Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

[A CASE OF RENAL CELL CARCINOMA WITH INFERIOR VENA CAVAL TUMOR THROMBUS WHICH THE REDUCTION OF TUMOR IN SPITE OF DRUG DISCONTINUANCE AFTER THE FULMINANT HEPATITIS ONSET WITH THE SUNITNIB].

A 70-year-old man presented with right renal cell carcinoma with inferior vena caval tumor thrombus into the right atrium. CT Scan presented local invasion and lymph node metastasis. We estimated inoperative case, so he was started sunitinib. After 5 month he had general fatigue and admitted to our hospital. He diagnosed serious adverse events of fulminant hepatitis and left ventricular systolic dysfunction and discontinued sunitnib. After drug discontinuance reduction of tumor and tumor thrombus were detected. 7-months later, we showed the increase of tumor and the improvement of the left ventricular systolic dysfunction. We performed right renal nephrectomy and it passes now in 14 months after surgery, but doses not show a recurrence, metastasis.

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.

BACKGROUND: Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. METHODS: Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. RESULTS: Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). CONCLUSION: Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

INITIAL EXPERIENCE OF THE ENZALUTAMIDE TREATMENT FOR CASTRATION-RESISTANT PROSTATE CANCER.

(Objective) Enzalutamide is an oral androgen-receptor inhibitor that prolongs survival in men with castration-resistant prostate cancer (CRPC). We retrospectively evaluated clinical efficacy and safety of enzalutamide in CRPC. (Patients and methods) We reviewed clinical records of 73 patients who had received enzalutamide for the CRPC at Showa University and affiliated 7 hospitals. Enzalutamide was given at a dose of 160 mg/day, but some patients were treated at lower dose because of there age or poor performance status. Prostrate-specific antigen (PSA) response, prior docetaxel use and the previously administered agents were evaluated retrospectively. (Results) The median patients age was 77 years, the median Gleason score was 9 and the median PSA level at baseline was 26.9 ng/ml. The patients who had prior docetaxel use were 29 (39.7%) and the median of total docetaxel dose was 460 mg/body. The median number of total prior treatments (anti-androgens, Estramustine and steroid) was 3. Twenty seven (61.4%) patients with docetaxel-naïve achieved over 50% reduction of PSA level from baseline, but only 7 (24.1%) in patients previously treated with docetaxel. The most common adverse events included fatigue (24.7%), anorexia (24.7%) and the nausea (16.4%). We found a small proportion of responders to enzalutamide experienced a PSA flare. (Conclusion) Our results of the use of Enzaltamide for CRPC were similar with previous reports. PSA flare was found in some patients with CRPC who responded to enzaltamide. It should be noted that this possible PSA flare phenomenon.

[A Case of Urachal Carcinoma Treated by TS-1/CDDP as Adjuvant Chemotherapy].

A 49-year-old female presented complaining of gross hematuria. Cystoscopy and magnetic resonance imaging revealed a papillary tumor on the bladder dome. At biopsy pathology the tumor was diagnosed as adenocarcinoma. We diagnosed the tumor as urachal adenocarcinoma and performed partial cystectomy of bladder dome with en-bloc resection of the urachal ligament up to the umbilicus. In surgical pathology, the tumor had invaded to the fat tissue around the urachal ligament with metastasis to the lymph node. Therefore the tumor was diagnosed as a stage IVA (Sheldon's category) urachal adenocarcinoma. After surgery, 6 cycles of chemotherapy with TS-1 and cisplatin (CDDP) were performed. There has been no relapse 5 years after surgery. This is the first report of successful adjuvant chemotherapy with TS-1/CDDP for advanced urachal adenocarcinoma.

[A case of mucinous tubular and spindle cell carcinoma of the kidney].

Mucinous tubular and spindle cell carcinoma (MTSCC) is a distinct entity in the World Health Organization classification of kidney tumors since 2004. Herein, we report a case of a patient with MTSCC of the kidney. A 48-year-man visited our hospital with a chief complaint of occult blood in his urine, confirmed by urine occult blood reaction. Computed tomography revealed a solid tumor in the right kidney. The tumor was 40×38 mm in length and was slightly enhanced (cT1aN0M0). Therefore, we performed radical nephrectomy. On analysis of the resected specimen, we found that the number of comparatively small malignant cells had increased markedly, forming branched tubular cuboidal cells. Further more, positive results were obtained on staining the stroma with both PAS and alcian blue stains characteristic of papillary renal cell carcinoma ; however, extracellular mucinous material was found to be depleted. Therefore, we needed to differentiate between papillary renal cell carcinoma and MTSCC. Finally, on the basis of the immunohistochemical staining results-vimentin (＋), CK34ßE12 (-), and CD10 (-)-MTSCC was confirmed.

[A case of vesical endometriosis that worsened during the early pregnancy period].

We report a case of vesical endometriosis that worsened during the early pregnancy period. A 37-year old woman had been under treatment for endometriosis (including vesical endometriosis) by a gynecologist during the past 10 years. She was treated for sterility 1 year ago, and became pregnant through in vitro fertilization. In her 8th gestational week, she complained of gross hematuria at our hospital. Cystoscopic findings revealed some tumors that appeared worse than the last findings two years ago. In order to deny malignancy, transurethral resection of the bladder tumor was performed in her 12th gestational week. The pathologic diagnosis was endometriosis. She was able to stay pregnant, and delivered a girl. After delivery, cystoscopic findings revealed reduction of tumors. In most cases pregnancy cures endometriosis ; however, in this case symptoms became worse during the early stage of pregnancy. The reason for this contrary event is discussed.

Testicular volume measurement: comparison of ultrasonography, orchidometry, and water displacement.

OBJECTIVES: To determine the accuracy of orchidometry and ultrasonography for measuring the testicular volume by comparing the resultant measurements with the actual testicular volume in humans. METHODS: The testicular volume of 40 testes from 20 patients with prostate cancer (mean age +/- SD 74.5 +/- 7.5 years) was measured using the Prader orchidometer and ultrasonography before therapeutic bilateral orchiectomy. The ultrasound measurements of testicular volume were calculated using three formulas: length (L) x width (W) x height (H) x 0.52, L x W2 x 0.52, and L x W x H x 0.71. The actual testicular volumes were determined by water displacement of the surgical specimen. RESULTS: The mean actual testicular volume of the 40 testes was 9.3 cm3 (range 2.5 to 23.0). A strong correlation was found between the testicular volume calculated by the three ultrasound formulas and the actual volume (r = 0.910 to 0.965, P <0.0001) and was stronger than the correlation with the Prader orchidometer (r = 0.818, P <0.0001). The smallest mean difference from the actual testicular volume was observed with the formula L x W x H x 0.71, which overestimated the actual volume by 0.80 cm3 (7.42%). The measurements using the Prader orchidometer correlated with the actual testicular volume and with the testicular volume calculated using the three ultrasound formulas (r = 0.801 to 0.816, P <0.0001). However, the orchidometer measurements had the largest mean difference from the actual testicular volume (6.68 cm3, 81.7%). CONCLUSIONS: The results of this study have shown that measuring the testicular volume by ultrasonography is more accurate than by the Prader orchidometer, and the formula L x W x H x 0.71 was the most accurate for calculating the testicular volume.

Testicular microlithiasis identified ultrasonographically in Japanese adult patients: prevalence and associated conditions.

OBJECTIVES: To determine the prevalence of testicular microlithiasis (TM) seen by testicular ultrasonography (US) in Japanese adult men referred for andrologic symptoms and evaluate associations of TM with pathologic conditions. METHODS: For 7 years, US was performed in 969 patients (mean age 40.9 years) at one institution. The patients were divided into groups with infertility (n = 550), unilateral testicular tumor (n = 46), or other andrologic conditions (n = 373). TM was identified as multiple small hyperechogenic foci. In the tumor group, only images of the tumor-free testis were reviewed. Patients with TM accompanying tumor or infertility completed follow-up questionnaires and US examinations. RESULTS: TM was diagnosed in 46 patients (mean age 38.5 years, range 23 to 75). The prevalence of TM was 17.4% in the tumor group, 5.6% in the infertility group, and 1.9% in the other-conditions group. TM was associated with testicular tumor and infertility, but not with other conditions. In patients with unilateral testicular germ cell tumor, the prevalence of carcinoma in situ in the contralateral testis was greater when TM was present in that testis (2 of 8 patients) than when TM was absent (0 of 32, P = 0.0037). No new testicular tumor developed subsequently. In the infertility group, the 31 patients with TM showed no subsequent testicular tumor development, and neither patient undergoing testicular biopsy had carcinoma in situ. CONCLUSIONS: TM, as demonstrated by US, was associated with infertility, as well as testicular tumor. TM in a testis contralateral to a unilateral testicular germ cell tumor may increase the risk of carcinoma in situ.

Color Doppler ultrasonography as a routine clinical examination in male infertility.

AIM: We assessed the value of scrotal color Doppler ultrasonography as a routine examination in infertile men. METHODS: Color Doppler ultrasonography was performed in 545 infertile men with a mean age of 35.8 years to detect intrascrotal abnormalities. Findings were compared with those of physical examination. RESULTS: Intrascrotal abnormalities were detected by ultrasonography in 65.3% of patients. Of 374 abnormalities, 58.3% were undetected by physical examination. Left varicocele was found in 313 patients (57.4%); testicular microlithiasis in 30 (5.5%); epididymal cyst in 21 (3.9%); right varicocele in 4 (0.8%); and testicular cysts in 3 (0.6%). One occurrence each (0.2%) was found for testicular tumor, intrascrotal hemangioma, and hydrocele of the spermatic cord. Compared to ultrasonography, sensitivity in detecting left varicocele by physical examination was 58.4%; specificity, 79.3%; accuracy, 67.3%; and positive predictive value, 79.3%. Venous diameters in the pampiniform plexus were 3 mm or more in 61.5% of 130 subclinical left varicoceles. Of 30 patients with testicular microlithiasis, 14 had varicocele, 2 had epididymal cyst,s 3 had a history of mumps orchitis, 1 had retractile testis, and 1 had a history of orchiectomy for contralateral testicular tumor. CONCLUSIONS: The routine Color Doppler ultrasonography is valuable for diagnosing scrotal abnormalities in infertile men, frequently detecting non-palpable lesions.

[The evaluation of urination status and International Prostate Symptom Score following radical prostatectomy].

PURPOSE: We evaluated condition of urination and International Prostate Symptom Score (I-PSS) after radical prostatectomy. PATIENTS AND METHODS: Forty-three men with prostatic cancer underwent radical prostatectomy between October 1993 and October 2002. Mean patients age was 66 years (range 56 to 79) and clinical follow-up averaged 38.6 months (range 3 to 84). Urodynamics studies including uroflowmetry, cystometry and evaluation of I-PSS were performed before and 1, 3, 6, 12 months after the operation. After 12 months these studies were performed every year. The status of postoperative urinary incontinence was based on patients' report. RESULTS: First desire to void, maximum desire to void and maximal flow rate was decrease temporarily after radical prostatectomy. However most patients had normal uroflowmetorogram and normal cystometrogram at 6 months. I-PSS and QOL index was improved during postoperative 12 months. CONCLUSIONS: Postoperatively urodynamics studies was improved from 3 to 6 months, but evaluation of I-PSS and QOL index was improved from 6 to 12 months. The difference was formed to both.