The inguinal lymph nodes as regional lymph nodes in anal canal adenocarcinomas: a nationwide database analysis in Japan.

PURPOSE: To establish if it is appropriate to treat the inguinal lymph node (LN) of anal canal adenocarcinoma (ACA) as the intermediate LN according to the Japanese classification. METHODS: The characteristics of 346 ACA patients were examined from the nationwide registry. The effect of LN dissection was evaluated using the therapeutic value index (TVI). Furthermore, the prognostic classification ability of N factors and stage was evaluated using Akaike's information criterion (AIC), the concordance index (C-index), and the 5-year overall survival (OS) rate. RESULTS: The rate of metastasis of the inguinal LN was 7.5% and the TVI was 3.05. Evaluation using AIC and the C-index showed better results when the inguinal LN was treated as the intermediate LN. The 5-year OS rate for 66 patients with perirectal or intermediate LN metastasis, 7 with inguinal LN metastasis, and 13 with inguinal and perirectal or intermediate LN metastasis were 49.2%, 68.6%, and 47.6%, respectively. When inguinal LN metastases were treated as N3, the 5-year OS rates were 66.7% for those with T1N3 and T2N3 disease, and 49.2% for those with T3N3 disease. CONCLUSIONS: The inguinal LN of ACA was evaluated and staged as the intermediate LN to devise an appropriate treatment strategy.

Enhanced Clinical Utility of Molecular Budding Signature as a Recurrence Risk Determinant in Stage II and III Colon Cancer Patients.

BACKGROUND: A molecular budding signature (MBS), which consists of seven tumor budding-related genes, was recently presented as a prominent prognostic indicator in colon cancer (CC) using microarray data acquired from frozen specimens. This study aimed to confirm the predictive power of MBS for recurrence risk based on formalin-fixed, paraffin-embedded (FFPE) materials. METHODS: This research utilized the same microarray data from a prior multicenter study using FFPE whole tissue sections, which retrospectively reviewed 232 stage II CC patients without adjuvant chemotherapy and 302 stage III CC patients with adjuvant chemotherapy. All patients underwent upfront curative surgery without neoadjuvant therapy between 2009 and 2012. An MBS score was calculated using the mean of log2 [each signal] of seven genes (MSLN, SLC4A11, WNT11, SCEL, RUNX2, MGAT3, and FOXC1) as described before. RESULTS: The MBS-low group exhibited a better relapse-free survival (RFS) than the MBS-high group in stage II (P = 0.0077) and in stage III CC patients (P = 0.0003). Multivariate analyses revealed that the MBS score was an independent prognostic factor in both stage II (P = 0.0257) and stage III patients (P = 0.0022). Especially among T4, N2, or both (high-risk) stage III patients, the MBS-low group demonstrated markedly better RFS compared with the MBS-high group (P = 0.0013). CONCLUSIONS: This study confirmed the predictive power of the MBS for recurrence risk by employing FFPE materials in stage II/III CC patients.

Influences of amino-terminal modifications on amyloid fibril formation of human serum amyloid A.

Human serum amyloid A (SAA) is a precursor protein involved in AA amyloidosis. The N-terminal region of the SAA molecule is crucial for amyloid fibril formation, and therefore modifications in this region are considered to influence the pathogenesis of AA amyloidosis. In the present study, using the N-terminal peptide corresponding to the putative first helix region of the SAA molecule, we investigated the influences of N-terminal modifications on amyloid fibril formation. Spectroscopic analyses revealed that carbamoylation of the N-terminal amino group delayed the onset of amyloid fibril formation. From transmission electron microscopic observations, the N-terminal carbamoylated aggregate showed remarkably different morphologies from the unmodified control. In contrast, acetylation of the N-terminal amino group or truncation of N-terminal amino acid(s) considerably diminished amyloidogenic properties. Furthermore, we also tested the cell toxicity of each peptide aggregate on cultured cells by two cytotoxic assays. Irrespective of carbamoylation or acetylation, MTT assay revealed that SAA peptides reduced the reductive activity of MTT on cells, whereas no apparent increase in LDH release was observed during an LDH assay. In contrast, N-terminal truncation did not affect either MTT reduction or LDH release. These results suggest that N-terminal modification of SAA molecules can act as a switch to regulate susceptibility to AA amyloidosis.

Chk1 is a histone H3 threonine 11 kinase that regulates DNA damage-induced transcriptional repression.

DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.

Characteristics of anal canal squamous cell carcinoma as an HPV-associated cancer in Japan.

The definition of the anal canal was revised in the TNM classification (8th edition). The Japanese Society for Cancer of the Colon and Rectum (JSCCR) conducted a retrospective multi-institutional study to clarify the characteristics of anal canal cancer (ACC) in Japan. The diagnoses of 1781 patients treated for ACC were squamous cell carcimoma (SCC; n = 428; 24.0%), adenosquamous cell carcinoma (n = 7; 0.4%), and adenocarcinoma (n = 1260; 70.7%). Anal carcinoma is associated with human papillomavirus (HPV) infection and is risk factor for anal SCC. Among 40 cases analyzed at Takano Hospital and 47 cases analyzed at National Cancer Center Hospital, 34 cases (85.0%) and 40 cases (85.1%), respectively were infected with HPV; HPV-16 was the most common genotype (79.4% and 82.5%). In the JSCCR retrospective multi-institutional study, the prognosis analysis by stage was performed for anal SCC cases (202 cases treated by CRT and 91 cases treated by surgery). The 5-year overall survival (OS) rates by stage did not differ between the two treatment groups to a statistically significant extent. Regarding the results of cancer treatment of patients who underwent HPV infection tests, although the 5-year OS rates by stage did not differ to a statistically significant extent due to the small number of cases, HPV-positive patients had better survival. While an HPV vaccine for anal canal SCC has already been approved internationally, HPV vaccination has already been implemented in Japan as a national immunization program for young women but not for men at present. An HPV vaccination for men is urgently needed.

Artificial Intelligence System to Determine Risk of T1 Colorectal Cancer Metastasis to Lymph Node.

BACKGROUND & AIMS: In accordance with guidelines, most patients with T1 colorectal cancers (CRC) undergo surgical resection with lymph node dissection, despite the low incidence (∼10%) of metastasis to lymph nodes. To reduce unnecessary surgical resections, we used artificial intelligence to build a model to identify T1 colorectal tumors at risk for metastasis to lymph node and validated the model in a separate set of patients. METHODS: We collected data from 3134 patients with T1 CRC treated at 6 hospitals in Japan from April 1997 through September 2017 (training cohort). We developed a machine-learning artificial neural network (ANN) using data on patients' age and sex, as well as tumor size, location, morphology, lymphatic and vascular invasion, and histologic grade. We then conducted the external validation on the ANN model using independent 939 patients at another hospital during the same period (validation cohort). We calculated areas under the receiver operator characteristics curves (AUCs) for the ability of the model and US guidelines to identify patients with lymph node metastases. RESULTS: Lymph node metastases were found in 319 (10.2%) of 3134 patients in the training cohort and 79 (8.4%) of /939 patients in the validation cohort. In the validation cohort, the ANN model identified patients with lymph node metastases with an AUC of 0.83, whereas the guidelines identified patients with lymph node metastases with an AUC of 0.73 (P < .001). When the analysis was limited to patients with initial endoscopic resection (n = 517), the ANN model identified patients with lymph node metastases with an AUC of 0.84 and the guidelines identified these patients with an AUC of 0.77 (P = .005). CONCLUSIONS: The ANN model outperformed guidelines in identifying patients with T1 CRCs who had lymph node metastases. This model might be used to determine which patients require additional surgery after endoscopic resection of T1 CRCs. UMIN Clinical Trials Registry no: UMIN000038609.

Prognosis of anal canal adenocarcinoma versus lower rectal adenocarcinoma in Japan: a propensity score matching study.

PURPOSE: Anal canal adenocarcinoma (AC) is rare and its surgical outcomes and prognostic factors (PFs) are not well understood. The aim of this retrospective study was to identify the characteristics and PFs of AC, using population-based data in Japan. METHODS: Patients with AC (n = 390) or lower rectal adenocarcinoma (LR) (n = 12,477) diagnosed between1991 and 2006 were enrolled in this study. We compared the clinical- and patient-related factors of the two diseases and then examined propensity score matching, overall survival (OS), and PFs. RESULTS: AC tended to develop more often in women and in patients of advanced age. Macroscopically, AC was of an unclassified type and microscopically, it was of high-grade histological types such as mucinous adenocarcinoma, poorly differentiated adenocarcinoma (por), or signet-ring cell carcinoma (sig), with a high frequency of inguinal node metastasis (P < 0.05). The 5 year OS rates were 56.9% for AC and 67.9% for LR (P = 0.002). The PFs of AC were a high-grade histological type (por/sig), T, N, and M. CONCLUSIONS: AC has a significantly worse prognosis than LR. Moreover, the AC lymph node metastatic sites for AC, especially the inguinal nodes, are different from those for LR.

Applications of Synthetic Polymer Discoidal Lipid Nanoparticles to Biomedical Research.

Liposomes are artificially prepared vesicular lipid nanoparticles with a bilayer structure, resembling cell membrane. Their ability to encapsulate various molecules along with excellent biocompatibility makes them ideal delivery vehicles for pharmaceuticals. They can also serve as platforms for membrane proteins to elucidate the structure and function in lipid membranes. Nascent high-density lipoproteins are discoidal lipid nanoparticles with a bilayer structure, which can be reconstituted with their constituents. Such reconstituted nanoparticles, nanodisks, were originally generated in terms of elucidation for mechanisms of lipoprotein metabolisms. At the same time, like liposomes, nanodisks have been developed as delivery vehicles and platforms for membrane proteins in structural biology. From a developmental background, apolipoproteins, their analogs, or fragment peptides were initially used as scaffolding molecules to wrap around the edge of the disk-shaped lipid bilayer. Since the discovery that styrene-maleic acid copolymers produce nanodisks instead of apolipoproteins, variously modified or novel polymers have been synthesized to broaden the applications of polymer nanodisks. This review provides an overview of the types of synthetic polymers used to produce nanodisks, and the biomedical applications of nanodisks to the developments of delivery vehicles and to the structural studies of membrane proteins.

Evaluation of a 55-gene classifier as a prognostic biomarker for adjuvant chemotherapy in stage III colon cancer patients.

BACKGROUND: Adjuvant chemotherapy reduces the risk of recurrence of stage III colon cancer (CC). However, more effective prognostic and predictive biomarkers are needed for better treatment stratification of affected patients. Here, we constructed a 55-gene classifier (55GC) and investigated its utility for classifying patients with stage III CC. METHODS: We retrospectively identified patients aged 20-79 years, with stage III CC, who received adjuvant chemotherapy with or without oxaliplatin, between the years 2009 and 2012. RESULTS: Among 938 eligible patients, 203 and 201 patients who received adjuvant chemotherapy with and without oxaliplatin, respectively, were selected by propensity score matching. Of these, 95 patients from each group were analyzed, and their 5-year relapse-free survival (RFS) rates with and without oxaliplatin were 73.7 and 77.1%, respectively. The hazard ratios for 5-year RFS following adjuvant chemotherapy (fluoropyrimidine), with and without oxaliplatin, were 1.241 (95% CI, 0.465-3.308; P = 0.67) and 0.791 (95% CI, 0.329-1.901; P = 0.60), respectively. Stratification using the 55GC revealed that 52 (27.3%), 78 (41.1%), and 60 (31.6%) patients had microsatellite instability (MSI)-like, chromosomal instability (CIN)-like, and stromal subtypes, respectively. The 5-year RFS rates were 84.3 and 72.0% in patients treated with and without oxaliplatin, respectively, for the MSI-like subtype (HR, 0.495; 95% CI, 0.145-1.692; P = 0.25). No differences in RFS rates were noted in the CIN-like or stromal subtypes. Stratification by cancer sidedness for each subtype showed improved RFS only in patients with left-sided primary cancer treated with oxaliplatin for the MSI-like subtype (P = 0.007). The 5-year RFS rates of the MSI-like subtype in left-sided cancer patients were 100 and 53.9% with and without oxaliplatin, respectively. CONCLUSIONS: Subclassification using 55GC and tumor sidedness revealed increased RFS in patients within the MSI-like subtype with stage III left-sided CC treated with fluoropyrimidine and oxaliplatin compared to those treated without oxaliplatin. However, the predictive power of 55GC subtyping alone did not reach statistical significance in this cohort, warranting larger prospective studies. TRIAL REGISTRATION: The study protocol was registered in the University Hospital Medical Education Network (UMIN) clinical trial registry (UMIN study ID: 000023879 ).

Diffuse large B-cell lymphoma originating from the rectum and diagnosed after rectal perforation during the treatment of ulcerative colitis: a case report.

BACKGROUND: Gastrointestinal lymphomas like diffuse large B-cell lymphoma (DLBCL) are rare complications of ulcerative colitis (UC), and only a few studies have reported intestinal ulcers caused by DLBCL, which got perforated during the treatment of UC. CASE PRESENTATION: A 43-year-old man with severe lower abdominal pain and an 8-year history of UC was admitted in our hospital. He was diagnosed UC since 8 years and received a maintenance oral dose of 5-aminosalicylic acid, and no other immunosuppressive drugs. A deep rectal ulcer was endoscopically diagnosed 10 months before admission, no malignancy or cytomegalovirus infection was detected on biopsy. After 7 months a further endoscopy with biopsies confirmed the finding and the absence of malignancy. Three months later the patient developed sudden abdominal pain and was admitted in our hospital. Rectal perforation was suspected on X-ray and computed tomography imaging, and an emergency surgery was performed. Surgical exploration revealed a perforation on the anterior wall of the rectum. A subtotal colectomy with temporary ileostomy was performed. Pathology examinations showed lymphocyte infiltration of all of the layers of the perforated site and an immunohistochemical evaluation revealed DLBCL. Clinical staging was stage IV, and the patient received a 6-months regimen of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy. Positron emission tomography restaging revealed disappearance of distant uptake and a slight uptake in the residual rectum, and completion proctectomy with ileal pouch-anal anastomosis was performed. No residual tumor in the specimen was found, and the patient was disease-free at 2 years follow-up. CONCLUSIONS: DLBCL may increase the frequency of perforation and is a poor prognostic risk factor for patients with UC. This case study emphasizes the importance of careful medical surveillance and repeated endoscopic biopsies during the treatment of UC.

Effect of mechanochemical inclusion of triamterene into sulfobutylether-ß-cyclodextrin and its improved dissolution behavior.

The formation of inclusion complexes between triamterene (TT) and cyclodextrins (CDs) to increase the water apparent solubility of TT was investigated. UV data showed that the binding constant of the TT/sulfobutylether-ß-cyclodextrin (SBE-ß-CD) inclusion complex was 510 L/mol. The phenyl ring of TT was inserted into the secondary hydroxy face of SBE-ß-CD, as demonstrated by 1H-1H rotating frame nuclear Overhauser effect spectroscopy NMR. Physicochemical properties of solid TT/SBE-ß-CD complexes prepared by physical mixing, kneading, freeze-drying, and mechanochemical methods were studied by X-ray diffraction and 13C cross-polarization and magic angle spinning NMR. With the mechanochemical method, the diffraction peak corresponding to TT disappeared, indicating the formation of an inclusion complex. The results of the dissolution test revealed that the solid complex obtained by the mechanochemical method improved the dissolution of TT. The water apparent solubility of TT can be improved by simple mechanical mixing without organic solvents, and improved bioavailability after oral administration is expected.

A Validation Study for Recurrence Risk Stratification of Stage II Colon Cancer Using the 55-Gene Classifier.

INTRODUCTION: DNA microarrays, such as the consensus molecular subtype (CMS) classification using >600 genes, are used to predict cancer patient prognosis. We recently constructed a simple 55-gene classifier (55GC) system to risk stratify colon cancer (CC). OBJECTIVE: Here, we validate the 55GC specifically for stage II CC and compare it with CMS categories. METHODS: Tissue sections from 232 stage II CC patients who underwent curative surgery without adjuvant chemotherapy between 2009 and 2012 were subjected to DNA microarray analysis. RESULTS: Based on the 55GC, patients were classified into microsatellite instability-like (27%), chromosomal instability-like (41%), and stromal (32%) subtypes with 5-year relapse-free survival (RFS) rates of 88.5, 83.3, and 71.2%, respectively (stromal vs. others: p = 0.0049). Multivariate analysis by Cox's proportional hazard model revealed that the stromal subtype, pT4, and the number of lymph nodes examined (<12) were independent poor prognostic factors. The overall concordance rate between 55GC and CMS was 72%, and 5-year RFS rates of patients with CMS1, CMS2, CMS3, and CMS4 cancers were 100, 85.5, 92.3, and 73.0%, respectively (p = 0.0113). CONCLUSIONS: We conclude that the 55GC is a useful and reproducible grading system for stage II CC recurrence risk stratification.

Preparation of Lipid Nanodisks Containing Apolipoprotein E-Derived Synthetic Peptides for Biocompatible Delivery Vehicles Targeting Low-Density Lipoprotein Receptor.

High-density lipoprotein (HDL) particles that are formed in vivo adopt a disk-shaped structure, in which the periphery of the discoidal phospholipid bilayer is surrounded by apolipoprotein. Such discoidal nanoparticles can be reconstituted with certain apolipoproteins and phospholipids and are commonly called lipid nanodisks. Apolipoprotein E (apoE), one of the HDL constituent proteins, serves as a ligand for the low-density lipoprotein (LDL) receptor. Thus, it is considered that biocompatible delivery vehicles targeting LDL receptors could be prepared by incorporating apoE as the protein component of lipid nanodisks. To enhance targeting efficiency, we designed lipid nanodisks with a large number of ligands using a peptide with the LDL receptor-binding region of apoE combined with a high lipid affinity sequence (LpA peptide). In our study, the LpA peptide spontaneously formed discoidal complexes (LpA nanodisks) of approximately 10 nm in size, equivalent to native HDL. LpA peptides on nanodisks adopted highly α-helical structures, a competent conformation capable of interacting with LDL receptors. As anticipated, the uptake of LpA nanodisks into LDL receptor-expressing cells (HepG2) was higher than that of apoE nanodisks, suggesting an enhanced targeting efficiency via the enrichment of LDL receptor-binding regions on the particle. Biodistribution studies using 111In-labeled LpA nanodisks showed little splenic accumulation and prolonged retention in blood circulation, reflecting the biocompatibility of LpA nanodisks. High accumulation of 111In-labeled LpA nanodisks was observed in the liver as well as in implanted tumors, which abundantly express LDL receptors. Thus, LpA nanodisks are potential biocompatible delivery vehicles targeting LDL receptors.

Correction to: Long-term results of intersphincteric resection for low rectal cancer in Japan.

In the original publication Fig. 2 and Table 4 were incorrectly published. The corrected figure and table are given in this Correction.

Long-term results of intersphincteric resection for low rectal cancer in Japan.

Intersphincteric resection (ISR) is the ultimate sphincter-preserving procedure for low rectal cancer. A questionnaire about the standardization of ISR was given to 2125 patients who underwent curative ISR for low rectal cancer between 2005 and 2012 at 127 affiliated institutions of the Japanese Society for Cancer of the Colon and Rectum (JSCCR), and the results were compared with the results of a systematic review. The findings revealed that although mortality and morbidity were relatively low and the survival rate after ISR was good, the rates of local recurrence and postoperative fecal incontinence were relatively high. The radicality of ISR was compared with that of abdominoperineal resection and low anterior resection using the propensity score matching prognosis analysis of patients in the JSCCR nationwide registry. The local recurrence rate was significantly higher after ISR, and especially high in patients with T3 (invasion into the external anal sphincter) and T4 disease. These results provide evidence about the factors related to fecal incontinence after ISR. As measures for the standardization of ISR, it is important to reconfirm that ISR is not indicated for patients with cT3 and cT4 disease and those with poor preoperative defecatory function, based on the ISR indication criteria.

Acceleration of amyloid fibril formation by carboxyl-terminal truncation of human serum amyloid A.

Human serum amyloid A (SAA) is a precursor protein of AA amyloidosis. Although the full-length SAA is 104 amino acids long, the C-terminal-truncated SAA lacking mainly residues 77-104 is predominantly deposited in AA amyloidosis. Nevertheless, the amyloid fibril formation of such truncated forms of human SAA has never been investigated. In the present study, we examined the effect of C-terminal truncation on amyloid fibril formation of human SAA induced by heparan sulfate (HS). Circular dichroism (CD) measurements demonstrated that the C-terminal truncation induces a reduced α-helical structure of the SAA molecule. HS-induced increases in thioflavin T fluorescence for SAA (1-76) peptide and less significant increases for full-length SAA were observed. CD spectral changes of SAA (1-76) peptide but not full-length SAA were observed when incubated with HS, although the spectrum was not typical for a ß-structure. Fourier transform infrared experiments clearly revealed that SAA (1-76) peptide forms a ß-sheet structure. Transmission electron microscopy revealed that short fibrillar aggregates of SAA (1-76) peptides, which became longer with increasing peptide concentrations, were observed under conditions in which full-length SAA scarcely formed fibrillar aggregates. These results suggested that the C-terminal truncation of human SAA accelerates amyloid fibril formation.

Direct and quantitative electrophoretic detection of covalently closed DNA circles formed by in vitro ligation.

The typical products of enzymatic circularization of DNA, using DNA ligase or recombinase, are covalently closed and mostly relaxed DNA circles. Because they are difficult to analyze on conventional gels, they are often converted to nicked circles prior to electrophoresis. Herein, we present a sensitive and quantitative procedure for directly analyzing ligated closed circle DNA on agarose gels without additional treatments. Specifically, inclusion of GelStar dye in the gel allowed detection of ligated closed circle DNAs, which were likely super-twisted by being intercalated by GelStar, as discrete bands with good separation from linear DNA of the same sizes.

Indium-111 labeling of high-density lipoprotein-mimicking phospholipid-styrene maleic acid copolymer complexes and its biodistribution in mice.

Discoidal lipid nanoparticles mimicking native high-density lipoproteins (HDL) are promising delivery vehicles of drugs and/or imaging agents. However, little is known about the in vivo biodistribution of such discoidal lipid nanoparticles compared to liposomes, clinically available spherical lipid nanoparticles. Recently, it has been reported that synthetic polymers instead of apolipoproteins can be complexed with phospholipid to form discoidal nanoparticles. In the present study, with the aim of developing phospholipid-synthetic polymer complexes for future clinical applications, the biodistribution of such particles in normal mice was investigated. Lipid nanoparticles comprising 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and styrene maleic acid copolymer (SMA), having sizes similar to native HDL, were prepared using the freeze-sonication method. POPC-SMA complexes remained stable at 37°C for at least 3 days in buffer. By devising ways to avoid detrimental effects accompanied by pH reduction and nonspecific binding of 111 In to SMA, POPC-SMA complexes were successfully labeled with 111 In without affecting particle integrity. The biodistribution of POPC-SMA complexes in normal mice was similar to that of discoidal lipid nanoparticles composed of POPC and apolipoprotein A-I, the major protein constituent of native HDL. Unlike liposomes, the accumulation of POPC-SMA complexes in the spleen was low, suggesting that these complexes are not recognized as foreign substances. To the best of our knowledge, this is the first in vivo study of HDL-mimicking phospholipid-synthetic polymer complexes.

Diversity of MHC class I alleles in Spheniscus humboldti.

The major histocompatibility complex locus (MHC) is a gene region related to immune response and exhibits a remarkably great diversity. We deduced that polymorphisms in MHC genes would help to solve several issues on penguins, including classification, phylogenetic relationship, and conservation. This study aimed to elucidate the structure and diversity of the so far unknown MHC class I gene in a penguin species. The structure of an MHC class I gene from the Humboldt penguin (Spheniscus humboldti) was determined by using an inverse PCR method. We designed PCR primers to directly determine nucleotide sequences of PCR products from the MHC class I gene and to obtain recombinant clones for investigating the diversity of the MHC class I gene in Humboldt penguins. A total of 24 MHC class I allele sequences were obtained from 40 individuals. Polymorphisms were mainly found in exons 2 and 3, as expected from the nature of MHC class I genes in vertebrate species including birds and mammals. Phylogenetic analyses of MHC class I alleles have revealed that the Humboldt penguin is closely related to the Red Knot (Calidris canutus) belonging to Charadriiformes.