Haploidentical HSCT: a 15-year experience at San Raffaele.

Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular-based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed.

Incidence, risk factors and clinical outcome of leukemia relapses with loss of the mismatched HLA after partially incompatible hematopoietic stem cell transplantation.

Genomic loss of the mismatched human leukocyte antigen (HLA) is a recently described mechanism of leukemia immune escape and relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Here we first evaluated its incidence, risk factors and outcome in 233 consecutive transplants from partially HLA-mismatched related and unrelated donors (MMRD and MMUD, respectively). We documented 84 relapses, 23 of which with HLA loss. All the HLA loss relapses occurred after MMRD HSCT, and 20/23 in patients with acute myeloid leukemia. Upon MMRD HSCT, HLA loss variants accounted for 33% of the relapses (23/69), occurring later than their 'classical' counterparts (median: 307 vs 88 days, P<0.0001). Active disease at HSCT increased the risk of HLA loss (hazard ratio (HR): 10.16; confidence interval (CI): 2.65-38.92; P=0.001), whereas older patient ages had a protective role (HR: 0.16; CI: 0.05-0.46; P=0.001). A weaker association with HLA loss was observed for graft T-cell dose and occurrence of chronic graft-versus-host disease. Outcome after 'classical' and HLA loss relapses was similarly poor, and second transplantation from a different donor appeared to provide a slight advantage for survival. In conclusion, HLA loss is a frequent mechanism of evasion from T-cell alloreactivity and relapse in patients with myeloid malignancies transplanted from MMRDs, warranting routine screening in this transplantation setting.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

Early molecular diagnosis of aspergillosis in a patient with acute myeloid leukaemia.

Diagnosis of invasive fungal infection remains challenging. Here we report a case of early diagnosis of invasive aspergillosis in a neutropenic patient affected by acute myeloid leukaemia, achieved through the detection of Aspergillus fumigatus species-specific ribonucleic acid sequences by a sensitive multiplex real-time polymerase chain reaction-based molecular assay. Thanks to the early diagnosis, targeted therapy was promptly established and the severe fungal infection controlled, allowing the patient to subsequently receive allogeneic hematopoietic stem cell transplantation from a haploidentical donor, her only curative option. Also in this instance, targeted secondary antifungal prophylaxis with voriconazole avoided any other fungal infection afterwards. This report suggests how the implementation of molecular assays in combination with routine diagnostic procedures, can improve microbiological diagnosis in sepsis, particularly in case of fungal infection, difficult to detect with standard microbiological culture methods.

Glomerular lesions in HIV-positive patients: a 20-year biopsy experience from Northern Italy.

AIM: Glomerular involvement in HIV-positive patients is quite heterogeneous. In the present paper we reviewed 73 renal biopsies performed during a period of more than 20 years in a single Nephrology Unit, Milan, Northern Italy, in order to evaluate the aspects of single types of glomerular lesions (including HIV associated nephropathy-HIVAN), grouped according to histological patterns and clinical presentation. Moreover, in the group of non-HIVAN patients, the possible differences in histological characteristics from non-HIV lesions were investigated. MATERIALS AND METHODS: Renal tissues were obtained by percutaneous biopsies and were studied by light microscopy, immunofluorescence and electron microscopy. For the histological description three histological groups were identified: HIVAN, immune complex glomerulonephritis (GN) and glomerulopathies not related to immune-mediated mechanisms (so-called "various" glomerulopathies). RESULTS: HIVAN was observed in 9 cases, immune complex GNs in 40 cases (10 mesangial proliferative GN, 8 membranoproliferative GN, 5 lupus-like GN, 4 "acute" GN, 2 crescentic GN, 4 IgA nephropathy, 4 membranous GN and 3 immunotactoid GN) and "various" glomerulopathies in 24 cases (13 non-collapsing focal segmental glomerulosclerosis, 3 minimal changes, 3 end-stage renal disease, 4 diabetic nephropathy and one amyloidosis). CONCLUSIONS: Our 20-year biopsy series of HIV-related glomerular involvement confirmed the heterogeneity of lesions. In our series, the vast majority of HIV-related GN are the so-called immune complex GNs, with some peculiar aspects, as multiple site location of deposits and a frequent tendency towards sclerosis, in agreement with experimental data regarding HIV and fibrosis.

Risk factors for fibrosis progression in HIV/HCV coinfected patients from a retrospective analysis of liver biopsies in 1985-2002.

OBJECTIVES: To identify predictive factors for moderate/severe liver fibrosis and to analyse fibrosis progression in paired liver biopsies from HIV-positive patients with chronic hepatitis C virus (HCV) infection. METHODS: HIV/HCV coinfected patients followed at the 2nd Department of Infectious Diseases of L. Sacco Hospital in Milan, Italy, with at least one liver biopsy specimen were retrospectively evaluated. RESULTS: A total of 110 patients were enrolled in the study. In a univariate analysis, predictive factors of Ishak-Knodell stage > or =3 were a history of alcohol abuse [odds ratio (OR) 3.6, P=0.004], alanine aminotransferase level >100 IU/L at biopsy (OR 2.4, P=0.05), necro-inflammatory grade > or =9 (OR 37.14, P<0.0001) and CD4 count <350 cells/microL at nadir (OR 5.3, P=0.05). In a multivariate analysis, age >35 years (OR 3.19, P=0.04) and alcohol abuse (OR 4.36, P=0.002) remained independently associated with Ishak-Knodell stage. Paired liver biopsies were available in 36 patients; 18 showed an increase of at least one stage in the subsequent liver biopsy. Either in a univariate or in a multivariate analysis, a decrease of CD4 cell count of more than 10% between two biopsies (OR 6.85, P=0.002) was significantly associated with liver fibrosis progression. CONCLUSION: Our findings highlight the relevance of encouraging a withdrawal of alcohol consumption in people with chronic HCV infection and of carrying out close follow-up of patients, especially if they are more than 35 years old. It is therefore mandatory to evaluate HIV/HCV coinfected patients for anti-HCV treatment and to increase CD4 cell count through antiretroviral therapy in order to reduce the risk of fibrosis progression and to slow the evolution of liver disease.

Ultrastructural study of renal involvement in two females with Anderson-Fabry disease.

Anderson-Fabry disease (AFD) is a rare X-linked lipid storage disorder due to a deficient lysosomal a-galactosidase A (a-Gal) activity. In males with the classic form of the disease the enzymatic defect leads to progressive accumulation of glycosphingolipids (GL) in different organs, mainly in the kidney, heart, and brain, causing severe multisystem failure. AFD is usually mild in heterozygous females, but severe cerebrovascular, renal, and cardiac manifestations have been rarely described. The aim of this study is to describe renal involvement of mild symptomatic female carriers by ultrastructural analysis focusing to microvascular lesions, considered to be one of the major causes of systemic disease in AFD. Resin-embedded renal biopsies from 2 sisters with isolated mild proteinuria and belonging to a family group with AFD were observed by light and electron microscopy. In spite of the mild clinical symptoms, diffuse GL storages were demonstrated in all types of glomerular cells and in interstitial endothelial cells. Moreover, platelets were frequently observed in glomerular vassels, a feature coherent with a possible role of prothrombotic state, and platelet activation, in early glomerular lesions.

Circulating ghrelin levels in patients with pancreatic and gastrointestinal neuroendocrine tumors: identification of one pancreatic ghrelinoma.

Ghrelin is a novel gastrointestinal hormone involved in several metabolic functions. Although the expression of ghrelin has been demonstrated in most gastrointestinal carcinoids and pancreatic tumors, the circulating levels of this peptide have been marginally assessed in patients with these disorders. We measured plasma ghrelin levels in 16 patients with gastrointestinal carcinoid (10 with midgut and 6 with gastric carcinoid), 24 patients with pancreatic tumor (8 with gastrinoma, 2 with insulinoma, 2 with vipoma, 1 with glucagonoma, and 11 with nonfunctioning tumor), and 35 healthy controls. Plasma ghrelin levels recorded in patients with gastroenteropancreatic tumors were similar to controls (mean +/- SE, 182.7 +/- 66.5 pM in patients vs. 329 +/- 32 pM in controls, P = not significant), and no significant difference between gastrointestinal and pancreatic, functioning and nonfunctioning, and metastatic and nonmetastatic tumors was observed. One patient with metastatic nonfunctioning pancreatic tumor had circulating ghrelin levels of 12,000 pM that were slightly reduced during chemotherapy and interferon therapy. Immunohistochemistry performed on peritoneal lesions showed an intense, focal cytoplasmic positivity for ghrelin. Despite the 50-fold increase in ghrelin concentrations, the patient had normal serum GH and IGF-I levels. In conclusion, the study showed that carcinoids and pancreatic tumors rarely cause ghrelin hypersecretion. However, in this series, 1 pancreatic ghrelinoma not associated with clinical features of acromegaly was identified.

Clinical aspects of the AIDS dementia complex in relation to histopathological and immunohistochemical variables.

To correlate cerebral histopathological and immunohistochemical changes in the neuroclinical features of the AIDS dementia complex (ADC), autopsy results of 28 ADC patients were related, in a retrospective analysis, to scores on a standardised neurological examination performed at neurologic onset. From a histopathological point of view, the cases were classified as follows: 9 cases of HIV leucoencephalopathy (HIVL; diffuse myelin damage and rare microglial nodules), 7 cases of HIV encephalitis (HIVE; several microglial nodules and no myelin damage) and 12 cases of mixed HIVL and HIVE (HIVL-E). The groups differed significantly with respect to symptoms and CD4 count at neurologic onset, survival and neurological impairment. Immunohistochemically, the interstitial component (p24-positive cells scattered singly within the white matter) was significantly more prevalent in HIVL, and the micronodular component (p24-positive cells confined within microglial nodules) in HIVE. Neurological damage was worse in cases with a high prevalence of interstitial component or a low prevalence of micronodular component. HIVE, HIVL and HIVL-E are distinct clinical forms of ADC. Neurological impairment is related to white matter damage.

Progressive multifocal leukoencephalopathy in a child with hyperimmunoglobulin E recurrent infection syndrome and review of the literature.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease due to infection with polyomavirus JC (JCV). PML occurs almost exclusively in immunocompromised patients, and although it has increased markedly in relation to AIDS, remains exceptional in children. We present the case of an immunocompromised child with hyperimmunoglobulin E recurrent infection syndrome (HIES) and pathologically-proven PML. HIES is a rare congenital immunodeficiency that to our knowledge has never before been reported in association with neurological complications. Following a recurrence of bronchopneumonia, the child's motor and cognitive functions deteriorated progressively in parallel with alterations on cerebral MRI. The neurological onset coincided with lymphocyte subset changes. PCR for JCV DNA did not detect the virus in CSF, and brain biopsy was required to secure the diagnosis. Antiviral treatment with cidofovir produced no benefit. Autopsy revealed the typical neuropathological findings of PML which were associated with inflammatory eosinophilic infiltrate (a marker of HIES). In accordance with the few pediatric PML cases reported and here reviewed, the child died five months after neurological onset.

Amplified in situ hybridization with peptide nucleic acid probes for differentiation of Mycobacterium tuberculosis complex and nontuberculous Mycobacterium species on formalin-fixed, paraffin-embedded archival biopsy and autopsy samples.

The aim of this study was to evaluate sensitivity and specificity of in situ hybridization (ISH) using peptide nucleic acid (PNA) probes and tyramide-based amplification for the differentiation between Mycobacterium tuberculosis (MTB) and mycobacteria other than tuberculosis (MOTT) on formalin-fixed, paraffin-embedded tissue samples. We performed ISH simultaneously with both probes on 86 specimens from different organs: 70 obtained at autopsy and 16 by biopsy, all with a histologic evidence of mycobacterial infection confirmed by Ziehl-Neelsen-positive staining. Taking culture as the "gold standard," the sensitivity and the specificity of the MTB probe were 100% (41/41) and 95% (38/40), respectively. In only 2 cases ISH failed to identify mycobacteria. Culture results were not available in 3 cases. We propose ISH as a relatively simple and rapid method to differentiate mycobacteria on formalin-fixed, paraffin-embedded specimens (it is more specific than usual histologic stains) and as an alternative to polymerase chain reaction, allowing the morphologic evaluation of positive bacilli.

Rantes distribution and cellular localization in the brain of HIV-infected patients.

OBJECTIVE: To study the immunochemical distribution ofRantes chemokine and its correlation with HIV-p24 expression, in brains with HIV-related lesions. MATERIAL AND METHODS: 17 HIV-positive cases of HIV-related brain lesions, 7 HIV-positive cases without cerebral HIV-related lesions (5 with opportunistic brain diseases), and 7 HIV-negative cases as controls (4 with brain lesion) were selected. RESULTS: High expression of Rantes was observed in the cases with inflammatory brain lesions (22/24 HIV-positive and 2/7 HIV-negative patients). Positivity was observed in the diffuse and nodular microglial cells and lymphocytes. In the patients with HIV-related lesions, the presence of Rantes-stained microglia did not correlate with that of HIV-p24-positive cells. Positive astrocytes were only found in the HIV-positive patients. Multinucleated giant cells were always Rantes-negative. CONCLUSIONS: Our results seem to demonstrate the role of Rantes chemokine in inducing inflammatory brain perivascular and microglial reactions both in HIV-positive and -negative patients.

[Lymphoepithelioma-like carcinoma of the esophagus: description of a case]. / Carcinoma simil linfoepitelioma (LELC) dell'esofago: descrizione di un caso.

The authors describe a case of lymphoepithelioma-like carcinoma (LELC) of the esophagus occurring in a 77-year-old man. To date, 13 cases have been described in the literature, all in natives of Japan. Histological features, immunohistochemical findings and differential diagnosis are discussed. In our case epithelial neoplastic cells were completely negative for the EBV genome.

Presence of gastric autoantibodies impairs gastric secretory function in patients with Helicobacter pylori-positive duodenal ulcer.

BACKGROUND: Although the association between Helicobacter pylori infection and gastric autoimmunity is now well established, to date little is known about the significance of anticanalicular autoantibodies in patients with duodenal ulcer (DU). We therefore investigated the prevalence of serum antiparietal cell autoreactivity in DU patients as well as the relationship between these autoantibodies, gastric histopathology and gastric secretory function in this setting. METHODS: Forty-one consecutive patients with H. pylori-positive DU were initially recruited. In all patients, basal (BAO) and pentagastrin stimulated acid output (PAO), fasting and meal-induced serum gastrin levels, as well as serum pepsinogen I concentrations, were measured. Antral and body gastritis was evaluated according to the Sydney system. Serum anticanalicular autoreactivity was determined by the indirect immunoperoxidase technique. RESULTS: Serum anticanalicular autoantibodies were found in 7 out of 34 patients (20%). The presence of these antibodies was associated with a significantly higher grade of body gastritis (activity: 1.9 versus 0.9) as well as with significantly higher fasting and meal stimulated gastrin levels (mean fasting gastrin, 76.4 (15.2) microg/ml versus 59.3 (20.5) microg/ml). In addition, PAO values were significantly lower in patients with gastric autoantibodies than in those without this autoreactivity (mean 0.35 (0.16) mmol kg(-1)h(-1) versus 0.49 (0.16)mmol kg(-1)h(-1)). In contrast, no significant differences were found between patients with and without anticanalicular autoantibodies as regards fasting serum pepsinogen I concentrations. CONCLUSIONS: Serum anticanalicular autoantibodies can be detected in 20% of patients with DU and are associated with a more severe pattern of body gastritis, higher gastrin levels and decreased peak acid secretion values. Their presence could account for the normal or reduced acid output which can be seen in a subset of DU patients.

Beta amyloid precursor protein and patterns of HIV p24 immunohistochemistry in different brain areas of AIDS patients.

OBJECTIVES: To evaluate the correlation between immunohistochemical positive patterns (globular and filamentous structures) of beta-amyloid precursor protein (beta-APP), used as a marker of axonal damage, and the different distribution of HIV p24 antigens, in three different brain areas of AIDS patients. METHODS: Eighteen AIDS patients with HIV-related brain lesions were included in the study. Forty-nine sections from basal ganglia, frontal cortex and hippocampus were selected. After microwave oven pre-treatment, the sections were incubated with anti-HIV p24 and anti-beta-APP monoclonal antibodies; the reactions were developed with peroxidase/3,3'diaminobenzidine. The positivity was graded by semi-quantitative scores. Double immunohistochemical staining was used to evaluate the co-localization of the antigens. RESULTS: HIV p24 immunohistochemistry was positive in 44 of 49 sections (89%), with a prevalence of interstitial positive cells and positive microglial nodules in 27 and 13 sections respectively. beta-APP-positive structures were demonstrated in 23 of 44 sections (52%) with HIV-related lesions, and were absent from the five sections without viral expression. Globular and filamentous lesions were observed in 21 of 23 sections and 10 of 23 lesions respectively. Moreover, a high grade of globular type lesion was related to an elevated presence of diffuse interstitial HIV p24-positive cells in basal ganglia; double immunohistochemical reactions demonstrated the co-localization of beta-APP globules and HIV p24 antigens. CONCLUSIONS: The data obtained confirm the coexpression of beta-APP and viral antigens in particular areas of the brain with HIV-related lesions; there is a strict correlation between beta-APP globules (indicating chronic cerebral damage) and the interstitial pattern of HIV p24 immunohistochemistry.