Kirsi Jahnukainen

Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.

Response-Guided Induction Therapy in Pediatric Acute Myeloid Leukemia With Excellent Remission Rate

PURPOSE To evaluate the early treatment response in children with acute myeloid leukemia (AML) using a response-guided induction strategy that includes idarubicin in the first course. PATIENTS AND METHODS All Nordic children with AML younger than 15 years (n = 151) were treated on the Nordic Society for Pediatric Hematology and Oncology (NOPHO) AML 2004 protocol. After the first course of idarubicin, cytarabine, etoposide, and 6-thioguanin, patients with good response were allowed hematologic recovery before the second course, whereas patients with a poor (≥ 15% blasts) or intermediate (5% to 14.9% blasts) were recommended to proceed immediately with therapy. Patients not in remission after the second course received fludarabine, cytarabine, and granulocyte colony-stimulating factor. Poor responders received allogeneic stem-cell transplantation (SCT) as consolidation. RESULTS Seventy-four percent of patients had good response, 17% had intermediate response, and 7% had poor response after the first course. The overall remission frequency was 97.4%, with 92% in remission after the second course. The rate of induction death was 1.3%. Patients with an intermediate response had a lower event-free survival of 35% compared with good (61%) and poor responders (82%). CONCLUSION The NOPHO-AML 2004 induction strategy gives an excellent remission rate with low toxic mortality in an unselected population. Outcome is worse in patients with intermediate response but may be improved by intensifying consolidation in this group using SCT.

Doxorubicin Induces Apoptosis in Germ Line Stem Cells in the Immature Rat Testis and Amifostine Cannot Protect against This Cytotoxicity

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined. As indicated by microscopy and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16- and 24-day-old rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult.

Increased Apoptosis Occurring During the First Wave of Spermatogenesis Is Stage-Specific and Primarily Affects Midpachytene Spermatocytes in the Rat Testis

Abstract The physiological apoptosis that occurs in immature testis appears to be necessary for the maturation of this tissue. Thus, inhibition of the early apoptotic wave associated with the first round of spermatogenesis is followed by accumulation of spermatogonia and infertility later in life. To identify the cell types undergoing apoptosis in immature rat testis and to characterize the relationship between this apoptosis and progression of the first wave of spermatogenesis, sequential viable segments of seminiferous tubules from 8-, 18-, and 26-day-old rats were examined under a phase-contrast microscope. One novel observation was the existence of pronounced stage-specificity during the peak of apoptosis at the very early postnatal ages of 18 and 26 days. Increased apoptosis of pachytene spermatocytes in stages VII–VIII was the major feature that distinguished immature spermatogenesis from the corresponding adult process. The frequency of apoptosis among type A spermatogonia in immature stages IX–I was also elevated in comparison to the corresponding mature stages. The age-related peak of apoptosis was mediated by caspase 3; furthermore, stage-dependent expression of Bax in midpachytene spermatocytes was observed in the 18- and 26-day-old testis. These observations suggest that this Bax-regulated, caspase 3-mediated, increased apoptosis of midpachytene spermatocytes during the first wave of immature spermatogenesis represents a major difference in comparison to apoptosis occurring in the mature testis, and it may play an important regulatory role in establishing spermatogenesis in the rat testis.

Testicular function and fertility preservation in male cancer patients

The testis has been shown to be highly susceptible to the toxic effects of cancer therapy at all stages of life. Young cancer survivors are approximately half as likely as their siblings to sire a pregnancy. Radiation therapy to the testes and high cumulative dose of alkylating agents are the major factors decreasing the probability of fertility. This review aims to present an overview of the current state of knowledge in mechanisms how human spermatogonia proliferate and differentiate and how cancer therapy affects germ cells, what are the options for fertility preservation and what are the clinical risks and limitations related to such procedures. This area of research is discussed in the context of the potential future options that may become available for preserving fertility in male cancer patients.

Testicular stem cells for fertility preservation: preclinical studies on male germ cell transplantation and testicular grafting.

Spermatogonial stem cells open novel strategies for preservation of testicular tissue and fertility preservation in boys and men exposed to gonadotoxic therapies. This review provides an update on the physiology of spermatogonial stem cells in rodent and primate testes. Species‐specific differences must be considered when new technologies on testicular stem cells are considered. Germ cell transplantation is presented as one novel and promising strategy. Whereas this technique has become an important research tool in rodents, a clinical application must still be regarded as experimental and many aspects of the procedure need to be optimized prior to a safe and efficient clinical application in men. Testicular grafting opens another exciting strategy for fertility preservation. Autologous and xenologous transfer of immature tissue revealed a high regenerative potential of immature testicular tissue. Grafting was applied in rodents and primates and resulted in the generation of sperm. Further research is needed before an application in humans can be considered safe and efficient. Despite the current limitations in regard to the generation of sperm from cryopreserved male germline cells and tissues, protocols for cryopreservation of testicular tissue are available and reveal a promising outcome. Since future improvements of germ cell transplantation and grafting approaches can be assumed, bioptic retrieval and cryopreservation of testicular tissue fragments should be performed in oncological patients at high risk of fertility loss since this is their only option to maintain their fertility potential. Pediatr Blood Cancer 2009;53:274–280.

A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys

STUDY QUESTION What clinical practices, patient management strategies and experimental methods are currently being used to preserve and restore the fertility of prepubertal boys and adolescent males? SUMMARY ANSWER Based on a review of the clinical literature and research evidence for sperm freezing and testicular tissue cryopreservation, and after consideration of the relevant ethical and legal challenges, an algorithm for the cryopreservation of sperm and testicular tissue is proposed for prepubertal boys and adolescent males at high risk of fertility loss. WHAT IS KNOWN ALREADY A known late effect of the chemotherapy agents and radiation exposure regimes used to treat childhood cancers and other non-malignant conditions in males is the damage and/or loss of the proliferating spermatogonial stem cells in the testis. Cryopreservation of spermatozoa is the first line treatment for fertility preservation in adolescent males. Where sperm retrieval is impossible, such as in prepubertal boys, or it is unfeasible in adolescents prior to the onset of ablative therapies, alternative experimental treatments such as testicular tissue cryopreservation and the harvesting and banking of isolated spermatogonial stem cells can now be proposed as viable means of preserving fertility. STUDY DESIGN, SIZE, DURATION Advances in clinical treatments, patient management strategies and the research methods used to preserve sperm and testicular tissue for prepubertal boys and adolescents were reviewed. A snapshot of the up-take of testis cryopreservation as a means to preserve the fertility of young males prior to December 2012 was provided using a questionnaire. PARTICIPANTS/MATERIALS, SETTING, METHODS A comprehensive literature review was conducted. In addition, survey results of testis freezing practices in young patients were collated from 24 European centres and Israeli University Hospitals. MAIN RESULTS AND THE ROLE OF CHANCE There is increasing evidence of the use of testicular tissue cryopreservation as a means to preserve the fertility of pre- and peri-pubertal boys of up to 16 year-old. The survey results indicate that of the 14 respondents, half of the centres were actively offering testis tissue cryobanking as a means of safeguarding the future fertility of boys and adolescents as more than 260 young patients (age range less than 1 year old to 16 years of age), had already undergone testicular tissue retrieval and storage for fertility preservation. The remaining centres were considering the implementation of a tissue-based fertility preservation programme for boys undergoing oncological treatments. LIMITATIONS, REASONS FOR CAUTION The data collected were limited by the scope of the questionnaire, the geographical range of the survey area, and the small number of respondents. WIDER IMPLICATIONS OF THE FINDINGS The clinical and research questions identified and the ethical and legal issues raised are highly relevant to the multi-disciplinary teams developing treatment strategies to preserve the fertility of prepubertal and adolescent boys who have a high risk of fertility loss due to ablative interventions, trauma or genetic pre-disposition.

Clinical Potential and Putative Risks of Fertility Preservation in Children Utilizing Gonadal Tissue or Germline Stem Cells

Rapid progress in the development of novel experimental strategies to generate fertile gametes from cryo-preserved ovarian and testicular tissue motivates oncologists to investigate ways in which gonadal tissue might be preserved. Childhood cancer patients remain the major pediatric group which can benefit from these techniques. Other potential candidates include patients with systemic diseases, which require gonadotoxic chemotherapy, patients undergoing gonadectomy, patients with Turner or Kleinefelters syndrome, and boys with cryptorchid testes. This review aims to present an overview of the current state of knowledge in experimental germ stem cell transplantation in higher primates including humans, and the clinical risks and limitations related to such procedures in children. This area of research is discussed in the context of the potential future options that may become available for preserving fertility in boys and girls.

Role of transforming growth factor β in testicular immunosuppression

Abstract The potential role of transforming growth factor β (TGFβ) in the regulation of the immunological milieu of the testis was investigated. Antibodies neutralizing TGFβ reversed the previously observed suppression of rat peripheral blood lymphocyte proliferation induced by rat abdominal testis extract. Recombinant TGF β 1 dose-dependently inhibited testicular interleukin-1-like factor-driven proliferation of murine thymocytes and ConA-stimulated rat peripheral blood mononuclear cells. Extracts of seminiferous tubules contained a M r ∼25 K TGFβ-like growth inhibitor of the CLL-64 mink lung epithelial cell line. The present findings suggest an important role for TGFβ in testicular immunosuppression.

Presence of FLT3-ITD and high BAALC expression are independent prognostic markers in childhood acute myeloid leukemia

Mutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information.