Lombardy diagnostic and therapeutic network of thrombotic microangiopathy.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA) requiring urgent treatment. Standardization of its diagnosis and optimal management is challenging. This study aimed to evaluate the role of centralized, rapid testing of ADAMTS13 in patients experiencing acute TMAs requiring plasma-exchange (PEX) and to estimate the incidence of TTP in a large Italian Region. METHODS: We perfomed a cohort study in the frame of the project "Set-up of a Lombardy network for the study and treatment of patients undergoing apheresis", including 11 transfusion centers in the Region. Consecutive patients referred from 2014 to 2016 with acute TMAs requiring PEX were enrolled. Centralized ADAMTS13 activity testing was performed at the Milan Hemophilia and Thrombosis Center within 24 h. RESULTS: Forty-three TMA patients (44 events) were enrolled, of whom 35 (81%) had severe ADAMTS13 deficiency. Patients with severe ADAMTS13 deficiency were younger, mainly women, with a higher prevalence of autoimmune disorders and a lower prevalence of cancer. Clinical and laboratory characteristics of patients with and without severe ADAMTS13 deficiency largely overlapped, with a lower platelet count being the only baseline marker that significantly differed between the two patient groups (ADAMTS13 activity < 10% vs ≥ 10%: median difference of -27 × 109/l, 95% CI - 37 to - 3). PEX treatment was initiated in all patients, but soon discontinued in cases without severe ADAMTS13 deficiency. In this group, the mortality rate was higher and no episode exacerbations or relapses within 6 months occured. The estimated average annual incidence of acute acquired TTP events was 1.17 [0.78-1.55] per million people. CONCLUSIONS: Severe ADAMTS13 deficiency distinguished two groups of patients with largely overlapping clinical features but different treatment and disease course. This study provides a feasible model implemented in a large Italian region for the practical clinical approach to TMAs and underlines the importance of urgent ADAMTS13 activity testing for an accurate differential diagnosis and therapeutic approach.

Depression and anxiety disorders in patients with multiple sclerosis: association with neurodegeneration and neurofilaments.

There is increasing evidence that neurofilament light chain (NF-L) can be considered as a biomarker for neuro-axonal damage. This polypeptide can be released into the cerebrospinal fluid (CSF) and the blood, where it can be quantified. The concentration of NF-L is elevated in patients with multiple sclerosis (MS) and psychiatric disorders. We aimed to investigate the NF-L levels in the CSF from treated MS patients and the relationship with depression or anxiety. The study involved three groups: control group (individuals without inflammation), the relapse-remitting multiple sclerosis (RRMS)-untreated group, and the RRMS-Fingo group (RRMS patients who were treated with fingolimod). MS disability was assessed by the Expanded Disability Status Scale, and depression and anxiety were evaluated by a neuropsychologist, using the Hospital Anxiety and Depression Scale, the Beck Depression Inventory-II, and the Beck Anxiety Inventory. Individual CSF samples were collected to measure NF-L levels. The results of the statistical analysis on levels of NF-L in the CSF of control subjects, RRMS-untreated patients, and RRMS-Fingo patients were significant. The relationship between depression and anxiety in RRMS-Fingo patients and NF-L levels was not statistically significant. In conclusion, MS events such as anxiety and depression appear to contribute to the onset of clinical relapses, subclinical cases, and neurodegeneration.

Depression and anxiety disorders in patients with multiple sclerosis: association with neurodegeneration and neurofilaments

There is increasing evidence that neurofilament light chain (NF-L) can be considered as a biomarker for neuro-axonal damage. This polypeptide can be released into the cerebrospinal fluid (CSF) and the blood, where it can be quantified. The concentration of NF-L is elevated in patients with multiple sclerosis (MS) and psychiatric disorders. We aimed to investigate the NF-L levels in the CSF from treated MS patients and the relationship with depression or anxiety. The study involved three groups: control group (individuals without inflammation), the relapse-remitting multiple sclerosis (RRMS)-untreated group, and the RRMS-Fingo group (RRMS patients who were treated with fingolimod). MS disability was assessed by the Expanded Disability Status Scale, and depression and anxiety were evaluated by a neuropsychologist, using the Hospital Anxiety and Depression Scale, the Beck Depression Inventory-II, and the Beck Anxiety Inventory. Individual CSF samples were collected to measure NF-L levels. The results of the statistical analysis on levels of NF-L in the CSF of control subjects, RRMS-untreated patients, and RRMS-Fingo patients were significant. The relationship between depression and anxiety in RRMS-Fingo patients and NF-L levels was not statistically significant. In conclusion, MS events such as anxiety and depression appear to contribute to the onset of clinical relapses, subclinical cases, and neurodegeneration.

The smart approach: feasibility of lentigo maligna superficial margin assessment with hand-held reflectance confocal microscopy technology.

BACKGROUND: Lentigo maligna may be challenging to clear surgically. OBJECTIVE: To evaluate feasibility of using superficial skin cuts as RCM imaging anchors for attaining negative surgical margins in lentigo maligna. METHODS: Included patients presented with lentigo maligna near cosmetically sensitive facial structures. We evaluated, with hand-held-RCM, microscopic clearance of melanoma beyond its dermoscopically detected edges. Evaluated margins were annotated using shallow skin cuts. If a margin was positive at 'first-step' RCM evaluation, we sequentially advanced the margin radially outward at that segment by 2-mm intervals until an RCM-negative margin was identified. Prior to final surgical excision, we placed sutures at the outmost skin cuts to allow comparison of RCM and histopathological margin assessments. Primary outcome measure was histopathological verification that RCM-negative margins were clear of melanoma. RESULTS: The study included 126 first-step margin evaluations in 23 patients, median age 70 years (range: 43-91). Seventeen patients (74%) had primary in-situ melanoma and six (26%) invasive melanoma, mean thickness 0.3 mm (range 0.2-0.4 mm). Six cases (26%) showed complete negative RCM margins on 'first-step', 11 (48%) were negative at 'second-step', and four (17%) at 'third-step'. In two additional cases (9%), margins clearance could not be determined via RCM due to widespread dendritic cells proliferation. The RCM-negative margins in all 21 cases proved clear of melanoma on histopathology. Of the 15 cases that returned at 1-year follow-up, none showed any residual melanoma on dermoscopic and RCM examinations. Interobserver reproducibility showed fair agreement between bedside RCM reader and blinded remote-site reader, with Spearman's rho of 0.48 and Cohen's kappa of 0.43; using bedside reader as reference, the remote reader's sensitivity was 92% and specificity 57% in positive margin detection. CONCLUSIONS: Margin mapping of lentigo maligna with hand-held-RCM, using superficial skin cuts, appears feasible. This approach needs validation by larger studies.

Dermoscopic and reflectance confocal microscopy features of cutaneous squamous cell carcinoma.

BACKGROUND: Squamous cell carcinoma (SCC) of the skin is a highly prevalent neoplasm. The management and the prognosis of this tumour are dependent on its invasiveness and its grade of differentiation. OBJECTIVES: To evaluate whether specific dermoscopic and reflectance confocal microscopy (RCM) criteria can predict the diagnosis of invasive SCC vs. in situ SCC and poorly differentiated compared with well- and moderately differentiated SCC. METHODS: Dermoscopic and RCM images of SCC were retrospectively evaluated for the presence of predefined criteria. RESULTS: Among 143 SCCs, 121 cases had a complete set of images and thus were included in the study set. The head and neck area was the most frequently involved body site (74/121; 61.1%) followed by extremities (36/121, 29.7%) and trunk (11/121, 9.1%). Seventy tumours were in situ (57.8%), while 51 were invasive (42.1%), of these 11 were poorly differentiated (21.5%), 16 were moderately differentiated (31.3%), and 24 were well differentiated (47.0%). Chi-squared analysis demonstrated that invasive SCCs were characterized by polymorphic vessels, erosion/ulceration, architectural disarrangement, speckled nucleated cells in the dermis, irregularly dilated vessels and absence of hyperkeratosis. Buttonhole vessels, white structureless areas and dotted or glomerular vessels were significantly associated with in situ lesions. Poorly differentiated SCCs were typified by red areas, erosion/ulceration and architectural disarrangement. Well- or moderately differentiated SCCs were associated with white areas and speckled nucleated cells in the epidermis. CONCLUSION: Clinical, dermoscopic and RCM images provide useful information that should be integrated in order to achieve the optimal therapeutic management for the patient.

Dermoscopic difficult lesions: an objective evaluation of reflectance confocal microscopy impact for accurate diagnosis.

BACKGROUND: Early detection of melanoma is the main objective to ensure a high survival rate. In some cases melanoma diagnosis still remain difficult and this leads to unnecessary excisions. OBJECTIVE: The aim of this study was to detect the most relevant Reflectance confocal microscopy (RCM) features for the detection of dermoscopic difficult melanomas. METHOD: A total of 322 lesions were selected from database and were evaluated on dermoscopy according to the 7-point checklist score, in blind from histological diagnosis. We classified the lesions into three categories: (i) 'featureless' lesions with score ranging between 0 and 2; (ii) 'positive-borderline' moles with score between 3 and 4 and (iii) 'positive-clear cut' lesions with score from 5 to 10. We evaluated confocal features of the 'featureless' lesions and of the 'positive-borderline' lesions. Evaluated confocal features were as follows: presence of pagetoid cells, cell shape (roundish or dendritic) and number (< 5 or >5 cells per mm(2) ), overall architecture (ringed, meshwork, clods and non-specific pattern); architectural disorder, presence of cytological atypia (>5 cells per mm(2) ) and cells arranged in nests. RESULTS: Among 322 lesions 70 were melanomas and 252 were nevi. According to the classification based on the 7-point checklist score, 130 'featureless lesions' (score 0-2) including six melanomas, and 102 'positive-borderline' moles (score 3-4) including 17 melanomas, were identified. Round pagetoid cells >5 cells per mm(2) and/or architectural disorder on RCM were found in all of six melanomas with featureless dermoscopy. Round pagetoid infiltration and five or more atypical cells at the DEJ were found in 16 positive 'borderline melanomas'. CONCLUSIONS: RCM represents a rapid non-invasive technique that can aid early diagnosis of dermoscopic difficult melanomas. Use of RCM on lesions with clinical and/or dermoscopic suspect of malignancy may reduce the number of unnecessary excision increasing the rate of accurate diagnoses.

Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Onygenales (Eurotiomycetes, Ascomycota) queratinofílicos en suelos de establecimientos educacionales urbanos y rurales de la V Región, Chile / Queratinophilic onygenales (Eurotiomycetes, Ascomycota) in urban and rural soils of eduacational center of V Region, Chile

Con la finalidad de estudiar la presencia de Onygenales queratinofílicas potencialmente patógenas para el hombre y los animales, se analizaron mediante la técnica del anzuelo queratínico (Marzo – Diciembre, 2006), suelos de establecimientos educacionales urbanos y rurales de la V Región, Chile. Se colectaron un total de 64 muestras, de las cuales, la mitad se obtuvieron en Valparaíso-Viña del Mar (urbano) y la otra en Olmué-Limache (en zonas rurales). En zona urbana se aislaron 112 cepas (7 géneros y 12 especies) y en la rural 147 (11 géneros y 18 especies). Los géneros de mayor prevalencia en la zona urbana y rural en orden decreciente fueron: Chrysosporium y su teleomorfo 32 por ciento versus 45 por ciento; Keratinomyces y su teleomorfo, 26 por ciento y 16 por ciento; Microsporum y su teleomorfo 23 por ciento y 16 por ciento y Myceliophthora y su teleomorfo con un 13 y 10 por ciento. Amauroascus mutatus, Auxarthron umbrinum, Gymnoascus reessii, Chrysosporium charmichaelii, Ch. merdarium, Ch. tropicum, Geomyces pannorum var. pannorum fueron detectados sólo en la zona rural; mientras que Malbranchea flava sólo en la zona urbana. Mediante la técnica de Takashio, se pudo identificar separadamente las especies del complex Microsporum gypseum, determinándose que M. gypseum (Arthrodema gypseum) y M.fulvum (A. fulvum) obtuvieron una frecuencia de aislamiento similar en ambas zonas, siendo el primero un oportunista potencialmente patógeno para el hombre y los animales.

With the purpose of studying the presence of keratinophilic Onygenales that are potentially pathogenous for man and animal, urban and rural soils from educational centers in the V Region were examined with the keratinic bait technique (march-december 2006). A total of 64 samples were collected, 32 of them being from Valparaíso-Viña del Mar (urban) while the rest in Olmué-Limache (in rural zones). One hundred and twelve strains (7 genera and 12 species) were collected in the urban zone and 147 strains in the rural zone (11genera and 18 species). Genera with the highest prevalence both in the urban and the rural zones were, in decreasing order: Chrysosporium and its teleomorph 32 per cent vs. 45 per cent; Keratinomyces and its teleomorph 26 per cent and 16 per cent; Microsporum and its teleomorph 23 per cent and 16 per cent and Myceliophthora and its. teleomorph with 13 per cent and 10 per cent. Amauroascus mutatus, Auxarthron umbrinum, Gymnoascus reessii, Chrysosporium charmichaelii, Ch. merdarium, Ch.tropicum, Geomyces pannorum var. pannorum were detected only in the rural zone, whereas Malbranchea flava only in the urban zone. Species of the complex Microsporum gypseum, could be identified separately by means of the Takashio technique, coming to the conclusion that M. gypseum (Arthrodema gypseum) and M.fulvum (A. fulvum) achieved a similar frequency of isolation in both zones and that the former is a potentially opportunistic pathogen for man and animal.

[Angiosarcoma of the nasal cavity. Report of a case and review of the literature]. / Angiosarcoma della cavità nasale. Descrizione di un caso e revisione della letteratura.

We report an unusual case of primary angiosarcoma of the nasal cavity (AS-nc). Clinical--monolateral epistaxis in a young person--, radiological--polypoid hemorrhagic tumor arising within the nasal cavity and expanding into paranasal sinuses--, pathological--a network of anastomosing channels and solid areas immunoreactive for CD31 and CD34--and prognostic features--patient alive and well 36 months after the original diagnosis--are superimposable to those of previously reported AS-nc, suggesting that this lesion should be considered as a peculiar variant of classical AS.

Closed-tube DNA extraction using a thermostable proteinase is highly sensitive, capable of single parasite detection.

Current DNA extraction methods for parasites are labour-intensive and usually involve several steps, increasing the potential for cross-contamination. We describe here a closed-tube DNA extraction procedure based upon the use of a thermostable proteinase that enabled sensitive amplification of target loci from parasites from diverse lineages including Apicomplexa, Sarcomastgophora and Nematoda. Moreover, this procedure is not subject to cross-contamination and is readily adaptable to automation.

Follicular fluid vascular endothelial growth factor concentrations are increased during GnRH antagonist/FSH ovarian stimulation cycles.

BACKGROUND: The aim of this study was to investigate the effect of GnRH antagonists (GnRH-ant) on follicular fluid vascular endothelial growth factor (FF VEGF). METHODS: Sixty women undergoing assisted reproduction were randomised (computer-generated randomisation list) and assigned to two different GnRH analogue regimens: GnRH agonist (GnRH-a) (Group A; n = 30) and GnRH-ant (Group B; n = 30). RESULTS: Mean (+/-S.D.) FF VEGF concentrations were 1598+/-612 pg/mL and 2906+/-1558 pg/mL for Groups A and B, respectively (p < 0.001). In the women treated with GnRH-ant, we found a statistically significant reduction in serum LH levels (1.72+/-0.74 IU/L in Group A versus 0.93+/-0.43 IU/L in Group B, p < 0.001), in serum oestradiol (E2) levels (1562.1+/-410.7 pg/mL in Group A versus 1214.67+/-779.9 pg/mL in Group B, p < 0.05), in FF E2 levels (1146+/-593 ng/mL in Group A versus 621+/-435 ng/mL in Group B, p < 0.05), and in FF androstenedione levels (136+/-55 ng/mL in Group A versus 78+/-31 ng/mL in Group B, p < 0.001), as well as a reduction in the number of pregnancies, though not statistically significant (23.3% in Group A versus 16.6% in Group B). CONCLUSION: The increase in FF VEGF levels in women treated with GnRH-ant might be explained by a suppression of LH and E2 levels.

Neonatal porencephaly in very low birth weight infants: ultrasound timing of asphyxial injury and neurodevelopmental outcome at two years of age.

OBJECTIVE: To investigate and diagnose the timing of asphyxial injury leading to cerebral cavitation with subsequent developing of neonatal porencephaly in the preterm VLBW infant. All newborns underwent careful neurodevelopmental outcome at 2 years of corrected age. METHODS: 250 consecutive VLBW infants (mean gestational age of 28 weeks and mean birthweight of 1150 g) have been study by means of weekly neonatal transfontanellae ultrasonography. Periventricular white matter necrosis was diagnosed when echolucencies were visible after day 3 from birth. RESULTS: Twelve cases of neonatal porencephaly were diagnosed by ultrasound. The timing of asphyxial insult leading to cerebral cavitation seems to have occurred in 33% of neonates during the antepartum period, in 42% during the peripartum period (antepartum + neonatal period) and 25% in the remaining neonatal period. Periventricular-intraventricular hemorrhage (PVH-IVH) was found in all cases and in 50% a severe IVH (grade III-IV) was diagnosed within 7 days neonatal period. Nine infants had evidence of cerebral palsy at 2 years neurological outcome. CONCLUSIONS: The ultrasound criteria of cerebral cavitation have been priorly selected in order to assure that the damage may have occurred before delivery. A comprehensive prenatal study of fetal brain, integrating ultrasound with high-velocity MRI, is also advocate. This will lead to a more detailed understanding of the underlying cerebral condition that is of critical importance for the clinician in planning the time and mode of delivery and have great deal with further medico-legal consideration.

Activity of a specific inhibitor, gefitinib (Iressa, ZD1839), of epidermal growth factor receptor in refractory non-small-cell lung cancer.

BACKGROUND: Gefitinib (Iressa(TM), ZD1839) is an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. Phase I studies showed that it is well tolerated, with evidence of tumor regression in patients with advanced non-small-cell lung cancer (NSCLC). Therefore, we aimed to assess the antitumor activity and tolerability of gefitinib in a series of patients with previously treated, advanced NSCLC, as a part of a compassionate use program. PATIENTS AND METHODS: To be eligible, all patients were required to have histologically or cytologically proven advanced or metastatic NSCLC, prior chemotherapy with at least one cisplatin-containing chemotherapy regimen or contraindication to cytotoxic drugs, Eastern Cooperative Oncology Group performance status < or =2, and adequate hematological, renal and hepatic parameters. All patients provided signed informed consent. Patient re-evaluation was performed every 4-6 weeks. RESULTS: Seventy-three consecutive patients were enrolled. Response rate, including complete and partial response, was 9.6%; an additional 43.8% of patients achieved stable disease, for an overall disease control of 53.4%. EGFR1 status was evaluated by immunocytochemistry in 25 patients. According to EGFR1 immunoreactivity all responses were observed with medium/strong imunoreactivity while three out of four responses were observed in high expressive patients. Median survival for all patients was 4 months while it reached 6 months for patients with disease control. The 1-year survival rate was 13.1% for the entire series and 23.2% for patients with disease control. Non-hematological toxicity was generally mild. CONCLUSION: Gefitinib has promising activity with a good toxicity profile in patients with progressive NSCLC who have received one or two prior chemotherapy regimens. A possible relationship within response and EGFR1 expression is suggested.

Application of the novel fluorescent dye Beljian red to the differentiation of Giardia cysts.

Beljian red (BR) is a novel long Stokes shift fluorescent dye that fluoresces orange when illuminated with UV or blue light. Due to its long Stokes shift, and the fact that it is excitable at 488 nm, BR has particular utility in multi-colour applications with short Stokes shift fluorophores such as fluorescein. Here we have demonstrated that BR can be used to discriminate Giardia cysts seeded into water samples from those naturally present in the sample. We show that the dye does not interfere with other staining methods such as DAPI, and is compatible with mAb-FITC staining in a multi-colour fluorescence technique. This should be useful in determining the specific recovery of protozoan parasites from environmental samples.

Leptin inhibits steroid biosynthesis by human granulosa-lutein cells.

Absence of leptin secretion compromises reproductive function and fertility in the ob/ob mouse which, when given leptin, shows a rise in serum LH levels and becomes fertile. Recently, the long and active isoform of the leptin receptor was detected in the ovary, indicating that leptin may also show direct gonad-related activity. To examine this, we studied the effect of graded doses of human leptin on estradiol (E2) and progesterone (P4) concentrations in the culture media of human granulosa-lutein cells obtained from follicular fluid of women undergoing in vitro fertilization. We also evaluated the mRNA expression of steroidogenic acute regulatory protein (StAR), aromatase, and cytochrome P450 17alpha (CYP17) in these cells at baseline and after exposure to leptin. Estradiol levels were significantly decreased in the media 24 hours after incubation of the cells with increasing hLeptin concentrations (10(-11) - 10(-7) mol/l). The maximal 30% decrease in E2 production was caused by the 10(-9) mol/l hLeptin concentration; however, P4 levels in the media were not influenced by leptin. Exposure of granulosa-lutein cells to 10(-9) mol/l hLeptin did not produce any measurable changes on StAR, aromatase, or CYP17 mRNA expression. When hLeptin (10(-9) mol/l) was co-incubated with increasing concentrations of hCG (1.25 - 10 mlU/ml), IGF-II (15-60 ng/ml) or 1-6 desaminated IGF-II (deslGF-II; 15-60 ng/ml), it did not modify the elevation of E2 concentrations caused by each of the different stimuli. We conclude that leptin suppresses E2 secretion by human granulosa-lutein cells but does not impair the stimulatory effects of hCG and IGFs on these cells. Leptin may play a minor, but direct regulatory role on unstimulated human ovarian steroidogenesis by interfering with either the translational or post-translational steps of the baseline CYP17 and/or aromatase synthesis and/or the activation of the enzymes.

Sweet's syndrome associated with monosomy 7 myelodysplastic syndrome.

Acute febrile neutrophilic dermatosis (Sweet's syndrome) is a reactive skin process frequently associated with inflammatory and neoplastic diseases, but particularly with hematologic malignancies. It usually precedes the underlying disorders for months or even years. Much of the evidence for this is based on a small series of case reports and reviews of the literature. Recently, immunological theories have suggested that helper T cell type 1 is involved in the pathogenesis of Sweet's syndrome. This process causes stimulation of the cytokine cascade, which may be responsible for the local and systemic activation of neutrophils and histiocytes. Clinically, Sweet's syndrome is characterized by an acute eruption of painful erythematous or violaceous plaques or nodules with fever, malaise, neutrophilic leukocytosis, and an elevated erythrocyte sedimentation rate. Peripheral blood neutrophilia is frequent and is one of the diagnostic criteria. However, 53% of patients with Sweet's syndrome linked to hematologic malignancies do not present any neutrophilia but rather granulocytopenia. Abnormal functioning of neutrophils is possible in many diseases. We report a case of a middle-aged male patient presenting Sweet's syndrome and granulocytopenia due to myelodysplasia and an anomalous chromosome seven (7-) with poor prognosis.