About 3 – 5% of all patients with testicular germ cell tumour (GCT) develop a contralateral cancer, the majority of which arise within 10–15 years. Little is known about the risk of second GCTs after more than two decades. Here we present 3 cases with very late presenting contralateral GCT and provide a summary of similar cases reported previously.
(1) This white Caucasian man underwent right-sided orchiectomy for a nonseminomatous GCT at the age of 22 years. Additional treatment consisted of retroperitoneal lymph node dissection (RPLND) and chemotherapy with 4 cycles of vinblastin / bleomycin. 36 years later, contralateral seminoma clinical stage 1 developed. Cure was achieved by orchiectomy. Histologically, testicular intraepithelial neoplasia (TIN; intratubular germ cell neoplasia) was detected in the tumour-surrounding tissue.
(2) This white Caucasian male had right-sided orchiectomy for nonseminomatous GCT at the age of 29 years. Pathological stage 1 was confirmed by RPLND. 25 years later, he received left sided orchiectomy for seminoma stage 1. Histologically, TIN was found in the tissue adjacent to seminoma. Two brothers had testicular GCT, too, one with bilateral GCT. (3) This 21 year old white Caucasian man underwent left-sided orchiectomy for nonseminomatous GCT. Pathological stage 1 was confirmed by RPLND. 21 years later, he received organ-preserving excision of a right-sided seminoma, followed by BEP chemotherapy for stage 3 disease. Histologically, TIN was found in the surrounding testicular tissue.
22 cases of bilateral GCT with intervals of 20 or more years have previously been reported, thereof three with intervals of more than 30 years, the longest interval being 40 years.
Apart from increased risks of cardiovascular diseases and non-testicular malignancies, patients with GCT face the specific probability of a second GCT in the long run. This risk persists life-long and is not eliminated by chemotherapy. Contralateral testicular biopsy can identify patients at risk by revealing precursor cells of GCT though false-negative biopsies may occur sporadically. However, in view of the multi-facetted late hazards of GCT patients, this minor surgical procedure might somewhat simplify the long-time care of these patients.
Testicular germ cell neoplasms; Bilateral tumours; Testicular biopsy; Seminoma; Familial germ cell tumours
Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP) protein is associated with T-shaped ribbons (“T-bars”) at presynaptic active zones (AZs). BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, we have identified a P-transposon insertion in gene CG11489 (location 79D) which shows high homology to mammalian genes for SR protein kinases (SRPKs). SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins). Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, we name CG11489 the Drosophila Srpk79D gene. We have characterized Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. We propose that the Drosophila Srpk79D gene characterized here may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the identification of its substrate and the detailed analysis of SRPK function for the maintenance of nervous system integrity.
Neurons communicate through release of neurotransmitters at specialized contacts called synapses. Modulation of synaptic transmission likely underlies all higher brain function including feature abstraction, learning and memory, and cognition. The complex molecular machinery that regulates neurotransmitter release has been conserved in evolution but is still incompletely understood. Using the genetic model organism Drosophila, we recently discovered a protein of the presynaptic ribbon (T-bar) that was called Bruchpilot (German for crash pilot) because flies with reduced amounts of this protein cannot fly. We now screened various Drosophila mutants for changes in tissue localization of Bruchpilot and discovered a gene that codes for an enzyme which is similar to mammalian kinases that phosphorylate splicing factors and may co-localize with Bruchpilot at the synapse. Larval nerves of mutants for this gene contain conspicuous accumulations of Bruchpilot that correspond to extensive electron-dense ribbon-like agglomerates surrounded by vesicles. While general axonal transport and basic synaptic transmission at larval nerve-muscle synapses are not affected, adult mutants show reduced life span and impaired flight and walking. The substrate for this kinase and its role in maintaining brain function must now be identified. Its discovery raises important questions about the function of homologous proteins in mammals including humans.
The aim of this study was to identify predictors of viable germ cell tumor (GCT) in postchemotherapeutic residual retroperitoneal masses.
Materials and Methods:
The pertinent clinical and pathologic data of 16 male patients who underwent postchemotherapeutic retroperitoneal lymph node dissection (PC-RPLND) at King Faisal Specialist Hospital and Research Centre between 1994 and 2005 were reviewed retrospectively. It was found that all patients received cisplatin-based chemotherapy for advanced testicular GCT.
Out of the 16 male patients, 2 (13%), 8 (50%), and 6 (37%) had viable GCT, fibrosis, and teratoma, respectively. Ten (10) of the patients with prechemotherapeutic S1 tumor markers did not have viable GCT, and two of the six patients who had prechemotherapeutic S2 tumor markers have viable GCT. All tumor marker levels normalized after chemotherapy even in patients with viable GCT. Four patients had vascular invasion without viable GCT. Furthermore, four patients had more than 60% embryonal elements in the original pathology, but only 1 had viable GCT at PC-RPLND. Four of the five patients with immature teratoma had teratoma at PC-RPLND but no viable GCT; however, out of the four patients with mature teratoma, one had viable GCT and two had teratoma at PC-RPLND. Of the two patients with viable GCT, one had 100% embryonal cancer in the original pathology, prechemotherapeutic S2 tumor markers, history of orchiopexy, and no vascular invasion; the other patient had yolk sac tumor with 25% embryonal elements and 40% teratoma in the original pathology, and prechemotherapeutic S2 tumor markers.
None of the clinical or pathological parameters showed a strong correlation with the presence of viable GCT in PC-RPLND. However, patients with ≥S2 may be at higher risk to have viable GCT. Further studies are needed to clarify this.
Chemotherapy; germ cell tumor; predictor; retroperitoneal lymph node dissection
Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown.
We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents.
Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT.
Pediatric germ cell tumors (GCT) are rare tumors: 80% are benign, 20% malignant (2-3% of all malignant pediatric tumors). The gonadal sites (ovary and testis) account for 40% of cases.
Represent 30% of GCTs and 70% of neoplastic ovarian masses, being the most common ovarian neoplasms in children and teenagers. Benign and immature forms (teratomas) constitute about 80% of all ovarian GCTs, malignant forms represent 20% increasing during adolescence. The most common malignant entity in children is the yolk sac tumors (YST); dysgerminoma is frequent during adolescence and being bilateral in 10% of cases. Presentation is similar in malignant and benign lesions; abdominal pain (70-80%) and lower abdominal mass are common symptoms. Evaluation of alpha-fetoprotein (αFP) or beta subunit of human chorionic gonadotropin (βHCG) is essential to address the nature of the tumors: Their elevation means presence of malignancy. Surgery includes intraoperative staging procedures and requires ovariectomy or ovarosalpingectomy for malignant lesions, but may be conservative in selected benign tumors. Since malignant GCTs are very chemosensitive, primary chemotherapy is recommended in metastatic or locally advanced tumors.
Represent 10% of pediatric GCT, and about 30% of malignant GCT with two age peaks: Children <3 years may experience mature teratoma and malignant GCTs, represented almost exclusively by YST, while adolescents may also show seminomas or other mixed tumors. The main clinical feature is a painless scrotal mass. Surgery represents the cornerstone of the management of testicular GCTs, with an inguinal approach and a primary high orchidectomy for malignant tumors, while a testis-sparing surgery can be considered for benign lesions. A retroperitoneal lymph node (LN) biopsy may be necessary to define the staging when the involvement of retroperitoneal LN is uncertain at imaging investigations.
Patients with gonadal malignant GCTs fare better than those with extragonadal mediastinal germ cell tumors (MGCTs) and survival rate exceeds 90% in localized forms. Chemotherapy has significantly improved the outcome of malignant forms since the introduction of platinum based regimens. The surgical procedure has to be performed in agreement with the ongoing protocols.
Children; germ cell tumors; gonadal
The development of sarcomatous component (SC) in testicular germ cell tumor (GCT) is an uncommon phenomenon. We searched our surgical pathology files from 1985 to 2007 and identified 33 cases of testicular GCTs with SC. The average age of patients was 31 years. All patients underwent radical orchiectomy, which demonstrated a GCT in all patients except for 3 patients who had received neoadjuvant chemotherapy. All testicular GCTs contained a teratomatous component. The GCTs were pure teratomas in 3 cases, and were mixed GCTs in the other cases. The SC was observed in primary testicular tumor (n = 19), in metastasis (n = 11), or in both primary testicular tumor and metastasis (n=3). The average percentage of the SC in the primary testicular GCT was 32% (range, 5% to 99%). The most common histologic type of SC was rhabdomyosarcoma (n = 24), followed by high-grade unclassified sarcoma (n = 5), rhabdomyosarcoma admixed with high-grade unclassified sarcoma (n = 2), angiosarcoma (n = 1), and low-grade myxoid sarcoma (n = 1). Clinical follow-up information was available for 27 patients. Of the 13 patients whose SC was limited to the testicular GCT, 2 died of GCT not otherwise specified (NOS) at 37 and 68 months, respectively; and 11 patients were free of disease at a mean of 46 months. Of the 14 patients with a SC in the metastasis, 7 patients died of GCT NOS at a mean of 95 months, and 7 patients were free of disease at a mean of 104 months. These results suggest that patients with a SC confined to the primary testicular GCT may not have a higher risk of mortality than those at a comparable stage without a SC. However, patients with a SC in the metastasis have an increased risk of mortality.
testicular germ cell tumor; sarcomatous component; rhabdomyosarcoma
Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.
Cisplatin (CDDP) resistance in testicular germ cell tumors (GCTs) is still a clinical challenge, and one associated with poor prognosis. The purpose of this work was to test pazopanib, an anti-tumoral and anti-angiogenic multikinase inhibitor, and its combination with lapatinib (an anti-ErbB inhibitor) in mouse orthotopic models of human testicular GCTs.
We used two different models of human testicular GCTs orthotopically grown in nude mice; a CDDP-sensitive choriocarcinoma (TGT38) and a new orthotopic model generated from a metastatic GCT refractory to first-line CDDP chemotherapy (TGT44). Nude mice implanted with these orthotopic tumors were treated with the inhibitors and the effect on tumoral growth and angiogenesis was evaluated.
TGT44 refractory tumor had an immunohistochemical profile similar to the original metastasis, with characteristics of yolk sac tumor. TGT44 did not respond when treated with cisplatin. In contrast, pazopanib had an anti-angiogenic effect and anti-tumor efficacy in this model. Pazopanib in combination with lapatinib in TGT38, an orthotopic model of choriocarcinoma had an additive effect blocking tumor growth.
We present pazopanib as a possible agent for the alternative treatment of CDDP-sensitive and CDDP-refractory GCT patients, alone or in combination with anti-ErbB therapies.
Pazopanib; Lapatinib; Testicular cancer; Germ-cell tumors; Cisplatin; Refractory
We previously reported a dose-finding and phase II trial of the TI-CE regimen (paclitaxel [T] plus ifosfamide [I] followed by high-dose carboplatin [C] plus etoposide [E] with stem-cell support) in germ cell tumor (GCT) patients predicted to have a poor prognosis with conventional-dose salvage therapy. We now report the efficacy of TI-CE with prognostic factors for disease-free survival (DFS) and overall survival (OS) in our full data set of 107 patients.
Patients and Methods
Eligible patients had advanced GCTs with progressive disease following chemotherapy and unfavorable prognostic features (extragonadal primary site, incomplete response [IR] to first-line therapy, or relapse/IR to ifosfamide-cisplatin–based conventional-dose salvage). Univariate and multivariate analyses (MVAs) of prognostic factors were performed. The predictive ability of the Einhorn and Beyer prognostic models was assessed.
Most patients were platinum refractory and had an IR to first-line chemotherapy. There were 54 (5%) complete and eight (8%) partial responses with negative markers; 5-year DFS was 47% and OS was 52% (median follow-up, 61 months). No relapses occurred after 2 years. Five (24%) of 21 primary mediastinal nonseminomatous GCTs are continuously disease free. On MVA, primary mediastinal site (P < .001), two or more lines of prior therapy (P < .001), baseline human chorionic gonadotropin ≥ 1,000 U/L (P = .01), and lung metastases (P = .02) significantly predicted adverse DFS. Poor-risk patients did worse than good- or intermediate-risk patients according to both Beyer (P < .002) and Einhorn (P < .05) models.
TI-CE is effective salvage therapy for GCT patients with poor prognostic features. Mediastinal primary site and two or more lines of prior therapy were most predictive of adverse DFS. Beyer and Einhorn models can assist in predicting outcome.
Alternatively spliced variants of several oncogenes and tumor suppressors have been shown to be important for their tumorigenicity. In the present study we have tested whether serine-arginine protein kinase 1 (SRPK1), a major regulator of splicing factors, is involved in ovarian cancer progression and plays a role in chemo-sensitivity. By Western blot analyses, SRPK1 protein was found to be overexpressed in 4 out of 6 ovarian cancer cell lines as compared with an immortalized ovarian surface epithelial cell line; and in 55% of ovarian tumor samples as compared with non-neoplastic ovarian tissue samples. Reduction of SRPK1 expression using small interfering RNA (siRNA) encoding small hairpin RNA in ovarian cancer cells led to (i) reduced cell proliferation rate, slower cell cycle progression and compromised anchorage-independent growth and migration ability in vitro, (ii) decreased level of phosphorylation of multiple serine-arginine proteins, and P44/42MAPK and AKT proteins, and (iii) enhanced sensitivity to cisplatin. Together, these results suggest that elevated SRPK1 expression may play a role in ovarian tumorigenesis and SRPK1 may be a potential target for ovarian cancer therapy.
Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1^E4 protein during the productive phase of infection. However, the precise mechanisms of how E1^E4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1^E4. We map critical residues within an arginine-rich domain of HPV1 E1^E4, and in a region known to facilitate E1^E4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1^E4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1^E4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1^E4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1^E4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1^E4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.
Human male germ cell tumors (GCTs) arise from undifferentiated primordial germ cells (PGCs), a stage in which extensive methylation reprogramming occurs. GCTs exhibit pluripotentality and are highly sensitive to cisplatin therapy. The molecular basis of germ cell (GC) transformation, differentiation, and exquisite treatment response is poorly understood.
To assess the role and mechanism of promoter hypermethylation, we analyzed CpG islands of 21 gene promoters by methylation-specific PCR in seminomatous (SGCT) and nonseminomatous (NSGCT) GCTs. We found 60% of the NSGCTs demonstrating methylation in one or more gene promoters whereas SGCTs showed a near-absence of methylation, therefore identifying distinct methylation patterns in the two major histologies of GCT. DNA repair genes MGMT, RASSF1A, and BRCA1, and a transcriptional repressor gene HIC1, were frequently methylated in the NSGCTs. The promoter hypermethylation was associated with gene silencing in most methylated genes, and reactivation of gene expression occured upon treatment with 5-Aza-2' deoxycytidine in GCT cell lines.
Our results, therefore, suggest a potential role for epigenetic modification of critical tumor suppressor genes in pathways relevant to GC transformation, differentiation, and treatment response.
Germ cell tumor; promoter hypermethylation; MGMT; RASSF1A; BRCA1; gene expression
Granulosa cell tumors constitute less than 5 % of all ovarian tumors. Unlike epithelial ovarian tumors, they occur in a younger age group, are usually detected in an early stage and often have features of hyperestrogenism. The presenting symptoms are usually nonspecific with abdominal pain or distension. They follow an indolent course and are characterized by a long natural history. Mutation of FOXL2 (402C->G) seen in 97 % of adult GCT may be pathognomonic for adult GCT. Only stage of the disease has been consistently shown in various studies to affect survival of patients with GCT. The initial management of patients, for whom fertility is not an issue, is total abdominal hysterectomy, bilateral salpingo-oophorectomy and removal of all gross disease. Nodal dissection is not a significant factor for survival and is not recommended in surgical staging of GCT. Fertility preserving surgery with unilateral salpingo-oophorectomy is feasible in young patients with stage Ia GCT. Patients with early stage disease (stage I and II) have a very good prognosis with 5 year DFS and OS of 89 % and 99 % respectively and these groups of patients usually don’t require any postoperative treatment. Patients with stage Ic disease associated with poor prognostic factors like large tumor size or high mitotic index and stage II, have a higher chance of relapse, and may benefit with postoperative treatment but role of chemotherapy is still debatable. In advanced stage disease (stage III and IV) the 5 year DFS and OS disease was 72 % and 80 % respectively hence the option of postoperative treatment with 6 cycles of BEP should be considered in this group. Recently paclitaxel is being investigated as an effective tool in GCT. The efficacy of radiation in GCT is not well defined but in optimally debulked cases postoperative radiation is a viable option. Due to high chance of recurrence even years after apparent clinical cure of the primary tumor, lifelong follow up with clinical examination and tumor markers like inhibin B is recommended. About 25 % GCT develop recurrence and the median time to recur is usually 4–5 years. Most recurrences are intraperitoneal and usually a complete debulking of the disease is feasible even in the recurrent setting. Postoperative chemotherapy (platinum based) is usually given after surgery more so in cases with widespread disease or after suboptimal cytoreduction. Recurrent chemoresistant, progressive non-responding GCT or patients with high surgical risk are ideal candidates for targeted therapy.
Granulosa cell tumor; Inhibin B
SRPK, a family of cell cycle regulated protein kinases, phosphorylate Serine/Arginine (SR) domain-containing proteins in nuclear speckles and mediate the pre-mRNA splicing. However, the physiological roles of this event in cell cycle are incompletely understood. Here we show that SRPK2 binds and phosphorylates acinus, an SR protein essential for RNA splicing, and redistributes it from the nuclear speckles to the nucleoplasm, resulting in cyclin A1 but not A2 upregulation. Acinus S422D, an SRPK2 phosphorylation mimetic, enhances cyclin A1 transcription, whereas acinus S422A, an unphosphorylatable mutant, blocks the stimulatory effect of SRPK2. Ablation of acinus or SRPK2 abrogates cyclin A1 expression in leukemia cells and arrest cells at G1 phase. Overexpression of acinus or SRPK2 increases leukemia cell proliferation. Further, both SRPK2 and acinus are overexpressed in some of human AML patients and correlate with elevated cyclin A1 expression levels, fitting with the oncogenic activity of cyclin A1 in leukemia. Thus, our findings establish a molecular mechanism by which SR splicing machinery regulates cell cycle and contributes to leukemia tumorigenesis.
SRPK2; Acinus; Cyclin A1; Phosphorylation; Cell proliferation
Splicing abnormalities frequently occur in cancer. A key role as splice site choice regulator is played by the members of the SR (Ser/Arg-rich) family of proteins. We recently demonstrated that SRSF2 is involved in cisplatin-mediated apoptosis of human lung carcinoma cell lines. In this study, by using immunohistochemistry, we demonstrate that the SR proteins SRSF1 and SRSF2 are overexpressed in 63% and 65% of lung adenocarcinoma (ADC) as well as in 68% and 91% of squamous cell lung carcinoma (SCC), respectively, compared to normal lung epithelial cells. In addition, we show that SRSF2 overexpression correlates with high level of phosphorylated SRSF2 in both ADC (p<0.0001) and SCC (p = 0.02), indicating that SRSF2 mostly accumulates under a phosphorylated form in lung tumors. Consistently, we further show that the SR-phosphorylating kinases SRPK1 and SRPK2 are upregulated in 92% and 94% of ADC as well as in 72% and 68% of SCC, respectively. P-SRSF2 and SRPK2 scores are correlated in ADC (p = 0.01). Using lung adenocarcinoma cell lines, we demonstrate that SRSF1 overexpression leads to a more invasive phenotype, evidenced by activation of PI3K/AKT and p42/44MAPK signaling pathways, increased growth capacity in soft agar, acquisition of mesenchymal markers such as E cadherin loss, vimentin and fibronectin gain, and increased resistance to chemotherapies. Finally, we provide evidence that high levels of SRSF1 and P-SRSF2 proteins are associated with extensive stage (III–IV) in ADC. Taken together, these results indicate that a global deregulation of pre-mRNA splicing regulators occurs during lung tumorigenesis and does not predict same outcome in both Non Small Cell Lung Carcinoma histological sub-types, likely contributing to a more aggressive phenotype in adenocarcinoma.
The aim of this study was to evaluate the risk of thrombo-embolic events (TEE) in patients with germ-cell tumours (GCT) who receive cisplatin-based chemotherapy, to compare this risk to that of a matched control group of non-GCT cancer patients, and to identify risk factors of TEE. The rate of TEE during the 6 months following the initiation of chemotherapy was assessed in 100 consecutive patients with GCT and in 100 controls with various neoplasms who were matched on sex and age, and who received first-line cisplatin-based chemotherapy during the same period of time at Institut Gustave Roussy, Villejuif, France. Data were subsequently tested on a validation group of 77 GCT patients treated in Lyon, France. A total of 19 patients (19%) (95% confidence interval (CI): 13–28) and six patients (6%) (95% CI: 3–13) had a TEE in the GCT group and the non-GCT control group, respectively (relative risk (RR): 3.4; P<0.01). Three patients from the GCT group died of pulmonary embolism. In multivariate analysis, two factors had independent predictive value for TEE: a high body surface area (>1.9 m2) (RR: 5 (1.8–13.9)) and an elevated serum lactate dehydrogenase (LDH) (RR: 6.4 (2.3–18.2)). Patients with no risk factor (n=26) and those with at least one risk factor (n=71) had a probability of having a TEE of 4% (95% CI: 1–19) and 26% (95% CI: 17–37), respectively. In the GCT validation set, 10 (13%) patients had a TEE; patients with no risk factor and those with at least one risk factor had a probability of having a TEE of 0 and 17% (95% CI: 10–29), respectively. Patients with GCT are at a higher risk for TEE than patients with non-GCT cancer while on cisplatin-based chemotherapy. This risk can be accurately predicted by serum LDH and body surface area. This predictive index may help to study prospectively the impact of thromboprophylaxis in GCT patients.
cancer of the testis; chemotherapy; cisplatin; germ-cell tumour; thrombosis
Germ cell tumors (GCT) are the most common solid tumors in adolescent and young adult males (age 15 and 35 years) and remain one of the most curable of all solid malignancies. However a subset of patients will have tumors that are refractory to standard chemotherapy agents. The management of this refractory population remains challenging and approximately 400 patients continue to die every year of this refractory disease in the United States.
Given the preclinical evidence implicating vascular endothelial growth factor (VEGF) signaling in the biology of germ cell tumors, we hypothesized that the vascular endothelial growth factor receptor (VEGFR) inhibitor sunitinib (Sutent) may possess important clinical activity in the treatment of this refractory disease. We proposed a Phase II efficacy study of sunitinib in seminomatous and non-seminomatous metastatic GCT’s refractory to first line chemotherapy treatment (ClinicalTrials.gov Identifier: NCT00912912). Next generation targeted exome sequencing using HiSeq 2000 (Illumina Inc., San Diego, CA, USA) was performed on the tumor sample of the unusual responder.
Five patients are enrolled into this Phase II study. Among them we report here the clinical course of a patient (Patient # 5) who had an exceptional response to sunitinib. Next generation sequencing to understand this patient’s response to sunitinib revealed RET amplification, EGFR and KRAS amplification as relevant aberrations. Oncoscan MIP array were employed to validate the copy number analysis that confirmed RET gene amplification.
Sunitinib conferred clinical benefit to this heavily pre-treated patient. Next generation sequencing of this ‘exceptional responder’ identified the first reported case of a RET amplification as a potential basis of sensitivity to sunitinib (VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor) in a patient with refractory germ cell tumor. Further characterization of GCT patients using biomarkers for clinical response and patient selection is warranted.
ClinicalTrials.gov Identifier: NCT00912912
Sunitinib; Germ cell tumor; Phase II trials; Targeted therapy; RET; EGFR; Next generation sequencing; Adolescent and young adult; Vascular endothelial growth factor receptor; VEGF; Unusual responder; Exceptional responder; Outlier responder
Germ cell tumors (GCTs) of the testis are rare, but are the most common cancer in young men. GCTs may consist of one predominant histologic pattern or may represent a mixture of multiple histologic types. For treatment purposes, two broad categories are recognized: 1) pure seminoma and 2) others, which together are termed nonseminomatous GCTs (NSGCTs). In general, seminoma tends to be less aggressive, to be diagnosed at an earlier stage, and to spread predictably along lymphatic channels to the retroperitoneum before spreading hematogenously to the lung or other organs. Compared with NSGCTs, seminoma is exquisitely sensitive to radiation therapy and platinum-based chemotherapy. NSGCTs are usually mixed tumors and teratoma often exists at the sites of metastasis with other GCT elements; cure often requires chemotherapy to kill the chemosensitive-components and surgery to remove the teratomatous components. The main factors contributing to excellent cure rates of GCTs are careful staging at diagnosis; adequate early treatment using chemotherapeutic combinations, with or without radiotherapy and surgery; and very strict follow-up and salvage therapy. We review several clinical studies and summarize the current trends in the management of GCTs.
Neoplasms; Testis; Therapeutics
Cisplatin-based chemotherapy has been commonly used for the treatment of intracranial germ cell tumors (IC-GCTs). However, this treatment exhibits some adverse effects such as renal problems and hearing difficulty. Carboplatin-based chemotherapy was administered to pediatric patients with IC-GCTs from August 2004 at the Samsung Medical Center. In this study, we assessed the responses and adverse effects of carboplatin-based chemotherapy in pediatric IC-GCTs patients according to the risk group, and compared the results with those of the previous cisplatin-based chemotherapy.
We examined 35 patients (27 men and 8 women) diagnosed with IC-GCTs between August 2004 and April 2008 and received risk-adapted carboplatin-based chemotherapy at the Samsung Medical Center. Patients were divided into either low-risk (LR) or high-risk (HR) groups and a retrospective analysis was performed using information from the medical records.
Although hematological complications were common, hearing difficulties or grade 3 or 4 creatinine level elevation were not observed in patients who underwent carboplatin-based chemotherapy. The frequency of febrile neutropenia did not differ between the risk groups. The overall survival was 100% and event-free survival (EFS) was 95.7%. The EFS rate was 100% in the LR group and 90% in the HR group, respectively.
Despite their common occurrence in high-risk patients, no lethal hematological complications were associated with carboplatin-based treatment. The current carboplatin-based chemotherapy protocol is safe and effective for the treatment of pediatric patients with IC-GCTs.
Intracranial germ cell tumor; Carboplatin; Adverse effects
Primary central nervous system (CNS) germ cell tumors (GCTs) are a rare heterogeneous group of lesions, which the clinicopathological features have a marked degree of heterogeneity comparing with that of gonadal GCTs. Accurately diagnosing CNS GCTs might be extremely difficult and requires immunohistochemical verification. This study was to investigate the biological feature of CNS GCTs and diagnostic value of immunohistochemical markers OCT3/4, C-kit, PLAP, and CD30 in CNS GCTs. A retrospective study was performed on 34 patients with CNS germ cell tumors between 1990 and 2014. 34 CNS GCTs account for 9.2% of all primary CNS neoplasms. The sellar region (35.3%) and pineal gland (17.6%) were the most common sites of intracranial GCTs. Hydrocephalus (82.4%) and diplopia (46.9%) were the two most common clinical presentations. The most common histological subtypes were germinoma (67.6%). PLAP, c-kit, OCT3/4 were highly expressed in gernimomas. CD30 and CK AE1/3 stainings were positive in embryonal carcinoma. Yolk sac tumor component showed positive staining for AFP and CK AE1/3. β-HCG staining was positive in choriocarcinoma and STGC. Patients with mature teratomas and germinomas had a better prognosis (a 5-year survival rate) than those with embryonal carcinoma and choriocarcinoma (a 5-year survival rates were 0). Our finding suggest that the incidences of primary CNS GCTs are higher in South China than in the West, but mixed GCTs are uncommon in our study. The judicious use of a panel of selected markers is helpful in diagnosing and predicting the prognosis for CNS GCTs.
Central nervous system; germ cell tumors; C-kit; PLAP; OCT3/4; CD30
Primary central nervous system germ cell tumors (CNS-GCTs) in children and adolescents have unique clinical features and methods of treatment compared with those in adults. There is little information about Chinese children and adolescents with CNS-GCTs. Therefore, in this study we retrospectively analyzed the clinical features and treatment outcome of Chinese children and adolescents with primary CNS-GCTs. Between January 2002 and December 2012, 57 untreated patients from a single institution were enrolled. They were diagnosed with CNS-GCTs after pathologic or clinical assessment. Of the 57 patients, 41 were males and 16 were females, with a median age of 12.8 years (range, 2.7 to 18.0 years) at diagnosis; 43 (75.4%) had non-germinomatous germ cell tumors (NGGCTs) and 14 (24.6%) had germinomas; 44 (77.2%) had localized disease and 13 (22.8%) had extensive lesions. Fifty-three patients completed the prescribed treatment, of which 18 underwent monotherapy of surgery, radiotherapy, or chemotherapy, and 35 underwent multimodality therapies that included radiotherapy combined with chemotherapy or surgery combined with chemotherapy and/or radiotherapy. PEB (cisplatin, etoposide, and bleomycin) protocol was the major chemotherapy regimen. The median follow-up time was 32.3 months (range, 1.2 to 139 months). Fourteen patients died of relapse or disease progression. The 3-year event-free survival (EFS) and overall survival rates for all patients were 72.2% and 73.8%, respectively. The 3-year EFS was 92.9% for germinomas and 64.8% for NGGCTs (P = 0.064). The 3-year EFS rates for patients with NGGCTs who underwent monotherapy and multimodality therapies were 50.6% and 73.5%, respectively (P = 0.042). Our results indicate that multimodality therapies including chemotherapy plus radiotherapy were better treatment option for children and adolescents with CNS-GCTs.
Primary central nervous system germ cell tumors; chemotherapy; radiotherapy; survival rate; children
SR proteins are essential splicing factors whose phosphorylation by the SRPK family of protein kinases regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, non-homologous spacer insert domain that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation. To investigate the conformation of the full-length kinase and the functional role of noncatalytic regions, hydrogen-deuterium exchange and steady-state kinetic experiments were performed on SRPK1. Unlike the kinase core, the large spacer insert domain lacks stable, hydrogen-bonded structure and may provide an intrinsically disordered region for chaperone interactions. Conversely, the N-terminus, which positively regulates SR protein binding, adopts stable structure when the insert domain is present and stabilizes a docking groove in the large lobe of the kinase domain. The N-terminus and spacer insert domain equally enhance SR protein turnover by altering the stability of several catalytic loop segments. These studies reveal that SRPK1 uses an N-terminal extension and a large, intrinsically disordered region juxtaposed to stable structure to facilitate high affinity SR protein interactions and phosphorylation rates.
kinetics; hydrogen-deuterium exchange; phosphorylation; splicing; SR protein
A significant proportion of the human genome is comprised of human endogenous retroviruses (HERVs). HERV transcripts are found in every human tissue. Expression of proviruses of the HERV-K(HML-2) family has been associated with development of human tumors, in particular germ cell tumors (GCT). Very little is known about transcriptional activity of individual HML-2 loci in human tissues, though.
By employing private nucleotide differences between loci, we assigned ~1500 HML-2 cDNAs to individual HML-2 loci, identifying, in total, 23 transcriptionally active HML-2 proviruses. Several loci are active in various human tissue types. Transcription levels of some HML-2 loci appear higher than those of other loci. Several HML-2 Rec-encoding loci are expressed in GCT and non-GCT tissues. A provirus on chromosome 22q11.21 appears strongly upregulated in pathologic GCT tissues and may explain high HML-2 Gag protein levels in GCTs. Presence of Gag and Env antibodies in GCT patients is not correlated with activation of individual loci. HML-2 proviruses previously reported capable of forming an infectious HML-2 variant are transcriptionally active in germ cell tissue. Our study furthermore shows that Expressed Sequence Tag (EST) data are insufficient to describe transcriptional activity of HML-2 and other HERV loci in tissues of interest.
Our, to date, largest-scale study reveals in greater detail expression patterns of individual HML-2 loci in human tissues of clinical interest. Moreover, large-scale, specialized studies are indicated to better comprehend transcriptional activity and regulation of HERVs. We thus emphasize the need for a specialized HERV Transcriptome Project.
Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing are among the best characterized proteins that contain RS domains. TwoSRprotein-specifickinases, SRPK1 and SRPK2, have been considered as highly specific for the phosphorylation of these proteins, thereby contributing to splicing regulation. However, despite the fact that SR proteins are more or less conserved among metazoa and have a rather ubiquitous tissue distribution we now demonstrate that SRPK1 is predominantly expressed in testis. In situ expression analysis on transverse sections of adult mouse testis shows that SRPK1 mRNA is abundant in all germinal cells but not in mature spermatozoa. RS kinase activity was found primarily in the cytosol and only minimal activity was detected in the nucleus. In a search for testis-specific substrates of SRPK1 we found that the enzyme phosphorylates human protamine 1 as well as a cytoplasmic pool of SR proteins present in the testis. Protamine 1 belongs to a family of small basic arginine-rich proteins that replace histones during the development of mature spermatozoa. The result of this progressive replacement is the formation of a highly compact chromatin structure devoid of any transcriptional activity. These findings indicate that SRPK1 may have a role not only in pre-mRNA splicing, but also in the condensation of sperm chromatin.
A neuronal serine-arginine protein kinase that localizes to the presynaptic active zone is required for kinase-dependent repression of active zone assembly.
Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc) that causes T-bar–like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.
Neurons communicate with each other through electrochemical impulses transmitted primarily at specialized intercellular junctions termed synapses. At each synapse, the primary site of synaptic vesicle fusion occurs at the active zone, an electron-dense presynaptic membrane with associated fibrillary matrix. Many active zones also possess one or more electron-dense cytosolic projections that are believed to facilitate vesicle mobilization to the active zone membrane and are required for normal synaptic transmission. These electron-dense projections are referred to as T-bars in Drosophila or ribbons in vertebrates. The molecular composition of these structures remains poorly characterized, and very little is known about how these structures are specifically assembled and stabilized at the presynaptic membrane. Here, we identify in Drosophila a neuronally expressed serine-arginine kinase called SRPK79D that localizes to the presynaptic active zone and that through its kinase activity appears to repress T-bar formation within peripheral axons. Our study thus provides evidence for kinase-dependent repression of active zone assembly, with implications for the development and growth of synaptic connections throughout the nervous system.