an attempt to reduce disease relapses, we have administered maintenance rituximab infusions to these patients. In this approach, all patients received rituximab infusions (375 mg/m for one day only) every 3 months starting Dþ 100 after ASCT for a total of eight infusions or until disease relapse. We reasoned that rituximab infusions given for only 1 day every 3 months may be sufficient to prevent disease relapse during this post-transplant period when any residual tumor bulk is likely low. We observed that low dose maintenance rituximab given to these patients over a 2-year period after ASCT was associated with severe delays in the immunoglobulin recoveries in these patients, during the 2-year periods when the patients were receiving the rituximab maintenance therapy [1]. However, it remained to be determined how long the hypogammaglobulinemia would persist when more patients are studied in longer follow-up periods. Here, we have analyzed the immunoglobulin recovery in the 15 consecutive patients (eight men, seven women) with high-risk B-cell NHL who have received the post-transplant maintenance rituximab therapy. Their diagnoses were advanced mantle cell lymphoma (MCL) in CR1 (n1⁄4 8), refractory advanced marginal zone lymphoma (n1⁄4 2), refractory follicular large cell lymphoma (n1⁄4 1), high risk T-cell rich B-cell NHL in CR1 (n1⁄4 1), Stage IV diffuse large cell lymphoma in CR1 (n1⁄4 1) and B-cell NHL in CR2 (n1⁄4 2). The median age was 59 years (range 38 – 72 years). CR was achieved using R-CHOP (n1⁄4 10) or R-DHAP/R-ICE (n1⁄4 5) and autologous stem cells harvested during hematopoietic recovery from the last course of R-CHOP or the first course of R-DHAP/R-ICE. Conditioning regimens for the transplant consisted of high dose melphalan (n1⁄4 14) or total body irradiation, cyclophosphamide and thiotepa (n1⁄4 1). In keeping with the preliminary observations we previously made in B-cell NHL [1] and multiple myeloma [2] and unlike patients who underwent ASCT without rituximab in whom B-cell recovery occurred between 3 and 6 months [3 – 5], severe delays in the immunoglobulin recoveries were observed in all 15 patients (Figure 1). In addition to our previous findings, these defects were observed beyond the rituxan maintenance periods. With a median immunoglobulin follow-up of 28 months (range 6 – 64), none of the 15 patients showed normalization of total IgG. Two patients normalized total IgA and two patients total IgM. The hypogammaglobulinemia persisted beyond the rituximab maintenance period. The median time from transplant to attainment of 75% normal IgG level was 36 months and 75% normal IgA level was 48 months.

A yeast two-hybrid system using Sp17 identified Ropporin as a novel cancer-testis antigen in hematologic malignancies.

Since most intracellular proteins are expressed with their ligands, ligands of cancer–testis (CT) antigens may also be CT in their distribution. Applying Sperm protein 17 (Sp17) as the bait in a yeast 2‐hybrid system of a testicular cDNA library, 17 interacting clones were isolated and all encoded Ropporin, a spermatogenic cell‐specific protein that serves as an anchoring protein for the A‐kinase anchoring protein, AKAP110. Ropporin showed a very restricted normal tissue gene expression, detected only in testis and fetal liver. Ropporin mRNA could also be detected in tumor cells from patients with multiple myeloma, chronic lymphocytic leukemia and acute myeloid leukemia. Interestingly, expression of Sp17 did not necessarily predict for the expression of Ropporin suggesting that their coexpression in these tumor cells was random rather than coordinated. Ropporin gene expression in tumor cells is associated with the presence of high titer IgG antibodies against Ropporin, suggesting the in vivo translation of the mRNA into protein and the immunogenicity of the protein to the autologous hosts. Using a CT antigen as the bait in a yeast 2‐hybrid system may, therefore, identify novel tumor antigen. Our results also suggest that Ropporin is a novel CT antigen in hematologic malignancies.

Zap-70 positive chronic lymphocytic leukemia co-existing with Jak 2 V671F positive essential thrombocythemia: A common defective stem cell?

Essential thrombocythemia (ET) co-existing with chronic lymphocytic leukemia (CLL) is extremely rare. We report two cases of ET with Jak 2 V617F in Zap-70+ CLL. ET is a myeloproliferative stem cell disease. Zap-70 expression in CLL correlates with non-mutated immunoglobulin genes. The occurrence of a less mature CLL in patients with a pluripotential stem cell disease raises the possibility that an initial trigger hit occurred in a pre-Jak 2 common early progenitor in these patients. Subsequent additional molecular events accumulated independently following myeloid and lymphoid differentiation, leading to the development of two diseases of likely identical origin but different lineages.

Clofarabine induces hypomethylation of DNA and expression of Cancer-Testis antigens.

In this study, treatment of lymphoid tumor cells with low dose clofarabine upregulated the expression of Sp17 and SPAN-Xb. This was associated with an increase in hypomethylated CpG dinucleotides and a decrease in global DNA methylation, as demonstrated by decreases in the percent of methylated Alu repeats. The most optimal concentration of clofarabine to induce DNA hypomethylation and CT antigen expression was between 1x10(-9) and 1x10(-8)M. Above this, clofarabine resulted in tumor cell growth inhibition and apoptosis. Our results provide the first evidence for the CT antigen-inducing and DNA hypomethylating property of low concentration clofarabine.

Semenogelin I expression in myeloma cells can be upregulated pharmacologically

Semenogelin (SEMG) I is a Cancer-Testis (CT) antigen that we have found to be expressed in myeloma cells. In this study, we set out to determine whether the expression of SEMG I could be upregulated pharmacologically. We found that SEMG I expression in myeloma cells can be upregulated by 5-azacytidine, IL-4 and IL-6. The mechanisms of SEMG I gene upregulation by 5-azacytidine is unclear since there was no correlation between the methylation of the single CpG dinucleotide at position -11 and SEMG I expression. Both IL-4 and IL-6 appeared to enhance SEMG I expression through increasing its promoter function.

A phase II study of low dose intravenous clofarabine for elderly patients with myelodysplastic syndrome who have failed 5-azacytidine

The treatment options for patients with myelodysplastic syndrome (MDS) who have failed DNA hypomethylating agents are limited. In this study, we set out to investigate the efficacy of low dose clofarabine in 10 patients with MDS (four intermediate-2/high risk disease) who had failed 5-azacytidine. The median age was 73 years (range 65–78) and median cycles of clofarabine received were 2 (range 1–4). Nine patients were evaluable for response. An overall response rate of 44% was observed (one CR, one PR, and two HI). All responders had low risk disease. The median duration of response was 12 months (range 6.5–15.5). Although the doses of clofarabine administered were only 12.5–25% of that used in other studies, significant hematologic toxicities were observed. Severe and prolonged pancytopenia occurred in all 10 patients. One patient who had a history of thrombocytopenic gastrointestinal bleed died due to an intracranial bleed despite aggressive platelet support. Low dose clofarabine may, therefore, induce response, but with significant toxicities, in patients with low risk MDS who fail 5-azacytidine. Future work involving a larger patient population is needed to establish the role of low dose clofarabine in low risk MDS.

Core promoter sequence of SEMG I spans between the two putative GATA-1 binding domains and is responsive to IL-4 and IL-6 in myeloma cells

Semenogelin (SEMG) I is a cancer-testis antigen expressed in myeloma cells. SEMG I expression is upregulated by IL-4, IL-6 and 5-azacytidine. In this study, we set out to define the core promoter sequence needed for the expression of SEMG I in myeloma cells. We found that nucleotide sequences spanning the two putative GATA-1 binding domains are vital for the primary regulation of SEMG I promoter function while the other parts of the promoter sequence are responsible for the fine adjustment of the core promoter function. The core promoter sequence is responsive to the enhancing effect of IL-4 and IL-6.

Reduced-intensity umbilical cord blood transplant for older adult patients.

recently addressed the role of unrelated UCBtransplantsforolderpatientsandreportedthattheresultsof UCB transplants carried out in 43 adult patients olderthan 55yearswerecomparabletothoseobtainedfrom 47matched related donor transplants.The 3-year probabilityof progression-free survival was 34% in UCB transplantscompared with30%in matched related donor transplantsand suggested that HLA-mismatched UCB could be analternative graft source for older patients who need atransplantbutdonothaveamatchedrelateddonor.We have carried out an analysis of the results of olderpatients who have undergone reduced-intensity mis-matched UCB transplant in our program. Nine olderpatients, median age 60 years (range 48–77 years) havereceivedmismatchedUCB;78%ofthesepatientsreceivedtwo UCB units to optimize the cell dose. Two patientsreceived one HLA-mismatched, four patients two HLA-mismatched and three patients three HLA-mismatchedgrafts. Clinical characteristics of these patients and theinfused UCB units are shown in Table 1. There were sixmale patients and three female patients. Their medianweight was 72kg (range 60–98kg). Eight patients hadAML,twowithprimaryrefractoryAML,andonepatienthadadvancedrefractoryCLL.Fourpatientshadhemato-poietic cell transplantation comorbidity scores of X2before transplant.

Early renal arterial stent thrombosis associated with the JAK2 V617F mutation

Myeloproliferative diseases (MPD) are a recognized risk factor for thrombotic events. Patients with MPD can sometimes present with thrombotic complications even prior to the diagnosis or manifestation of any clinical features of MPD, although the exact incidence of such a mode of presentation of MPD remains unclear, since it had been difficult to make a diagnosis of early or occult MPD without the use of in vitro bone marrow colony assays not commonly available in the routine laboratory. The recent identification of an association between the JAK2 V617F mutation and MPD enables the convenient early diagnosis in these patients using a molecular approach. JAK2 V617F is a gain-of-function mutation that results in a constitutive activation of the JAK-STAT signaling pathway and leads to the expansion of the affected hematopoietic precursor cells, with skewing towards the erythroid lineage. JAK2 V617F mutation has been found in more than 90% of patients with polycythemia rubra vera but with a lesser frequency in the other MPD. Recently, a strong association has been found between JAK2 V617F mutation and idiopathic Budd-Chiari syndrome or splanchnic thrombosis. A study that involved 139 cases of non-cirrhotic splanchnic vein thrombosis identified the JAK2 V617F mutation in 27 (21%) of these patients, 14 of whom had overt MDS, 3 developed MDS during follow up (median 41 months) and 11 were still disease free at the time of reporting (1). Similar studies have been carried out in other common thrombotic diseases but no correlation was found between JAK2 V617F mutation and deep venous thrombosis, pulmonary embolus, stroke or early myocardial infarction (2). It has, therefore, been proposed that the JAK2 V617F mutation should be tested in patients with non-cirrhotic Budd-Chiari syndrome and thrombosis of the splanchnic circulation, even in the absence of any clinical features of MPD. The routine use of this test for other forms of thromboembolic diseases, however, remains debatable. n nAlthough the association between splanchnic thrombosis and early MPD is well recognized, the occurrence of arterial stent thrombosis in patients with occult MPD has not previously been reported. We report in this paper the first case of a patient with an occult MPD unmasked by a bilateral early renal arterial stent thrombosis. The diagnosis of an occult MPD was made possible molecularly by the detection of the JAK2 V617F mutation. n nA 72 years old lady with a history of hypertension and renal impairment was found to have a bilateral renal artery stenosis by CT scan. Her right renal artery was 80–85% and her left renal artery 85–90% stenosed. She underwent a percutaneous bilateral renal arterial angioplasty with insertion of the Dynalink nitinol 6-Fr stents. The success of the procedure was confirmed by a repeat CT scan that showed 100% patency of the stents. The procedure also resulted in an improved renal function. Ten days after the procedure, the patient presented with abdominal and loin pain. Repeat CT scan showed total occlusion of the renal arterial stents by thrombus and the development of renal infarction. The unfortunate and unusual early adverse thrombotic event triggered the investigations for a thrombophilic tendency. A careful examination did not reveal any established genetic risk factors. She did not have any family history of thromboembolic disease. The genetic workup was unremarkable, without any evidence for Factor V Leiden mutation, Prothrombin gene mutation or methyltetrahydrofolate reductase gene mutation. Her serum homocysteine level was not elevated. Serum Protein C, Protein S and antithrombin III were all within limits. Her only risk factor for thromboembolic disease was a history of smoking half a pack of cigarette per day. Antiphospholipid antibody screening was also negative. She had six previous pregnancies without any thromboembolic complications. Her peripheral blood was checked for the JAK2 V617F mutation by PCR because she had a mild leukocytosis. The white cell count of 11 was associated with a normal hemoglobin and platelet count, although her white cell count was normal just before insertion of the renal arterial stents. She was found to be strongly positive for the JAK2 V617F mutation by qualitative PCR on two separate occasions. With further follow-up 3 months, her leukocytosis has resolved spontaneously and she has continued to have a normal complete blood count without developing any clinical features of MPD. n nEarly thrombosis of the arterial stents (<30 days after the procedure) is observed with both bare metal as well as drug-eluting stents and it occurs at the same frequency of 0.7% for both types of stents (3). It is an uncommon but serious complication of interventional angioplasty. The risk of early stent thrombosis is correlated with the local hemodynamic conditions (4), thrombophilia associated with malignancies (5), hypersensitivity drug reaction in drug eluting stents (6), smoking and younger age (7). The in-stent thrombus is triggered by pro-thrombotic local conditions and facilitated by the local pro-inflammatory reactions that result in platelets aggregation (8). Changes in the practice of interventional cardiology and radiology have led to the increasing use of stents for patients with arterial diseases. It is, therefore, anticipated that the number of cases of arterial stent thrombosis will increase in the next few years. n nThe case presented here raises an important point. Occult MPD may be a previously unrecognized predisposing factor for arterial stent thrombosis. With the availability of molecular approach to the diagnosis of MPD, it may now be possible to carry out a perspective study to determine the exact impact of occult MPD on this rare complication of interventional cardiology and radiology. Early identification of an occult MPD in these patients may suggest that a more aggressive antithrombotic approach is needed in these patients following the stenting procedure.