Boosting cytotoxicity of adoptive allogeneic NK cell therapy with an oncolytic adenovirus encoding a human vIL-2 cytokine for the treatment of human ovarian cancer.

Despite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.

Improving the cytotoxic response of tumor-infiltrating lymphocytes towards advanced stage ovarian cancer with an oncolytic adenovirus expressing a human vIL-2 cytokine.

While the presence of tumor-infiltrating lymphocytes (TILs) associates with improved survival prognosis in ovarian cancer (OvCa) patients, TIL therapy benefit is limited. Here, we evaluated an oncolytic adenovirus coding for a human variant IL-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, as an immunotherapeutic strategy to enhance TIL responsiveness towards advanced stage OvCa tumors. Fragments of resected human OvCa tumors were processed into single-cell suspensions, and autologous TILs were expanded from said samples. OvCa tumor specimens were co-cultured with TILs plus vIL-2 virus, and cell killing was assessed in real time through cell impedance measurement. Combination therapy was further evaluated in vivo through a patient-derived xenograft (PDX) ovarian cancer murine model. The combination of vIL-2 virus plus TILs had best cancer cell killing ex vivo compared to TILs monotherapy. These results were supported by an in vivo experiment, where the best OvCa tumor control was obtained when vIL-2 virus was added to TIL therapy. Furthermore, the proposed therapy induced a highly cytotoxic phenotype demonstrated by increased granzyme B intensity in NK cells, CD4+ T, and CD8+ T cells in treated tumors. Our results demonstrate that Ad5/3-E2F-d24-vIL2 therapy consistently improved TILs therapy cytotoxicity in treated human OvCa tumors.

Genomic prediction of relapse in recipients of allogeneic haematopoietic stem cell transplantation.

Allogeneic haematopoietic stem cell transplantation currently represents the primary potentially curative treatment for cancers of the blood and bone marrow. While relapse occurs in approximately 30% of patients, few risk-modifying genetic variants have been identified. The present study evaluates the predictive potential of patient genetics on relapse risk in a genome-wide manner. We studied 151 graft recipients with HLA-matched sibling donors by sequencing the whole-exome, active immunoregulatory regions, and the full MHC region. To assess the predictive capability and contributions of SNPs and INDELs, we employed machine learning and a feature selection approach in a cross-validation framework to discover the most informative variants while controlling against overfitting. Our results show that germline genetic polymorphisms in patients entail a significant contribution to relapse risk, as judged by the predictive performance of the model (AUC = 0.72 [95% CI: 0.63-0.81]). Furthermore, the top contributing variants were predictive in two independent replication cohorts (n = 258 and n = 125) from the same population. The results can help elucidate relapse mechanisms and suggest novel therapeutic targets. A computational genomic model could provide a step toward individualized prognostic risk assessment, particularly when accompanied by other data modalities.

Description of four new HLA alleles in the Finnish population: A*03:283N, A*68:167, C*03:327, C*03:361.

New HLA alleles found in the Finnish population: A*03:283N, A*68:167, C*03:327 and C*03:361.

Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia.

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naïve CD56bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.

Conflicting HLA assignment by three different typing methods due to the apparent loss of heterozygosity in the MHC region.

Loss of heterozygosity (LOH) has been reported to cause false human leukocyte antigen (HLA) homozygous typing results in pre-transplant patients suffering from haematological malignancies, who in fact are HLA heterozygous. This poses a challenge for histocompatibility testing, as a stem cell graft from a genuinely HLA homozygous donor to a mistyped patient may lead to acute life-threatening graft-vs-host disease. LOH in the HLA region on chromosome 6 is known to be quite common in solid tumours, helping malignant cells to escape T-cell surveillance, but the incidence in haematological malignancies is less well known and the estimates vary. Here we report LOH in the HLA region of five patients with haematological malignancy. We found considerable differences in sensitivity between the three different HLA typing methods used in our laboratory: SSP was clearly the most sensitive method for detecting the lost haplotype, followed by rSSO, while SBT was the least sensitive technique. A subsequent, retrospective genotyping of 65 HLA homozygous haematological patients by SSP method showed no mistyped LOH cases in our laboratory in the past 10 years. The frequency of HLA homozygosity was found to be similar between haematological patients and control groups. It is important for an HLA laboratory to be aware of the differences in various HLA typing techniques' sensitivity for detecting an under-represented haplotype between HLA typing techniques when genotyping patients with haematological diseases. It is advisable for HLA laboratories to have at least two different methods with different sensitivities in their repertoire to be able to retype samples when a false homozygous result is suspected.

Difficulty in making contact with others and social withdrawal as early signs of psychosis in adolescents--the Northern Finland Birth Cohort 1986.

AIM: Social withdrawal is among the first signs of the prodromal state of psychosis seen in clinical samples. The aim of this prospective study was to find out whether difficulty in making contact with others and social withdrawal precede first episode psychosis in the young general population. METHODS: The members of the Northern Finland Birth Cohort 1986 (n=6274) completed the PROD-screen questionnaire in 2001-2002. The Finnish Hospital Discharge Register was used to detect both new psychotic and non-psychotic disorders requiring hospitalisation during 2003-2008. RESULTS: Twenty-three subjects developed psychosis and 89 developed a non-psychotic mental disorder requiring hospitalisation during the follow-up. Of those who developed psychosis, 35% had reported difficulty or uncertainty in making contact with others and 30% social withdrawal in adolescence. In hospitalised non-psychotic disorder, the corresponding precentages were 10 and 13% and in the control group without hospital-treated mental disorder 9 and 11%. The differences between psychotic and non-psychotic hospitalised subjects (P<0.01) as well as controls (P<0.001) were statistically significant regarding difficulty or uncertainty in making contact with others. CONCLUSIONS: In this general population-based sample self-reported difficulty or uncertainty in making contact with others in adolescence preceded psychosis specifically compared to hospitalised non-psychotic mental disorders and controls.

HLA antigen, allele and haplotype frequencies and their use in virtual panel reactive antigen calculations in the Finnish population.

The human leukocyte antigen (HLA) antigen, allele and haplotype frequencies of the Finnish population are quite unique because of a rather restricted and homogeneous gene pool. This has a strong influence on finding suitable donors for transplant patients; hence knowledge about the HLA frequencies of the patient population is essential. Here we report the HLA antigen frequencies for a large population sample and show high resolution HLA allele frequencies for 11 loci, including the rarely typed DPA1 and DQA1 loci. Furthermore, the most common Finnish high resolution haplotypes are presented for five HLA loci. The study shows that there are fewer HLA haplotypes in the Finnish population compared with mixed populations, and the common Finnish HLA haplotypes are more frequent. Using HLA antibody identification and panel reactive antibody calculations we show that a virtual population-specific panel, combined with single antigen testing, gives a more accurate and reliable estimate of the reactivity of the recipient serum against potential solid organ donors within the Finnish population. The results can be directly used to improve donor search for patients waiting for stem cell transplantation and to allocate highly immunised patients accurately to acceptable mismatch programs.

Adapting spa typing for national laboratory-based surveillance of methicillin-resistant Staphylococcus aureus.

Laboratory-based surveillance of methicillin-resistant Staphylococcus aureus (MRSA) monitors the baseline occurrence of different genotypes and identifies strains and transmission chains responsible for outbreaks. The consequences of substituting pulsed-field gel electrophoresis (PFGE) with spa typing as a first-line typing method were analyzed by typing 589 strains isolated between 1997 and 2006, with a focus on both short- and long-term correspondence between the PFGE and spa typing results. The study, covering these ten years, included all Finnish MRSA blood isolates and representatives of the two most prevalent MRSA strains (PFGE types FIN-4 and FIN-16) in Finland. In addition, all sporadic isolates from 2006 were included. spa typing was more expensive but approximately four times faster to perform than PFGE. Nearly 90% of FIN-4 and FIN-16 isolates showed consistent spa types, t172 and t067, respectively. spa typing predicted the PFGE result of the blood isolates by a Wallace coefficient of 0.9009, recognized internationally successful strains (t041, t067) to be common also in Finland, and identified a separate cluster of isolates, also related in time and place among the FIN-4 strains. Additional typing by another method was needed to provide adequate discrimination or to characterize isolates with a newly recognized spa type in Finland.

First isolations of KPC-2-carrying ST258 Klebsiella pneumoniae strains in Finland, June and August 2009.

The first two Klebsiella pneumoniae carbapenemase-producing (KPC) type 2 strains carrying ST258 were detected in Finland in June and early August 2009. They were found colonising two patients transferred from the Mediterranean; one patient referred from a hospital in Greece where isolates were first found in 2007 and another from Italy where the first isolates have been described only very recently.

Characterization a novel HLA-B40 allele with serological Bw4 motif, HLA-B*4047, in the Finnish population and confirmation of B*270503 allele.

We describe a novel HLA-B*40 allele assigned as B*4047*. The B*4047 allele was detected in a Finnish patient awaiting kidney transplantation. The patient had a "short" B60-like serological specificity with Bw4 association. After sequencing, the B*4047 allele was found to be identical to B*4001, except having five amino acid changes in exon 2, including the entire motif corresponding to Bw4 and w6 specificity. As a result of recombination or gene conversion, B*4047 has the Bw4 motif instead of expected Bw6. Screening of B40 alleles in the Finnish population revealed no other cases with this pattern, suggesting that this allele is rare. The sequence of B*270503 presented here provides the complete sequence for exons 2 and 3 for this allele. B*270503 allele differs from B*270502 by a single synonymous nucleotide substitution at non-variable position 489 in exon 3.

Neonatal thrombocytopenia in two of six human platelet alloantigen (HPA) 5a-positive children of an HPA-5a-immunized mother.

We describe a human platelet alloantigen (HPA) 5a-alloimmunized HPA-5b5b mother. The children were obligatory heterozygotes for HPA-5a but despite IgG class maternal anti-HPA-5a antibodies only two (second and fifth) of the six children developed neonatal thrombocytopenia. Throughout the 4-year follow-up the mother had anti-HPA-5a antibodies (confirmed in the 8th Platelet serology workshop of International Society of Blood Transfusion in 1996). Antibodies against glycoproteins (GP) IIbIIIa or IbIX were not detected. Differences in the children's HPA type (HPA-1, -2, -3, -5) did not correlate with thrombocytopenia. We hypothesized that different expression of GPIaIIa recently associated with two silent polymorphisms (C807T and G873A) of GPIa could explain the unpredictable recurrence pattern of neonatal alloimmune thrombocytopenia (NAIT). Both parents were homozygous for the silent polymorphisms (C807 and G873) associated with the low expression of GP Ia. Thus, the inheritance pattern of the silent polymorphisms (C807T and G873A) did not help in predicting the recurrence risk of thrombocytopenia in the offspring. More detailed comprehension of the natural history of NAIT would be necessary to enable directing fetal blood sampling to the cases at the highest risk of thrombocytopenia.

Expression of CYP2A genes in human liver and extrahepatic tissues.

Members of the human cytochrome P450 2A (CYP2A) subfamily are known to metabolize several promutagens, procarcinogens, and pharmaceuticals. In this study, the expression of the three genes found in the human CYP2A gene cluster was investigated in the liver and several extrahepatic tissues by gene-specific reverse transcriptase-polymerase chain reaction (RT-PCR). All three transcripts (CYP2A6, CYP2A7, and CYP2A13) were found to be present in liver. Quantitative RT-PCR analysis showed that CYP2A6 and CYP2A7 mRNAs were present at roughly equal levels in the liver, while CYP2A13 was expressed at very low levels. Two putative splicing variants of CYP2A7 were found in the liver. Nasal mucosa contained a low level of CYP2A6 and a relatively high level of CYP2A13 transcripts. Kidney, duodenum, lung, alveolar macrophages, peripheral lymphocytes, placenta, and uterine endometrium were negative for all transcripts. This survey gives a comprehensive picture of the expression pattern of CYP2A genes in liver and extrahepatic tissues and constitutes a basis for a search for functional CYP2A forms and their roles in chemical toxicity in liver and nasal mucosa.

Variant Bernard-Soulier syndrome due to homozygous Asn45Ser mutation in the platelet glycoprotein (GP) IX in seven patients of five unrelated Finnish families.

Bernard-Soulier syndrome (BSS), a rare bleeding disorder with macrothrombocytopenia, is caused by a defect of the platelet glycoprotein (GP) Ib/IX/V complex. Here we report a variant form of BSS in eleven patients of five unrelated families who originate from a particular area of Finland. The differential diagnosis from idiopathic thrombocytopenic purpura was difficult. Bleeding symptoms were epistaxis and haematomas debuting in childhood, but no spontaneous, severe bleeding episodes were reported. The platelet count varied from 43 to 81x10(9)/l. Screening the entire GP Ibalpha, GP Ibbeta, GP IX and GP V genes revealed a recurrent homozygous Asn45Ser mutation in GP IX in all probands. Flow cytometry showed markedly reduced expression of GP Ib (<10%), and only moderately reduced expression of GP IX (24-36%) and GP V (38-49%). The expression of subunits seemed to vary independently from the normal polymorphisms. Heterozygotes did not differ significantly from controls by their GP Ib/IX/V expression. Since the Asn45Ser mutation has also been reported in three other kindreds of northern and central European origin, this study reveals that instead of being a mutation hot spot, it may be ancient and scattered in Europe. Moderate, chronic thrombocytopenia should be carefully studied to diagnose variant BSS correctly from treatment resistant idiopathic thrombocytopenia.

Molecular characterization of two mutations in platelet glycoprotein (GP) Ib alpha in two Finnish Bernard-Soulier syndrome families.

Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder and macrothrombocytopenia which is caused by a defect in the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the receptor for von Willebrand factor and thrombin. Here we report the molecular basis of the classical form of BSS in two unrelated Finnish patients, both with a life-long history of severe bleeding. Flow cytometry and immunoblotting showed no expression of GP Ib/IX, GP Ib alpha, GP Ib beta or GP IX (less than 10%) in the patients' platelets. No expression of GP V (< 10%) was observed in propositus 1, but a residual amount was found in propositus 2 (24%). DNA sequencing analysis revealed that propositus 1 was compound heterozygous for a two-base-pair deletion at Tyr505(TAT) and a point mutation Leu129(CTC)Pro(CCC) in the GP Ib alpha gene. Propositus 2 was homozygous for the Tyr505(TAT) deletion. The nine relatives who were heterozygous for either of the mutations also had low levels of GP Ib alpha (74-90%). Hence, Bernard-Soulier patients homozygous or compound heterozygous for Tyr505(TAT) are severely affected. Interestingly, both mutations have independently been found in three other families in previous reports, suggesting their ancient age or mutational 'hot spot'.

Genetic polymorphism in human platelet glycoprotein GP Ib/IX/V complex is enriched in GP V (CD42d).

Platelet glycoproteins Ib beta (CD42c), IX (CD42a), and V (CD42d), together with GP Ib alpha (CD42b), form a receptor whose interaction with the von Willebrand factor is essential in the initial stages of haemostasis. Genetic variation in these proteins can cause alloimmunization leading to neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. Defective mutations cause a rare bleeding disorder, Bernard-Soulier syndrome. Only two antigenic polymorphisms have thus far been established in these proteins: the HPA-2 in GP Ib alpha and the rare Iy variant in GP Ib beta. Recently, we reported that only a limited degree of polymorphism can be found in the GP Ib alpha gene; the level of variation in the other components is not known. We therefore systematically screened polymorphism in the GP Ib beta, GP IX, and GP V genes in 50 unrelated Finnish blood donors. Nine polymorphic sites were found in the GP V gene, of which four changed the amino acid code and five were silent. The gene frequencies for substitutions Asp114Tyr, Met273Ile, Gly341Arg, and Leu397Arg were 1%, 1%, 2%, and 1% respectively. The five silent polymorphisms also had low frequencies, 1-4%. No polymorphism was found in the GP Ib beta gene and only one mutation was found in the 3' untranslated region of the GP IX gene. Our results indicate that genetic variation in the GP Ib/IX/V complex is mostly tolerated in the GP V protein--whose function in the complex is not clear whereas the other components have only very limited genetic polymorphism.

Screening of inherited oligosaccharidurias among mentally retarded patients in northern Finland.

Urinary oligosaccharides were screened with thin-layer chromatography among 1058 mentally retarded patients of a geographically defined area with an approximate population of 200,000. Aspartylglucosaminuria was detected in 26 cases and Salla disease in 24 cases. All patients with aspartylglucosaminuria had the correct diagnosis before the screening, whereas two new cases of Salla disease were detected. No other inherited metabolic diseases were found. The results demonstrate the accuracy of the TLC method, and indicate a high gene frequency of those two lysosomal storage diseases in the population of northern Finland.

The incidence of Down syndrome in northern Finland with special reference to maternal age.

The incidence of live-born children with Down syndrome was found to be 1.73/1000 (1:578) in northern Finland over the years 1965 to 1979. Despite a marked reduction in the proportion of older mothers, no significant change in the incidence was observed. Instead, an age-specific rise in the incidence for mothers aged 25 to 29 years could be shown during the last five-year period in years 1975 to 1979.

Metabolism of collagen in aspartylglycosaminuria: urinary excretion of hydroxyproline.

Aspartylglycosaminuria (AGU) is a lysosomal storage disorder of glycoprotein degradation characterized by severe mental retardation and connective tissue alterations. We have previously described low collagen production in skin fibroblast cultures from AGU patients. In the present work we showed that the urinary excretion of hydroxyproline (total, non-dialysable and free hydroxyproline as indicators of collagen metabolism) was reduced in young AGU patients in comparison with age-matched controls. In adult patients no significant difference was detected. The results support the view that reduced collagen production is associated with the connective tissue abnormalities in this disorder.

Salla disease: a new lysosomal storage disorder with disturbed sialic acid metabolism.

Salla disease is a lysosomal storage disorder associated with increased urinary excretion of free sialic acid. The main clinical features in 34 patients were severe psychomotor retardation of early onset, ataxia, athetosis, rigidity, spasticity, and impaired speech. Growth retardation, thick calvarium, and exotropia were present in about half the patients. The amplitude of EEG decreased progressively with increasing age. Life span appears to be normal; the age range of the patients was 3 to 63 years. Genealogic studies suggest an autosomal mode of inheritance. A thin-layer method is described for the detection of increased urinary free sialic acid excretion. The basic defect is so far unknown.