Grazia Bossi's research while affiliated with Policlinico San Matteo Pavia Fondazione IRCCS and other places

Background Rare cases of primary thrombocytosis are related to inherited genetic abnormalities and are called hereditary thrombocytosis (HT). HT has clinical features resembling sporadic essential thrombocythemia (ET) but has usually an autosomal dominant (AD) inheritance with variable penetrance. To date, germline mutation involving the MPL, THPO, JAK2 genes have been associated with HT. Methods The study included 933 patients evaluated at our Division because of persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Patients were screened for the three canonical somatic driver mutations associated with myeloproliferative neoplasms: JAK2 V617F mutation, CALR exon 9 mutations, MPL exon 10 mutations. Triple-negative cases with unexplained isolated thrombocytosis which also resulted negative for non-canonical exon 10 MPL mutations were analyzed using next generation sequencing (NGS) to test a panel of 80 myeloid genes. Results Of 933 patients with isolated thrombocytosis screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated and 68 triple-negative. Two MPL-mutated patients carried a non-canonical mutation located in exon 10: MPL W515* and MPL V501A. Of the 68 triple-negative patients, 32 were evaluated by NGS and one of them showed another non-canonical mutation of MPL located outside exon 10: MPL R102P. These 3 non-canonical MPL mutations resulted to be germline as they were detected also in DNA extracted from T lymphocytes and from hair roots. Due to the germline nature of the mutations, a study of the family was carried out. In 2 out of 3 pedigrees at least a second relative carried the same germline mutation discovered in the proband; these 2 pedigrees are shown in the figure. Patient MPC17_15 was an 80 years-old patient with a history of thrombocytosis. She resulted JAK2- and CALR- wild-type while HRM screening was positive for exon 10 MPL mutations; Sanger sequencing identified the MPL W515* mutation in DNA from granulocytes, T-lymphocytes and hair roots, confirming its germline nature. Her children showed a normal complete blood count (CBC) and were MPL wild-type, thus confirming a segregation between mutation and clinical phenotype. Patient MPC14_674 (panel A) was a 36 years-old women with a history of mild thrombocytosis never investigated before. She was referred to our clinic by Pediatricians after the delivery because her baby (MPC17_167) showed persistent isolated thrombocytosis (826 x10 9/L) during the first months of life despite the exclusion of the most common reactive causes of thrombocytosis. Sanger sequencing identified the MPL V501A mutation in granulocyte and in T-lymphocyte in both cases. To evaluate the segregation of MPL V501A with the disease phenotype, we evaluated the CBC and MPL mutation in the other relatives. Both proband's father (MPC23_597) and sister (MPC23_602) carried germline heterozygous MPL V501A mutation associated with a PLT count above or at the upper limit of normal range (570 x10 9/L and 355 x10 9/L, respectively). Conversely, the husband (MPC17_495), the second son (MPC18_43) and the mother (MPC23_603) of the proband showed a normal CBC and were MPL wild-type, confirming an AD inheritance. Patient MPC07_130 (panel B) was a 44 years-old women with triple-negative isolated thrombocytosis lasting since 2007. NGS analysis showed the presence of a heterozygous germline MPL R102P mutation. As this mutation is located in exon 3, it was not detected through HRM and Sanger sequencing which target only exon 10. Her son (MPC13_672), 18 years-old, was completely asymptomatic but the CBC showed mild thrombocytosis (472 x 10 9/L). He tested negative for the 3 driver mutations but Sanger sequencing of MPL exon 3 identified the heterozygous germline MPL R102P mutation. Conclusions We identified 3 non-canonical germline MPL mutations in 3 different unrelated subjects with isolated thrombocytosis: MPL W515*, MPL R102P, and MPL V501A. Germline MPL mutations may underly HT and should be evaluated by sequencing of the whole exon 10 of MPL gene or by NGS in cases with isolated thrombocytosis negative for the three canonical somatic driver mutations. Namely, patients with HT might present as sporadic cases, and in the initial workup they might be inappropriately diagnosed with triple-negative ET. A correct diagnosis is pivotal to avoid unnecessary treatments.

Background Pediatric Mastocytosis is a rare and heterogeneous disease, characterized by accumulation of mast cells in the skin (Cutaneous Mastocytosis) and/or, less frequently, in other organs, mainly liver, spleen, bone marrow, lymph nodes and gastrointestinal tract (Systemic Mastocytosis). Patients affected by Systemic Mastocytosis show symptoms caused by a massive release of mast cell mediators: itching, flushing, abdominal pain, generalized weakness, fatigue and neuropsychiatric disorders. Moreover, children with Systemic Mastocytosis are at greater risk of anaphylactic/anaphylactoid reactions, often poorly controlled by the conventional therapy with antihistamines, mast cells stabilizers and steroids. As a result, children affected by Systemic Mastocytosis have a poor quality of life and suffer the consequence of prolonged steroidal treatment. Case presentation A child with Systemic Mastocytosis and severe symptoms, refractory to symptomatic and steroidal therapy, has been successfully treated with Omalizumab, an anti-IgE monoclonal antibody usually employed in allergic patients with severe asthma and orticaria. The onset of clinical benefit of Omalizumab therapy was extraordinarily rapid, but proved to be strictly dependent on drug administration. The child has become completely and steadily asymptomatic. No other anaphylactic episodes have been reported. Steroid treatment could be definitively withdrawn after the second dose of Omalizumab, and all the other medications were later reduced. Twenty months after beginning, Omalizumab therapy is still ongoing with good symptomatology control; no side effects have been observed so far. Conclusions In our experience, Omalizumab is an effective treatment for children affected by Systemic Mastocytosis not responding to conventional medical treatments. The main strengths of this therapy are its rapid and extraordinary efficacy to control the severe mast cells mediator-related symptoms, the lack of side effects and its steroid-sparing effect. However, more extensive and controlled studies in pediatric patients affected by Systemic Mastocytosis are needed to substantiate these promising findings.

Background. Pediatric Mastocytosis is a rare and heterogeneous disease, characterized by accumulation of mast cells in the skin (Cutaneous Mastocytosis) and/or, less frequently, in other organs, mainly liver, spleen, bone marrow, lymph nodes and gastrointestinal tract (Systemic Mastocytosis). Patients affected by Systemic Mastocytosis complaint symptoms caused by the massive release of mast cell mediators: itching, flushing, abdominal pain, generalized weakness, fatigue and neuropsychiatric disorders. Moreover, children with Systemic Mastocytosis are at greater risk of anaphylactic/anaphylactoid reactions, often poorly controlled by the conventional therapy with antihistamines, mast cells stabilizers and steroids. As a result, children affected by Systemic Mastocytosis have a poor quality of life and suffer the consequence of prolonged steroidal treatment. Case presentation. A child with Systemic Mastocytosis and severe symptoms, refractory to symptomatic and steroidal therapy, has been successfully treated with Omalizumab, an anti-IgE monoclonal antibody usually employed in allergic patients with severe asthma and orticaria. The onset of clinical benefit of omalizumab therapy was extraordinarily rapid but proved to be strictly dependent on drug administration. The child has become completely and steadily asymptomatic. No other anaphylactic episodes have been reported. Steroid treatment could be definitively withdrawn and all the other medications were reduced. Omalizumab was well tolerated and no side effects have been observed. Conclusions. In our experience Omalizumab is an effective treatment for children affected by Systemic Mastocytosis not responding to conventional medical treatments. The main strengths of this therapy are its rapid and extraordinary efficacy to control the severe mast cells mediator-related symptoms, the good safety profile and its steroid-sparing effect. However, more extensive and controlled studies in pediatric patients affected by Systemic Mastocytosis are needed to substantiate these promising findings.

Background : Intravenous administration of zidovudine (ZDV) during labour is a key step for vertical HIV transmission (VT) prevention, but there is no evidence of benefit when maternal HIV-RNA at delivery is <50 copies/mL. The aim of this study is evaluating the appropriateness of intrapartum ZDV use in Italy. Methods : Observational study including mother-infant pairs with perinatal HIV exposure during 2002-2019, enrolled in the Italian Register for HIV Infection in Children. Univariable and multivariable logistic regression were used to evaluate factors associated with VT. Results : A total of 3,861 infants, born from 3,791 pregnancies were included. The frequency of ZDV use was 79.9%, 92.1%, 93.7% and 92.8% when HIV-RNA was not available, ≥400 copies, between 50 and 399 copies, and <50 copies/mL. Thirty-three out of 3861 (0.85%) infants were subsequently diagnosed with HIV, 25/3861 (0.6%) of them born to mothers receiving intrapartum ZDV, and 31 (93.9%) to mothers with HIV-RNA ≥50 copies/mL or not available. In women with HIV-RNA < 50 copies/mL, ART discontinuation during pregnancy was the strongest risk factor for VT (odds ratio, OR, 23.1, 95%CI 2.4-219.3), while a higher gestational age (OR 0.6, 95%CI 0.4-0.8) and PEP administration to the newborn (aOR 0.004, 95%CI <0.0001-0.4) were protective factors. Intrapartum ZDV administration did not influence the final outcome in this group. Conclusions : In ART era, more transmission events may occur in utero, limiting value of intrapartum ZDV, particularly for women with suppressed HIV-RNA load. More attention to the HIV-RNA testing of mothers before delivery may avoid unnecessary ZDV use.

Background: Coronavirus disease 2019 (COVID-19) affects people of any age with high mortality and morbidity in adults older than 65 years. Reports on pediatric cases highlighted those children generally develop milder symptoms than adults or are asymptomatic. We aimed to assess the epidemiological and clinical data of children and adolescents with SARS-CoV-2 infection to improve pediatric COVID-19. Methods: We retrospectively analyzed clinical and epidemiological features of patients with SARS-CoV-2 infection hospitalized at the Pediatric Hospital of Pavia, Italy, between February 1, 2020, to April 30, 2021. Results: 71 patients aged 0-16 years were included; 33 (46%) females and 38 (54 %) males. Thirty-three (46%) patients had comorbidities, such as obesity and hematological diseases. Thirty-one children (44%) were exposed to COVID-19-positive household members. Nine (12.7 %) patients were asymptomatic, whereas 57 (80.3%) had a mild-moderate disease. Only five (7%) showed a severe or critical disease, and two patients required ICU admission. The most frequent symptoms were fever (76%), loss of appetite (26%), gastrointestinal symptoms (19%), and cough (19%). Chest X-ray was performed in 42 patients showing lung abnormalities in more than half of symptomatic patients. The most common laboratory features were lymphopenia and eosinopenia associated with high levels of inflammation markers. Conclusions: This study confirmed that COVID-19 has a mild course in children compared to adults. Most of the enrolled children were asymptomatic or had a mild-moderate disease. Patients with comorbidities were more prone to develop clinical complications.

Kawasaki disease (KD) is rare in infants less than 3 months of age, and its recurrence is exceptional. Infants with KD are at higher risk of severe clinical presentation, therapy failure, complications and coronary aneurysms (CAAs), and this is the reason they deserve more aggressive therapy and a strict clinical follow-up. We report a 2-month-old male with KD, complicated by Macrophage Activation Syndrome (MAS). Despite timely and aggressive therapy with immunoglobulins, steroids and aspirin, multiple CAAs developed. Two-month therapy with anakinra completely reverted all the aneurysms. After six months, the infant experienced KD relapse and was successfully re-treated with immunoglobulins, steroids and aspirin. A strict echocardiographic follow-up did not show recurrence of aneurysms. Two years later, the child is healthy, without cardiac sequelae. In our experience, anakinra was effective in reverting multiple aneurysms and its effect proved to be long-lasting, even in front of KD recurrence. Based on this evidence, it seems reasonable to hypothesize not to limit the use of anakinra as rescue therapy for complicated or refractory KD, but to consider the possibility of adding it to first-line therapies for some subgroups of very-high-risk patients, in order to strengthen the prevention of CAAs.

Acute hematogenous osteomyelitis (AHOM) is a rare pathology in pediatric population. The aim of this study is to analyse the epidemiological data and the management, compared to European Society for Paediatric Infectious Disease (ESPID, European Society for Pediatric Infectious Diseases) guidelines 2017 of 216 children with AHOM, divided in three cohorts (neonatal-onset osteomyelitis, those with vertebral involvement and other types of osteomyelitis). We conducted a retrospective single center study, evaluating data from all the children (aged 0–18 years) consecutively admitted to the Meyer Children’s Hospital, during a period of ten years (1 January 2010–31 December 2019). Isolation of pathogen was possible in 65 patients and S. aureus was the most frequently involved (43/65 children). Magnetic Resonance Imaging (MRI, magnetic resonance imaging) was performed in 201/216 cases and was compatible with osteomyelitis in 185/201 of these children (92.03%). In the neonatal-onset osteomyelitis group the percentage of diagnostic ultrasound for osteomyelitis was 36.36% significantly higher than the other groups. The median duration of total antibiotic therapy was 37.5 days. In total, 186/199 children recovered without complications. The present study delineates three heterogeneous cohorts of patients. S. aureus is confirmed as the first pathogen for isolation in all three groups analysed. MRI represent a gold standard for diagnosis. Longer duration of antibiotics treatment was performed in neonatal and spondylodiscitis group, compared to the other types of osteomyelitis.

Toxocarosis is the consequence of human infection by Toxocara spp. larvae and is one of the most common ascarioses, not only in developing countries, but also in the European region, where its prevalence reaches 14%. Due to their particular behavior, children are at higher risk of this parasitic infection, whose clinical features depend on the localization of the Toxocara larvae. Neurotoxocariasis is very uncommon in children and may take different forms depending on the underlying physiopathologic process: immune reaction against the parasite antigens, vasculitis, treatment complications, or, very rarely, brain localization of Toxocara spp. larvae. The association between neurotoxocariasis and the onset of childhood epilepsy has been postulated but is still debated. Moreover, a Toxocara spp. abscess causing epileptic seizures in children has been rarely described, especially in western countries. Hereby we present a 9-year-old patient with a new diagnosis of epilepsy definitely secondary to brain abscess due to the localization of Toxocara canis larvae. Diagnosis was confirmed by neuroimaging and serological test. The successful treatment with albendazole and steroids was documented with a close and long-term clinical and neuroradiological follow-up. Our experience confirms that every case of cryptogenetic epilepsy in children deserves a neuroimaging study and, in case of cystic images, Toxocara serology is mandatory to avoid further unnecessary invasive diagnostic investigations and to set the specific drug therapy.

Italy is one of the most exposed countries worldwide to COVID-19, and Lombardy is the most affected region in Italy. In this context, Fondazione IRCCS Policlinico San Matteo in Pavia, one of the largest University hospitals in the region, has been involved in the management of the outbreak since its inception. Immediately after the communication of the first Italian COVID-19+ patient, the Pediatric Unit has been completely reorganized to face the approaching outbreak. The optimization of the Pediatric Unit resources for COVID-19 emergency is reported as an example to safely preserve health activity during the pandemic.