Mutational Profile in 75 Patients With Anti-Myelin-Associated Glycoprotein Neuropathy: Clinical and Hematologic Therapy Response and Hints on New Therapeutic Targets.

BACKGROUND AND OBJECTIVES: Neuropathy with antibodies to myelin-associated glycoprotein (MAG) is the most common paraproteinemic IgM neuropathy. Recently, the mutational profile of the MYD88 and CXCR4 genes has been included in the diagnostic workup of IgM monoclonal gammopathies. The objective of our study was to assess the prevalence of MYD88 L265P and CXCR4 S338X gene variants in patients with anti-MAG antibody neuropathy. Secondary aims were to evaluate possible correlations between the mutational profile and neuropathy severity, antibody titers, and treatment response. METHODS: Seventy-five patients (47 men, mean age at molecular analysis 70.8 ± 10.2 years; mean disease duration 5.1 ± 4.9 years) with anti-MAG antibody neuropathy were recruited. Among them, 38 (50.7%) had IgM monoclonal gammopathy of undetermined significance, 29 (38.7%) Waldenstrom macroglobulinemia (WM), and 8 (10.6%) chronic lymphocytic leukemia/marginal zone lymphoma/hairy cell leukemia variant. Molecular analysis was performed on DNA from the bone marrow mononuclear cells in 55 of 75 patients and from peripheral mononuclear cells in 18 of 75 patients. Forty-five patients were treated with rituximab, 6 with ibrutinib, 2 with obinutuzumab-chlorambucil, and 3 with venetoclax-based therapy. All the patients were assessed with the Inflammatory Neuropathy Cause and Treatment (INCAT) Disability Scale, INCAT Sensory Sum Score, and MRC Sum Score at baseline and follow-up. We considered as responders, patients who improved by at least 1 point in 2 clinical scales. RESULTS: Fifty patients (66.7%) carried the MYD88L265P variant, with a higher frequency in WM and naive patients (77.2% vs 33.3%, p = 0.0012). No patients harbored the CXCR4S338X variant. There were no significant differences in hematologic data (IgM levels, M protein, and anti-MAG antibody titers), neuropathy severity, or response to rituximab in MYD88-altered and MYD88 wild-type patients. Nine of 11 (81.8%) patients treated with novel targeted drug, according to the MYD88 status, responded to treatments. DISCUSSION: MYD88L265P variant has a high prevalence (66.7%) in anti-MAG antibody neuropathy representing a potential effective mutational target for Bruton tyrosine kinase inhibitors. MYD88L265P variant, however, does not seem to be a prognostic factor of neuropathy severity or response to rituximab. In patients not responding or becoming refractory to rituximab, a tailored therapy with new effective target therapies should be considered.

The Bruton tyrosine kinase inhibitor ibrutinib improves anti-MAG antibody polyneuropathy.

OBJECTIVE: To assess whether neuropathy with anti-myelin-associated glycoprotein (MAG) antibody may improve after treatment with ibrutinib, an oral inhibitor of Bruton tyrosine kinase, we prospectively treated with ibrutinib a cohort of 3 patients with anti-MAG neuropathy and Waldenström macroglobulinemia (WM). METHODS: All 3 patients underwent bone marrow biopsy showing WM, with MYD88 L265P mutated and CXCR4S338X wild type, and were started on ibrutinib 420 mg/die. Patients were assessed at baseline, at 3-6-9 months, and at 12 months in 2 patients with a longer follow-up, using Inflammatory Neuropathy Cause and Treatment (INCAT) Disability Score, INCAT sensory sum score, and Medical Research Council sum score. The modified International Cooperative Ataxia Rating Scale was performed in 2 patients, whereas it was not used in the patient with Parkinson disease as a major comorbidity. Responders were considered the patients improving by at least one point in 2 clinical scales. RESULTS: All the patients reported an early and subjective benefit, consistent with the objective improvement, especially of the sensory symptoms as shown by clinical scales. Treatment was well tolerated. CONCLUSION: These preliminary data point to a possible efficacy of ibrutinib in anti-MAG antibody neuropathy, which is the most common disabling paraproteinemic neuropathy, where active treatment is eagerly needed. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that for patients with anti-MAG antibody neuropathy, ibrutinib improves neuropathy symptoms.

CAL2 monoclonal antibody is a rapid and sensitive assay for the detection of calreticulin mutations in essential thrombocythemia patients.

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.

Genetic, Epigenetic, and Immunologic Profiling of MMR-Deficient Relapsed Glioblastoma.

PURPOSE: In-depth characterization of recurrent glioblastoma (rGBM) might contribute to a better understanding of the mechanisms behind tumor progression and enable rGBM treatment with targeted drugs.Experimental Design: In this study, GBM samples were collected at diagnosis and recurrence from adult patients treated with Stupp protocol. Expression of mismatch repair (MMR) proteins was evaluated by IHC, followed by whole exome sequencing (WES) of tumor samples showing loss of MSH6 reactivity. Established genetic, epigenetic, and immunologic markers were assessed by standard methods and correlated with loss of MMR proteins and patient survival. RESULTS: Expression of MMR proteins was partially or completely lost in 25.9% rGBM samples. Specifically, 12 samples showed partial or total MSH6 expression reduction. Conversely, 96.4% of GBM samples at diagnosis expressed MMR markers. WES disclosed lack of variants in MMR genes in primary samples, whereas two MSH6-negative rGBM samples shared a c.3438+1G>A* splicing MSH6 variant with a potential loss of function effect. MSH6-negative rGBM specimens had high tumor mutational burden (TMB), but no microsatellite instability. In contrast, GBM samples with partial loss of MMR proteins disclosed low TMB. MMR-deficient rGBM showed significant telomere shortening and MGMT methylation and are characterized by highly heterogeneous MHC class I expression. CONCLUSIONS: Multilevel profiling of MMR-deficient rGBM uncovered hypermutated genotype uncoupled from enriched expression of immune-related markers. Assessment of MHC class I expression and TMB should be included in protocols aiming to identify rGBM patients potentially eligible for treatment with drugs targeting immune-checkpoint inhibitors.

LKB1 Expression Correlates with Increased Survival in Patients with Advanced Non-Small Cell Lung Cancer Treated with Chemotherapy and Bevacizumab.

Purpose: LKB1 is a key sensor of metabolic stress, including hypoxia and glucose deprivation, two features of the tumor microenvironment exacerbated by antiangiogenic therapy. We investigated the role of LKB1 as a potential predictive marker of sensitivity to bevacizumab in advanced non-small cell lung cancer (aNSCLC).Experimental design: We retrospectively analyzed LKB1 expression by IHC in 98 samples from 125 patients with aNSCLC, including 59 patients treated with chemotherapy and 39 treated with chemotherapy plus bevacizumab. IHC intensity was recoded in two classes (negative/weak vs. moderate/intense) and correlated with outcome according to treatment arm. Patient-derived tumor xenografts (PDXs) were used to investigate mechanisms involved in preclinical models.Results: In the whole study population (125), median OS and PFS were 11.7 [95% confidence interval (CI), 9.1-15.3] and 6.7 (95% CI, 5.7-7.2) months, respectively. Differential impact of the marker on outcome of the 98 patients was highlighted according to the treatment. Patients with negative/weak LKB1 status did not have a statistically significant benefit from bevacizumab added to chemotherapy (HR for patients treated with bevacizumab: 0.89; 95% CI, 0.51-1.56; P = 0.6803), whereas patients expressing moderate/intense LKB1 and receiving bevacizumab had significant lower risk of death compared with those not receiving bevacizumab (HR, 0.26; 95% CI, 0.10-0.64; P = 0.0035). Loss of LKB1 was associated with reduced AMPK activation in PDXs and increased tumor necrosis following bevacizumab administration, highlighting impaired control of the metabolic stress caused by this antiangiogenic drug.Conclusions: Our data hint at a possible predictive impact of LKB1 expression in patients with aNSCLC treated with chemotherapy plus bevacizumab. Clin Cancer Res; 23(13); 3316-24. ©2017 AACR.

Adrenocortical tumors in Italian children: analysis of clinical characteristics and P53 status. Data from the national registries.

AIM: Adrenocortical tumors are very rare in children. The distinction between adenoma and carcinoma is complex because of their clinical/histological characteristics. The analysis of the cases registered in two consecutive Italian Studies is described, in order to provide additional insight into their nature and possibly identify benign and malignant lesions. MATERIALS AND METHODS: The analysis includes patients registered from?? 1.1982 to 6.2011 into two consecutive Italian protocols. RESULTS: Fifty-eight children (age 2-210months) were evaluated. Endocrine manifestations were the most frequent symptoms. Stage distribution at diagnosis was: ST I 35, ST II 17, ST III 1, ST IV 5. Treatment consisted in mitotane for ST II, mitotane+chemotherapy for ST III/IV. Forty-four patients are alive without evidence of disease, 1 is alive with disease, 12 died of disease and 1 because of cardiomyopathy. The Wienecke score system was applied in 24 patients with good significance. A p53 mutation was found in 7 cases, and it was diagnostic for Li-Fraumeni syndrome in 2 benign tumors. CONCLUSIONS: The results highlight the importance of a complete excision to obtain the cure of patients. The efficacy of chemotherapy is controversial, however it was able to control the disease in 4 patients in ST II. The value of the Wienecke score system in predicting patients' outcome was confirmed. p53 mutation was more frequent in malignant tumors and represented the sentinel of the Li-Fraumeni syndrome.

C-Kit SCF receptor (CD117) expression and KIT gene mutation in conjunctival pigmented lesions.

PURPOSE: To investigate the presence of KIT gene mutations and immunoreactivity in 85 conjunctival melanocytic tumours and to clarify the role of KIT as a potential therapeutic target in this group of patients. METHODS: Eighty-five conjunctival pigmented tumours [27 melanomas, 12 primary acquired melanosis (PAMs) and 46 nevi] were immunostained for KIT. Intensity and pattern of expression were evaluated. Molecular analysis to identify KIT mutations was performed in 15 selected cases (tumour-rich areas >50%). KIT immunostaining score and pattern were statistically related to patients' age, sex, diagnostic category, presence of relapse, disease-free survival, presence of metastases, metastasis-free survival, limbal versus nonlimbal tumour location and thickness of melanomas. RESULTS: KIT stains were documented in 48% of melanomas, 50% of PAMs and 24% of nevi. The mean score of KIT staining in the melanomas/PAMs group was significantly different from nevi (p = 0.0076). No statistically significant differences were detected between either c-kit immunostaining score or pattern and each of the other clinico-pathologic parameters considered. No KIT gene mutations were detected in melanomas and nevi. A silent mutation/polymorphism in KIT exon 13 was found in one PAM. CONCLUSIONS: Despite the high level of KIT immunostains in PAMs and melanomas, this parameter seems not to be a good predictor of the presence of molecular mutations. KIT-activating mutations should be considered an uncommon event in this tumour.

Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes.

Interstitial cells of the adult rat heart were characterized with respect to i) expression of cardiac markers of commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We demonstrate for the first time that fibroblasts and a proportion of pericytes in the adult rat heart express the transcription factor GATA4. This appears to be a peculiar property of the heart. Fibroblasts that are also derived from the splanchnopleuric mesoderm, such as those of the gut, or fibroblasts of different embryological origin, such as those of skin and skeletal muscle, lack this property. Of note, a nestin+/GATA4+ putative stem cell population is also detected in the adult heart. GATA4+ cardiac interstitial cells do not display myogenic potential in vitro. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as demonstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone H3 (H3P) and incorporating 5-bromodeoxiuridine (BrdU) in the co-cultures. In conclusion, cardiac fibroblasts have peculiar expression of myogenic transcription factors, a property that may have an impact for reprogramming these cells to the myogenic differentiation. In addition, they are able to modulate the behavior of adult cardiomyocytes, a property that may be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium.