Continuous T cell receptor signals maintain a functional regulatory T cell pool.

Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR.

PD-1+ regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer.

PD-1 blockade is a cancer immunotherapy effective in various types of cancer. In a fraction of treated patients, however, it causes rapid cancer progression called hyperprogressive disease (HPD). With our observation of HPD in ∼10% of anti-PD-1 monoclonal antibody (mAb)-treated advanced gastric cancer (GC) patients, we explored how anti-PD-1 mAb caused HPD in these patients and how HPD could be treated and prevented. In the majority of GC patients, tumor-infiltrating FoxP3highCD45RA-CD4+ T cells [effector Treg (eTreg) cells], which were abundant and highly suppressive in tumors, expressed PD-1 at equivalent levels as tumor-infiltrating CD4+ or CD8+ effector/memory T cells and at much higher levels than circulating eTreg cells. Comparison of GC tissue samples before and after anti-PD-1 mAb therapy revealed that the treatment markedly increased tumor-infiltrating proliferative (Ki67+) eTreg cells in HPD patients, contrasting with their reduction in non-HPD patients. Functionally, circulating and tumor-infiltrating PD-1+ eTreg cells were highly activated, showing higher expression of CTLA-4 than PD-1- eTreg cells. PD-1 blockade significantly enhanced in vitro Treg cell suppressive activity. Similarly, in mice, genetic ablation or antibody-mediated blockade of PD-1 in Treg cells increased their proliferation and suppression of antitumor immune responses. Taken together, PD-1 blockade may facilitate the proliferation of highly suppressive PD-1+ eTreg cells in HPDs, resulting in inhibition of antitumor immunity. The presence of actively proliferating PD-1+ eTreg cells in tumors is therefore a reliable marker for HPD. Depletion of eTreg cells in tumor tissues would be effective in treating and preventing HPD in PD-1 blockade cancer immunotherapy.

T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development.

The transcription factor Foxp3 is essential for the development of regulatory T (Treg) cells, yet its expression is insufficient for establishing the Treg cell lineage. Here we showed that Treg cell development was achieved by the combination of two independent processes, i.e., the expression of Foxp3 and the establishment of Treg cell-specific CpG hypomethylation pattern. Both events were induced by T cell receptor stimulation. The Treg cell-type CpG hypomethylation began in the thymus and continued to proceed in the periphery and could be fully established without Foxp3. The hypomethylation was required for Foxp3(+) T cells to acquire Treg cell-type gene expression, lineage stability, and full suppressive activity. Thus, those T cells in which the two events have concurrently occurred are developmentally set into the Treg cell lineage. This model explains how Treg cell fate and plasticity is controlled and can be exploited to generate functionally stable Treg cells.

Impact of genetic risk loci for multiple sclerosis on expression of proximal genes in patients.

Despite advancements in genetic studies, it is difficult to understand and characterize the functional relevance of disease-associated genetic variants, especially in the context of a complex multifactorial disease such as multiple sclerosis (MS). As a large proportion of expression quantitative trait loci (eQTLs) are context-specific, we performed RNA-Seq in peripheral blood mononuclear cells from MS patients (n = 145) to identify eQTLs in regions centered on 109 MS risk single nucleotide polymorphisms and 7 associated human leukocyte antigen variants. We identified 77 statistically significant eQTL associations, including pseudogenes and non-coding RNAs. Thirty-eight out of 40 testable eQTL effects were colocalized with the disease association signal. As many eQTLs are tissue specific, we aimed to detail their significance in different cell types. Approximately 70% of the eQTLs were replicated and characterized in at least one major peripheral blood mononuclear cell-derived cell type. Furthermore, 40% of eQTLs were found to be more pronounced in MS patients compared with non-inflammatory neurological diseases patients. In addition, we found two single nucleotide polymorphisms to be significantly associated with the proportions of three different cell types. Mapping to enhancer histone marks and predicted transcription factor binding sites added additional functional evidence for eight eQTL regions. As an example, we found that rs71624119, shared with three other autoimmune diseases and located in a primed enhancer (H3K4me1) with potential binding for STAT transcription factors, significantly associates with ANKRD55 expression. This study provides many novel and validated targets for future functional characterization of MS and other diseases.

Pericyte-fibroblast transition promotes tumor growth and metastasis.

Vascular pericytes, an important cellular component in the tumor microenvironment, are often associated with tumor vasculatures, and their functions in cancer invasion and metastasis are poorly understood. Here we show that PDGF-BB induces pericyte-fibroblast transition (PFT), which significantly contributes to tumor invasion and metastasis. Gain- and loss-of-function experiments demonstrate that PDGF-BB-PDGFRß signaling promotes PFT both in vitro and in in vivo tumors. Genome-wide expression analysis indicates that PDGF-BB-activated pericytes acquire mesenchymal progenitor features. Pharmacological inhibition and genetic deletion of PDGFRß ablate the PDGF-BB-induced PFT. Genetic tracing of pericytes with two independent mouse strains, TN-AP-CreERT2:R26R-tdTomato and NG2-CreERT2:R26R-tdTomato, shows that PFT cells gain stromal fibroblast and myofibroblast markers in tumors. Importantly, coimplantation of PFT cells with less-invasive tumor cells in mice markedly promotes tumor dissemination and invasion, leading to an increased number of circulating tumor cells and metastasis. Our findings reveal a mechanism of vascular pericytes in PDGF-BB-promoted cancer invasion and metastasis by inducing PFT, and thus targeting PFT may offer a new treatment option of cancer metastasis.

Hypermethylation of MIR21 in CD4+ T cells from patients with relapsing-remitting multiple sclerosis associates with lower miRNA-21 levels and concomitant up-regulation of its target genes.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system caused by genetic and environmental factors. DNA methylation, an epigenetic mechanism that controls genome activity, may provide a link between genetic and environmental risk factors. OBJECTIVE: We sought to identify DNA methylation changes in CD4+ T cells in patients with relapsing-remitting (RR-MS) and secondary-progressive (SP-MS) disease and healthy controls (HC). METHODS: We performed DNA methylation analysis in CD4+ T cells from RR-MS, SP-MS, and HC and associated identified changes with the nearby risk allele, smoking, age, and gene expression. RESULTS: We observed significant methylation differences in the VMP1/MIR21 locus, with RR-MS displaying higher methylation compared to SP-MS and HC. VMP1/MIR21 methylation did not correlate with a known MS risk variant in VMP1 or smoking but displayed a significant negative correlation with age and the levels of mature miR-21 in CD4+ T cells. Accordingly, RR-MS displayed lower levels of miR-21 compared to SP-MS, which might reflect differences in age between the groups, and healthy individuals and a significant enrichment of up-regulated miR-21 target genes. CONCLUSION: Disease-related changes in epigenetic marking of MIR21 in RR-MS lead to differences in miR-21 expression with a consequence on miR-21 target genes.

VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism and serves as a marker of poor prognosis for cancer patients.

The biological functions of VEGF-B in cancer progression remain poorly understood. Here, we report that VEGF-B promotes cancer metastasis through the remodeling of tumor microvasculature. Knockdown of VEGF-B in tumors resulted in increased perivascular cell coverage and impaired pulmonary metastasis of human melanomas. In contrast, the gain of VEGF-B function in tumors led to pseudonormalized tumor vasculatures that were highly leaky and poorly perfused. Tumors expressing high levels of VEGF-B were more metastatic, although primary tumor growth was largely impaired. Similarly, VEGF-B in a VEGF-A-null tumor resulted in attenuated primary tumor growth but substantial pulmonary metastases. VEGF-B also led to highly metastatic phenotypes in Vegfr1 tk(-/-) mice and mice treated with anti-VEGF-A. These data indicate that VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism. High expression levels of VEGF-B in two large-cohort studies of human patients with lung squamous cell carcinoma and melanoma correlated with poor survival. Taken together, our findings demonstrate that VEGF-B is a vascular remodeling factor promoting cancer metastasis and that targeting VEGF-B may be an important therapeutic approach for cancer metastasis.

Genetic and epigenetic basis of Treg cell development and function: from a FoxP3-centered view to an epigenome-defined view of natural Treg cells.

Naturally occurring regulatory T (nTreg) cells, which specifically express the transcription factor Forkhead box protein P3 (FoxP3), are indispensable for the maintenance of immunological self-tolerance and homeostasis. Recent studies have shown that developing nTreg cells in the thymus acquire a Treg-specific and stable hypomethylation pattern in a limited number of genes, which encode key molecules including FoxP3, essential for Treg cell function. This epigenetic change is acquired via T-cell receptor (TCR) stimulation, beginning prior to FoxP3 expression. The Treg-specific DNA hypomethylated regions generally act as gene enhancers in steady state nTreg cells, contributing to the stable expression of Treg function-associated key genes including Ctla4, Il2ra, and Ikzf4 in addition to Foxp3. Upon TCR stimulation of mature nTreg cells, FoxP3 strongly represses many genes including Il2, contributing to Treg suppressive activity. Thus, the Treg-specific epigenome alteration can determine the heritable Treg-specific gene network including Foxp3 regulation. Considering physiological presence of non-suppressive FoxP3(+) T cells in the immune system and loss of FoxP3 in Treg cells under certain immunological conditions, functional nTreg cells can be more accurately defined as a T-cell subpopulation possessing the Treg-type epigenome, rather than FoxP3(+) T cells. This epigenome-based definition of Treg cells would enable better understanding of functional stability, plasticity, and heterogeneity of Treg cells.

Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation.

Naturally occurring regulatory T (Treg) cells, which specifically express the transcription factor forkhead box P3 (Foxp3), are engaged in the maintenance of immunological self-tolerance and homeostasis. By transcriptional start site cluster analysis, we assessed here how genome-wide patterns of DNA methylation or Foxp3 binding sites were associated with Treg-specific gene expression. We found that Treg-specific DNA hypomethylated regions were closely associated with Treg up-regulated transcriptional start site clusters, whereas Foxp3 binding regions had no significant correlation with either up- or down-regulated clusters in nonactivated Treg cells. However, in activated Treg cells, Foxp3 binding regions showed a strong correlation with down-regulated clusters. In accordance with these findings, the above two features of activation-dependent gene regulation in Treg cells tend to occur at different locations in the genome. The results collectively indicate that Treg-specific DNA hypomethylation is instrumental in gene up-regulation in steady state Treg cells, whereas Foxp3 down-regulates the expression of its target genes in activated Treg cells. Thus, the two events seem to play distinct but complementary roles in Treg-specific gene expression.

Construction of self-recognizing regulatory T cells from conventional T cells by controlling CTLA-4 and IL-2 expression.

Thymus-produced CD4(+) regulatory T (Treg) cells, which specifically express the transcription factor forkhead box p3, are potently immunosuppressive and characteristically possess a self-reactive T-cell receptor (TCR) repertoire. To determine the molecular basis of Treg suppressive activity and their self-skewed TCR repertoire formation, we attempted to reconstruct these Treg-specific properties in conventional T (Tconv) cells by genetic manipulation. We show that Tconv cells rendered IL-2 deficient and constitutively expressing transgenic cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) were potently suppressive in vitro when they were preactivated by antigenic stimulation. They also suppressed in vivo inflammatory bowel disease and systemic autoimmunity/inflammation produced by Treg deficiency. In addition, in the thymus, transgenic CTLA-4 expression in developing Tconv cells skewed their TCR repertoire toward higher self-reactivity, whereas CTLA-4 deficiency specifically in developing thymic Treg cells cancelled their physiological TCR self-skewing. The extracellular portion of CTLA-4 was sufficient for the suppression and repertoire shifting. It interfered with CD28 signaling to responder Tconv cells via outcompeting CD28 for binding to CD80 and CD86,or modulating CD80/CD86 expression on antigen-presenting cells. Thus, a triad of IL-2 repression, CTLA-4 expression, and antigenic stimulation is a minimalistic requirement for conferring Treg-like suppressive activity on Tconv cells, in accordance with the function of forkhead box p3 to strongly repress IL-2 and maintain CTLA-4 expression in natural Treg cells. Moreover, CTLA-4 expression is a key element for the formation of a self-reactive TCR repertoire in natural Treg cells. These findings can be exploited to control immune responses by targeting IL-2 and CTLA-4 in Treg and Tconv cells.

Exploring the Potential Use of Natural Products Together with Alkalization in Cancer Therapy.

Cancer treatment is a significant focus in medicine, owing to the increasing global incidence of cancers. Patients with advanced cancers that do not respond to conventional therapies have limited options and an unfavorable prognosis. Consequently, researchers are investigating complementary approaches to conventional treatments. One such approach is alkalization therapy, which aims to neutralize the acidic tumor microenvironment (TME) by increasing its pH level. The acidic TME promotes inflammation, tumor progression, and drug resistance. Alkalization therapy has been demonstrated to be effective for various cancers. In addition, natural products, such as triterpenoids, parthenolides, fulvic acid, Taxus yunnanensis, and apple pectin have the potential to alleviate symptoms, maintain physical fitness, and improve treatment outcomes of cancer patients through their anti-inflammatory, antioxidant, and anticancer properties. In this review, we focus on the effects of alkalization therapy and natural products on cancer. Furthermore, we present a case series of advanced cancer patients who received alkalization therapy and natural products alongside standard treatments, resulting in long-term survival. We posit that alkalization therapy together with supplementation with natural products may confer benefits to cancer patients, by mitigating the side effects of chemotherapy and complementing standard treatments. However, further research is warranted to validate these clinical findings.

Corrigendum: Effects of alkalization therapy on hepatocellular carcinoma: a retrospective study.

[This corrects the article DOI: 10.3389/fonc.2023.1179049.].

Effects of alkalization therapy on hepatocellular carcinoma: a retrospective study.

Background: In hepatocellular carcinoma (HCC) patients, is difficult to prevent recurrence even when remission is achieved. In addition, even with the advent of drugs that are effective for the treatment of HCC, a satisfactory extension of patient survival has not been achieved. To overcome this situation, we hypothesized that the combination of alkalization therapy with standard treatments will improve the prognosis of HCC. We here report the clinical results of HCC patients treated with alkalization therapy at our clinic. Patients and methods: Patients with HCC treated at Karasuma Wada Clinic (in Kyoto, Japan), from January 1, 2013, to December 31, 2020 were analyzed. Overall survival (OS) from both the time of diagnosis and the start of alkalization therapy for each patient was compared. The mean urine pH was also calculated as a surrogate marker of tumor microenvironment pH, and OS from the start of alkalization therapy was compared between patients with a mean urine pH of ≥ 7.0 and those with a mean urine pH of < 7.0. Results: Twenty-three men and six women were included in the analysis, with a mean age at diagnosis of 64.1 years (range: 37-87 years). Seven of the 29 patients had extrahepatic metastases. Patients were divided into two groups according to their mean urine pH after the initiation of alkalization therapy: 12 of the 29 patients had a mean urine pH of ≥ 7.0, and 17 had a mean urine pH of < 7.0. The median OS from diagnosis was 95.6 months (95% confidence interval [CI] = 24.7-not reached), and from the start of alkalization therapy was 42.3 months (95% CI = 8.93-not reached). The median OS from the start of alkalization therapy in patients with a urine pH of ≥ 7.0 was not reached (n = 12, 95% CI = 3.0-not reached), which was significantly longer than that in patients with a pH of < 7.0 (15.4 months, n = 17, 95% CI = 5.8-not reached, p < 0.05). Conclusions: The addition of alkalization therapy to standard therapies may be associated with more favorable outcomes in HCC patients with increased urine pH after alkalization therapy.

Potential of Alkalization Therapy for the Management of Metastatic Pancreatic Cancer: A Retrospective Study.

Current treatments for patients with pancreatic cancer offer limited benefits. In this study, we applied alkalization therapy, which was efficacious for other solid tumors at our clinic, to stage 4 pancreatic cancer patients, and investigated its effect on disease prognosis. Patients with metastatic pancreatic cancer who were treated at Karasuma Wada Clinic in Kyoto, Japan, between January 2011 and April 2022, were included in the study. All patients received alkalization therapy (a combination of an alkaline diet, bicarbonate, and citric acid administration), alongside standard chemotherapy. Urine samples were collected to assess urine pH as a marker of whole-body alkalization. In the 98 patients analyzed, the median overall survival (OS) from the time of diagnosis was 13.2 months. Patients with a mean urine pH of 7.5 or greater had a median OS of 29.9 months, compared with 15.2 months for those with a mean urine pH of 6.5 to 7.5, and 8.0 months for those with a mean urine pH of less than 6.5, which suggests a trend of a longer OS in patients with a higher urine pH (p = 0.0639). Alkalization therapy may offer a viable approach to extending the survival of stage 4 pancreatic cancer patients, who typically have an unfavorable prognosis.

Construction of a T cell receptor signaling range for spontaneous development of autoimmune disease.

Thymic selection and peripheral activation of conventional T (Tconv) and regulatory T (Treg) cells depend on TCR signaling, whose anomalies are causative of autoimmunity. Here, we expressed in normal mice mutated ZAP-70 molecules with different affinities for the CD3 chains, or wild type ZAP-70 at graded expression levels under tetracycline-inducible control. Both manipulations reduced TCR signaling intensity to various extents and thereby rendered those normally deleted self-reactive thymocytes to become positively selected and form a highly autoimmune TCR repertoire. The signal reduction more profoundly affected Treg development and function because their TCR signaling was further attenuated by Foxp3 that physiologically repressed the expression of TCR-proximal signaling molecules, including ZAP-70, upon TCR stimulation. Consequently, the TCR signaling intensity reduced to a critical range generated pathogenic autoimmune Tconv cells and concurrently impaired Treg development/function, leading to spontaneous occurrence of autoimmune/inflammatory diseases, such as autoimmune arthritis and inflammatory bowel disease. These results provide a general model of how altered TCR signaling evokes autoimmune disease.

Meaning and Significance of "Alkalization Therapy for Cancer".

Objectives of the Study: Our research aims to answer the following questions. Can cancer progression be stopped by changing the body condition of person with cancer? Can cancer be cured?If cancer progression can be stopped, what is the underlying mechanism? Theoretical Rationale for Alkalization Therapy: Almost 70 years ago, Goldblatt H. & Cameron G. reported on the idea of alkalization therapy. Before that, Otto Warburg had been studying the metabolism of cancer and had discovered the essential nature of cancer. He published a review in Science in 1956 under the title "On the origin of cancer cells". From his phenomena described above, we established the theoretical rationale for alkalization therapy, based on the question of "How does cancer form and what is its nature"? Limitations of Deductive Methods and Inductive Approaches: In this paper, we describe a method to reconstruct the limitations and weaknesses of modern cancer medicine as Science-based Medicine using an inductive method, and to present a new vision of cancer therapy. How should we treat cancer? (Case presentation): Using a specific clinical case, we present patients in whom were successfully treated with no or few anticancer drugs. Summary: The biggest weakness of current cancer treatments is that they only treat the cancer and not the actual patient. The "alkalization therapy" that we advocate does not compete with any of the current standard treatments, but improves the effectiveness of standard treatments, reduces side effects, and lowers medical costs.

Clinical review of alkalization therapy in cancer treatment.

One of the most unique characteristics of cancer metabolism is activated aerobic glycolysis, which is called the "Warburg effect", and is a hallmark of cancer. An acidic tumor microenvironment (TME) resulting from activated anaerobic glycolysis is associated with cancer progression, multi-drug resistance, and immune escape. Several in vitro and in vivo studies reported that neutralization of the acidic TME by alkalizing agents, such as bicarbonate, resulted in the suppression of cancer progression and a potential benefit for anti-cancer drug responses. In clinical settings, alkalizing effects were achieved not only by alkalizing agents, but also by a following a particular diet. An epidemiological study demonstrated that more fruits and vegetables and less meat and dairy products are associated with an increase in urine pH, which may reflect the alkalizing effect on the body. However, it remains unclear whether alkaline dietary intervention improves the effects of cancer treatment. Moreover, there are few clinical reports to date regarding cancer treatments being performed on patients together with alkalization therapy. In this review, we investigated whether alkalization therapy, which includes an alkaline diet and/or alkalizing agents, improves cancer treatment.

Improved Chemotherapy Outcomes of Patients With Small-cell Lung Cancer Treated With Combined Alkalization Therapy and Intravenous Vitamin C.

Background/Aim: This study aimed to investigate the effects of the combination of alkalization therapy (an alkaline diet and bicarbonate therapy) and intravenous vitamin C treatment on chemotherapy outcomes in patients with small-cell lung cancer (SCLC) (study registration: UMIN000043056). Patients and Methods: Twelve patients with SCLC in the intervention group (receiving both alkalization therapy and vitamin C treatment together with chemotherapy) were retrospectively compared to 15 patients with SCLC in the control group (receiving chemotherapy only). Results: The mean urine pH of the intervention group was significantly higher than that of the control group (7.32±0.45 vs. 6.44±0.74, respectively; p<0.005). The median overall survival for the intervention group was 44.2 months (95% confidence interval=22.0-not reached), as compared with 17.7 months for the control group (95% confidence intervaI=13.5-not reached; p<0.05). Conclusion: The combination of alkalization therapy and intravenous vitamin C treatment may be associated with favorable outcomes in patients with SCLC receiving chemotherapy.

Effects of Alkalization Therapy on Chemotherapy Outcomes in Advanced Pancreatic Cancer: A Retrospective Case-Control Study.

BACKGROUND/AIM: Neutralization of the acidic tumor microenvironment, which is associated with both progression and drug resistance of cancer cells, may be a new treatment option for progressing forms of cancer. We conducted a case-control study to investigate the effects of alkalization therapy, consisting of an alkaline diet with supplementary oral sodium bicarbonate, in patients with metastatic or recurrent pancreatic cancer (study registration no.: UMIN000036126). PATIENTS AND METHODS: Thirty-six patients in the alkalization group (Karasuma Wada Clinic; alkalization therapy plus chemotherapy) were retrospectively compared to 89 patients in the control group (Kyoto University Hospital; chemotherapy only). RESULTS: The median overall survival (OS) in the alkalization group was significantly longer than that in the control group (15.4 vs. 10.8 months; p<0.005). In the alkalization group, mean urine pH was significantly increased after alkalization therapy [6.38±0.85 (before) vs. 6.80±0.71 (after); p<0.05]. Furthermore, the median OS of patients with increased urine pH (pH>7.0 or ΔpH>1.0) in the alkalization group was significantly longer than that of the control group. CONCLUSION: Alkalization therapy may enhance the effects of chemotherapy in patients with advanced pancreatic cancer.

Immunometabolic Network Interactions of the Kynurenine Pathway in Cutaneous Malignant Melanoma.

Dysregulation of the kynurenine pathway has been regarded as a mechanism of tumor immune escape by the enzymatic activity of indoleamine 2, 3 dioxygenase and kynurenine production. However, the immune-modulatory properties of other kynurenine metabolites such as kynurenic acid, 3-hydroxykynurenine, and anthranilic acid are poorly understood. In this study, plasma from patients diagnosed with metastatic cutaneous malignant melanoma (CMM) was obtained before (PRE) and during treatment (TRM) with inhibitors of mitogen-activated protein kinase pathway (MAPKIs). Immuno-oncology related protein profile and kynurenine metabolites were analyzed by proximity extension assay (PEA) and LC/MS-MS, respectively. Correlation network analyses of the data derived from PEA and LC/MS-MS identified a set of proteins that modulate the differentiation of Th1 cells, which is linked to 3-hydroxykynurenine levels. Moreover, MAPKIs treatments are associated with alteration of 3-hydroxykynurenine and 3hydroxyanthranilic acid (3HAA) concentrations and led to higher "CXCL11," and "KLRD1" expression that are involved in T and NK cells activation. These findings imply that the kynurenine pathway is pathologically relevant in patients with CMM.