Spaln3: improvement in speed and accuracy of genome mapping and spliced alignment of protein query sequences.

MOTIVATION: Spaln is the earliest practical tool for self-sufficient genome mapping and spliced alignment of protein query sequences onto a mammalian-sized eukaryotic genomic sequence. However, its computational speed has become inadequate for the analysis of rapidly growing genomic and transcript sequence data. RESULTS: The dynamic programming calculation of Spaln has been sped up in two ways: (i) the introduction of the multi-intermediate unidirectional Hirschberg method and (ii) SIMD-based vectorization. The new version, Spaln3, is ∼7 times faster than the latest Spaln version 2, and its gene prediction accuracy is consistently higher than that of Miniprot. AVAILABILITY AND IMPLEMENTATION: https://github.com/ogotoh/spaln.

Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genomic features of Helicobacter pylori-naïve diffuse-type gastric cancer.

Helicobacter pylori (HP) is a major etiologic driver of diffuse-type gastric cancer (DGC). However, improvements in hygiene have led to an increase in the prevalence of HP-naïve DGC; that is, DGC that occurs independent of HP. Although multiple genomic cohort studies for gastric cancer have been conducted, including studies for DGC, distinctive genomic differences between HP-exposed and HP-naïve DGC remain largely unknown. Here, we employed exome and RNA sequencing with immunohistochemical analyses to perform binary comparisons between 36 HP-exposed and 27 HP-naïve DGCs from sporadic, early-stage, and intramucosal or submucosal tumor samples. Among the samples, 33 HP-exposed and 17 HP-naïve samples had been preserved as fresh-frozen samples. HP infection status was determined using stringent criteria. HP-exposed DGCs exhibited an increased single nucleotide variant burden (HP-exposed DGCs; 1.97 [0.48-7.19] and HP-naïve DGCs; 1.09 [0.38-3.68] per megabase; p = 0.0003) and a higher prevalence of chromosome arm-level aneuploidies (p < 0.0001). CDH1 was mutated at similar frequencies in both groups, whereas the RHOA-ARHGAP pathway misregulation was exclusive to HP-exposed DGCs (p = 0.0167). HP-exposed DGCs showed gains in chromosome arms 8p/8q (p < 0.0001), 7p (p = 0.0035), and 7q (p = 0.0354), and losses in 16q (p = 0.0167). Immunohistochemical analyses revealed a higher expression of intestinal markers such as CD10 (p < 0.0001) and CDX2 (p = 0.0002) and a lower expression of the gastric marker, MUC5AC (p = 0.0305) among HP-exposed DGCs. HP-naïve DGCs, on the other hand, had a purely gastric marker phenotype. This work reveals that HP-naïve and HP-exposed DGCs develop along different molecular pathways, which provide a basis for early detection strategies in high incidence settings. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genomic determinants impacting the clinical outcome of mogamulizumab treatment for adult T-cell leukemia/lymphoma.

In order to identify genomic biomarkers for the outcome of mogamulizumab-containing treatment, an integrated molecular analysis of adult T-cell leukemia/lymphoma (ATL) was conducted on 64 mogamulizumab-naïve patients. Among driver genes, CCR4 and CCR7 alterations were observed in 22% and 11% of the patients, respectively, both consisting of single nucleotide variants (SNV)/insertion-deletions (indels) in the C-terminus. Patients with CCR4 alterations or without CCR7 alterations exhibited a more favorable clinical response (complete response [CR] rate 93%, 13/14; P=0.024, and CR rate 71%, 40/56; P=0.036, respectively). Additionally, TP53, CD28, and CD274 alterations were identified in 35%, 16%, and 10% of the patients, respectively. TP53 alterations included SNV/indels or copy number variations (CNV) such as homozygous deletion; CD28 alterations included SNV, CNV such as amplification, or fusion; CD274 alterations included CNV such as amplification, or structural variants. Univariate analysis revealed that TP53, CD28 or CD274 alterations were associated with worse overall survival (OS) (hazard ratio [HR]: 2.330, 95% confidence interval [CI]: 1.183-4.589; HR: 3.191, 95% CI: 1.287- 7.911; HR: 3.301, 95% CI: 1.130-9.641, respectively) but that CCR4 alterations were associated with better OS (HR: 0.286, 95% CI: 0.087-0.933). Multivariate analysis indicated that in addition to performance status, TP53, CCR4 or CD274 alterations (HR: 2.467, 95% CI: 1.197-5.085; HR: 0.155, 95% CI: 0.031-0.778; HR: 14.393, 95% CI: 2.437-85.005, respectively) were independently and significantly associated with OS. The present study contributes to the establishment of precision medicine using mogamulizumab in ATL patients.

Immunogenic characteristics of microsatellite instability-low esophagogastric junction adenocarcinoma based on clinicopathological, molecular, immunological and survival analyses.

Microsatellite instability (MSI) is categorized by mutation frequency: high MSI (MSI-H), low MSI (MSI-L) and microsatellite stable (MSS). MSI-H tumors have a distinct immunogenic phenotype, with immunotherapies using checkpoint inhibitors already approved for the treatment of MSI-H gastroesophageal adenocarcinoma (GEA); this is not observed for MSI-L or MSS. Here, we tested the hypothesis that MSI-L tumors are also a distinct phenotype and potentially immunogenic. MSI-PCR assays (BAT25, BAT26, BAT40, D2S123, D5S346 and D17S250) were performed on 363 Epstein-Barr virus-negative, surgically resected esophagogastric junction (EGJ) adenocarcinoma samples. Tumors were characterized as MSI-H (≥2 markers), MSI-L (1 marker) or MSS (0 markers). CD8+ cell counts, PD-L1 and HER2 expression levels, TP53 mutations, epigenetic alterations and prognostic significance were also examined. All pathological and molecular experiments were conducted using serial, whole-tumor sections of chemo-naïve surgical specimens. MSI-H and MSI-L were assigned to 28 (7.7%) and 24 (6.6%) cases, respectively. Compared to MSS cases, MSI-L cases had significantly higher intratumoral CD8+ cell infiltration (P = .048) and favorable EGJ cancer-specific survival (multivariate hazard ratio = 0.35, 95% CI, 0.12-0.82; P = .012). MSI-L tumors were also significantly associated with TP53-truncating mutations as compared to MSI-H (P = .009) and MSS (P = .012) cases, and this trend was also observed in GEA data from The Cancer Genome Atlas (TCGA). Indel mutational burden among TCGA MSI-L tumors was significantly higher than that of MSS tumors (P = .016). These results suggest that MSI-L tumors may have a distinct tumor phenotype and be potentially immunogenic in EGJ adenocarcinoma.

Pathogenicity assessment of variants for breast cancer susceptibility genes based on BRCAness of tumor sample.

Genes involved in the homologous recombination repair pathway-as exemplified by BRCA1, BRCA2, PALB2, ATM, and CHEK2-are frequently associated with hereditary breast and ovarian cancer syndrome. Germline mutations in the loci of these genes with loss of heterozygosity or additional somatic truncation at the WT allele lead to the development of breast cancers with characteristic clinicopathological features and prominent genomic features of homologous recombination deficiency, otherwise referred to as "BRCAness." Although clinical genetic testing for these and other genes has increased the chances of identifying pathogenic variants, there has also been an increase in the prevalence of variants of uncertain significance, which poses a challenge to patient care because of the difficulties associated with making further clinical decisions. To overcome this challenge, we sought to develop a methodology to reclassify the pathogenicity of these unknown variants using statistical modeling of BRCAness. The model was developed with Lasso logistic regression by comparing 116 genomic attributes derived from 37 BRCA1/2 biallelic mutant and 32 homologous recombination-quiescent breast cancer exomes. The model showed 95.8% and 86.7% accuracies in the training cohort and The Cancer Genome Atlas validation cohort, respectively. Through application of the model for variant reclassification of homologous recombination-associated hereditary breast and ovarian cancer causal genes and further assessment with clinicopathological features, we finally identified one likely pathogenic and five likely benign variants. As such, the BRCAness model developed from the tumor exome was robust and provided a reasonable basis for variant reclassification.

T-cell complexity and density are associated with sensitivity to neoadjuvant chemoradiotherapy in patients with rectal cancer.

Emerging evidence suggests that an increased density of pre-treatment CD8+ tumor-infiltrating lymphocytes (TILs) is associated with good response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer. However, the significance of T-cell complexity in the clinical setting remains unknown. High-throughput T-cell receptor (TCR) ß sequencing was applied to quantify the TCR repertoire of pre-treatment biopsies from 67 patients with advanced rectal cancer receiving preoperative CRT. Diversity index was used to represent the complexity of the TCR repertoire in a tumor. Pre-treatment CD8+ TIL densities were assessed by immunohistochemistry. Changes in TCR repertoire before and after CRT were also analysed in 23 patients. Diversity indices were significantly higher for good responders than for non-responders (P = 0.031). The multivariate analysis revealed that both CD8+ TIL density and TCR diversity index were independently associated with good response to CRT (P < 0.001 and P = 0.049, respectively). Patients who were high for both CD8+ TIL density and TCR diversity (double-high) had markedly better responses to CRT than double-low patients (84.2% vs 16.7%, P < 0.0001). Larger changes in TCR repertoires before and after CRT were correlated with better recurrence-free survival (P = 0.027). The complexity and dynamic change in the TCR repertoire might serve as a useful indicator of response to CRT in combination with CD8+ TIL density in patients with rectal cancer.

Two Distinct Tumorigenic Processes in Endometrial Endometrioid Adenocarcinoma.

Endometrial endometrioid adenocarcinoma (EEA) is conventionally considered to be a single pathologic entity that develops through a hyperplasia-carcinoma sequence under the influence of estrogen. Previously, another EEA subtype was described and proposed to arise directly from normal endometrium. These conventional and de novo subtypes are designated groups 1 and 2, respectively. To identify the molecular mechanisms of these distinct tumorigenic processes, we conducted comprehensive integrated analyses of genomic data with hormonal status for group 1 paired carcinoma and hyperplasia and group 2 carcinoma samples. Although group 1 carcinomas mostly exhibited genomically stable characteristics and the activation of estrogen signaling, group 2 EEAs showed enriched hypermutator and CpG island methylator phenotypes. Pairwise comparisons of hyperplasia and carcinoma, along with time-course analyses of the hyperplasia-carcinoma sequence, revealed the acquisition of driver mutations in the evolutionary process of hyperplasia but not in the transition from hyperplasia to carcinoma. The current study confirms the existence of two different histopathologic programs during EEA development that harbor distinct molecular bases and demonstrates the biological relevance of these differential tumorigenic processes.

Genomic alterations in gynecological malignancies: histotype-associated driver mutations, molecular subtyping schemes, and tumorigenic mechanisms.

There are numerous histological subtypes (histotypes) of gynecological malignancies, with each histotype considered to largely reflect a feature of the "cell of origin," and to be tightly linked with the clinical behavior and biological phenotype of the tumor. The recent advances in massive parallel sequencing technologies have provided a more complete picture of the range of the genomic alterations that can persist within individual tumors, and have highlighted the types and frequencies of driver-gene mutations and molecular subtypes often associated with these histotypes. Several large-scale genomic cohorts, including the Cancer Genome Atlas (TCGA), have been used to characterize the genomic features of a range of gynecological malignancies, including high-grade serous ovarian carcinoma, uterine corpus endometrial carcinoma, uterine cervical carcinoma, and uterine carcinosarcoma. These datasets have also been pivotal in identifying clinically relevant molecular targets and biomarkers, and in the construction of molecular subtyping schemes. In addition, the recent widespread use of clinical sequencing for the more ubiquitous types of gynecological cancer has manifested in a series of large genomic datasets that have allowed the characterization of the genomes, driver mutations, and histotypes of even rare cancer types, with sufficient statistical power. Here, we review the field of gynecological cancer, and seek to describe the genomic features by histotype. We also will demonstrate how these are linked with clinicopathological attributes and highlight the potential tumorigenic mechanisms.

Immunogenomic landscape of gynecologic carcinosarcoma.

OBJECTIVE: Carcinosarcoma (CS) of the uterus or ovary is a rare, biphasic tumor comprising epithelial and mesenchymal elements, and exhibits more aggressive clinical features than its carcinoma counterpart. Four molecular subtypes of CS were recently established based on genomic aberration profiles (POLE, MSI, CNH, and CNL) and shown to be associated with multiple clinicopathological parameters, including patient outcomes. However, the role of the immune microenvironment in CS remains unclear. Here, we investigated the influence of the immune cells that infiltrate CS to better understand the immunological status of gynecological CS. METHODS: Tumor immune microenvironmental analyses on CS samples were performed using immune cell profiling with RNA-seq, transcriptomic subtyping with microenvironmental genes, and T-cell receptor repertoire assay. Carcinoma and sarcoma elements from CS samples were also assessed separately. RESULTS: Relying on estimations of tumor-infiltrating cell types from RNA-seq data, POLE and MSI (hypermutator) tumors showed an enrichment of M1 macrophages, plasma cells and CD8+ T cells, whereas CNH and CNL (non-hypermutator) tumors had high levels of M2 macrophages. Further subclassification by immune-related, non-cancer genes identified a fraction of tumors with distinct patient outcomes, particularly those with the CNH genomic aberration subtype. T-cell heterogeneity was independently correlated with prolonged progression-free survival. Differential analysis of carcinoma and sarcoma elements identified many shared mutations but there was little overlap in the T-cell receptor repertoire between the two elements. CONCLUSIONS: Tumor immune microenvironmental analyses could offer potential clinical utility in the stratification of gynecological CS above classification by genomic aberration subtype alone.

Modeling one thousand intron length distributions with fitild.

Motivation: Intron length distribution (ILD) is a specific feature of a genome that exhibits extensive species-specific variation. Whereas ILD contributes to up to 30% of the total information content for intron recognition in some species, rendering it an important component of computational gene prediction, very few studies have been conducted to quantitatively characterize ILDs of various species. Results: We developed a set of computer programs (fitild, compild, etc.) to build statistical models of ILDs and compare them with one another. Each ILD of more than 1000 genomes was fitted with fitild to a statistical model consisting of one, two, or three components of Frechet distributions. Several measures of distances between ILDs were calculated by compild. A theoretical model was presented to better understand the origin of the observed shape of an ILD. Availability and implementation: The C++ source codes are available at https://github.com/ogotoh/fitild.git/. Supplementary information: Supplementary data are available at Bioinformatics online.

A multiplex RNA quantification method to determine the absolute amounts of mRNA without reverse transcription.

We have developed a highly sensitive microarray-based method that determines the absolute amounts of mRNA in a total RNA sample in a multiplex manner without reverse transcription. This direct mRNA measurement promotes high-throughput testing and reduces bias in transcriptome analyses. Furthermore, quantification of the absolute amount of mRNA allows transcriptome analysis without common controls or additional, complicated normalization. The method, called Photo-DEAN, was validated using chemically synthesized RNAs of known quantities and mouse liver total RNA samples. We found that the absolute amounts of mRNA were successfully measured without the cDNA synthesis step, with a sensitivity of 15 zmol achieved in 7 h.

The apoptotic initiator caspase-8: its functional ubiquity and genetic diversity during animal evolution.

The caspases, a family of cysteine proteases, play multiple roles in apoptosis, inflammation, and cellular differentiation. Caspase-8 (Casp8), which was first identified in humans, functions as an initiator caspase in the apoptotic signaling mediated by cell-surface death receptors. To understand the evolution of function in the Casp8 protein family, casp8 orthologs were identified from a comprehensive range of vertebrates and invertebrates, including sponges and cnidarians, and characterized at both the gene and protein levels. Some introns have been conserved from cnidarians to mammals, but both losses and gains have also occurred; a new intron arose during teleost evolution, whereas in the ascidian Ciona intestinalis, the casp8 gene is intronless and is organized in an operon with a neighboring gene. Casp8 activities are near ubiquitous throughout the animal kingdom. Exogenous expression of a representative range of nonmammalian Casp8 proteins in cultured mammalian cells induced cell death, implying that these proteins possess proapoptotic activity. The cnidarian Casp8 proteins differ considerably from their bilaterian counterparts in terms of amino acid residues in the catalytic pocket, but display the same substrate specificity as human CASP8, highlighting the complexity of spatial structural interactions involved in enzymatic activity. Finally, it was confirmed that the interaction with an adaptor molecule, Fas-associated death domain protein, is also evolutionarily ancient. Thus, despite structural diversity and cooption to a variety of new functions, the ancient origins and near ubiquitous distribution of this activity across the animal kingdom emphasize the importance and utility of Casp8 as a central component of the metazoan molecular toolkit.

Assessment and refinement of eukaryotic gene structure prediction with gene-structure-aware multiple protein sequence alignment.

BACKGROUND: Accurate computational identification of eukaryotic gene organization is a long-standing problem. Despite the fundamental importance of precise annotation of genes encoded in newly sequenced genomes, the accuracy of predicted gene structures has not been critically evaluated, mostly due to the scarcity of proper assessment methods. RESULTS: We present a gene-structure-aware multiple sequence alignment method for gene prediction using amino acid sequences translated from homologous genes from many genomes. The approach provides rich information concerning the reliability of each predicted gene structure. We have also devised an iterative method that attempts to improve the structures of suspiciously predicted genes based on a spliced alignment algorithm using consensus sequences or reliable homologs as templates. Application of our methods to cytochrome P450 and ribosomal proteins from 47 plant genomes indicated that 50 ~ 60 % of the annotated gene structures are likely to contain some defects. Whereas more than half of the defect-containing genes may be intrinsically broken, i.e. they are pseudogenes or gene fragments, located in unfinished sequencing areas, or corresponding to non-productive isoforms, the defects found in a majority of the remaining gene candidates can be remedied by our iterative refinement method. CONCLUSIONS: Refinement of eukaryotic gene structures mediated by gene-structure-aware multiple protein sequence alignment is a useful strategy to dramatically improve the overall prediction quality of a set of homologous genes. Our method will be applicable to various families of protein-coding genes if their domain structures are evolutionarily stable. It is also feasible to apply our method to gene families from all kingdoms of life, not just plants.

Benchmarking spliced alignment programs including Spaln2, an extended version of Spaln that incorporates additional species-specific features.

Spliced alignment plays a central role in the precise identification of eukaryotic gene structures. Even though many spliced alignment programs have been developed, recent rapid progress in DNA sequencing technologies demands further improvements in software tools. Benchmarking algorithms under various conditions is an indispensable task for the development of better software; however, there is a dire lack of appropriate datasets usable for benchmarking spliced alignment programs. In this study, we have constructed two types of datasets: simulated sequence datasets and actual cross-species datasets. The datasets are designed to correspond to various real situations, i.e. divergent eukaryotic species, different types of reference sequences, and the wide divergence between query and target sequences. In addition, we have developed an extended version of our program Spaln, which incorporates two additional features to the scoring scheme of the original version, and examined this extended version, Spaln2, together with the original Spaln and other representative aligners based on our benchmark datasets. Although the effects of the modifications are not individually striking, Spaln2 is consistently most accurate and reasonably fast in most practical cases, especially for plants and fungi and for increasingly divergent pairs of target and query sequences.

High-risk pathogenic germline variants in blood relatives of BRCA1/2 negative probands.

BACKGROUND: Tailored, preventive cancer care requires the identification of pathogenic germline variants (PGVs) among potentially at-risk blood relatives (BRs). Cascade testing is carried out for BRs of probands who are positive for PGVs of an inherited cancer but not for negative probands. This study was conducted to examine the prevalence of PGVs for BRs of PGV-negative probands. METHODS: PGV prevalence was assessed for 682 BRs of 281 probands with BRCA1/BRCA2 wild-type hereditary breast and ovarian cancer (HBOC) syndrome. RESULTS: PGVs were discovered in 22 (45.8%) of the 48 BRs of the PGV-positive probands and in 14 (2.2%) of 634 BRs of the PGV-negative probands. Eleven PGVs on high-risk BRCA1, BRCA2, and TP53 genes were present only in BRs and not in the probands (probands vs BRs in Fisher exact test; p = 0.0104; odds ratio [OR] = 0.000 [0.000-0.5489 of 95% confidence interval]), partly due to the nature of the selection criteria. The enrichment of high-risk PGVs among BRs was also significant as compared with a non-cancer East Asian population (p = 0.0016; OR = 3.0791 [1.5521-5.6694]). PGV prevalence, risk class of gene, and genotype concordance were unaffected by the cancer history among BRs. CONCLUSION: These findings imply the necessity to construct a novel testing scheme to complement cascade testing.

Large-scale motif discovery using DNA Gray code and equiprobable oligomers.

MOTIVATION: How to find motifs from genome-scale functional sequences, such as all the promoters in a genome, is a challenging problem. Word-based methods count the occurrences of oligomers to detect excessively represented ones. This approach is known to be fast and accurate compared with other methods. However, two problems have hampered the application of such methods to large-scale data. One is the computational cost necessary for clustering similar oligomers, and the other is the bias in the frequency of fixed-length oligomers, which complicates the detection of significant words. RESULTS: We introduce a method that uses a DNA Gray code and equiprobable oligomers, which solve the clustering problem and the oligomer bias, respectively. Our method can analyze 18 000 sequences of ~1 kbp long in 30 s. We also show that the accuracy of our method is superior to that of a leading method, especially for large-scale data and small fractions of motif-containing sequences. AVAILABILITY: The online and stand-alone versions of the application, named Hegma, are available at our website: http://www.genome.ist.i.kyoto-u.ac.jp/~ichinose/hegma/ CONTACT: ichinose@i.kyoto-u.ac.jp; o.gotoh@i.kyoto-u.ac.jp

Multiplex cDNA quantification method that facilitates the standardization of gene expression data.

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h.

Transcriptomic Analyses of Pretreatment Tumor Biopsy Samples, Response to Neoadjuvant Chemoradiotherapy, and Survival in Patients With Advanced Rectal Cancer.

Importance: Neoadjuvant chemoradiotherapy (CRT) is the standard of care for advanced rectal cancer. Yet, estimating response to CRT remains an unmet clinical challenge. Objective: To investigate and better understand the transcriptomic factors associated with response to neoadjuvant CRT and survival in patients with advanced rectal cancer. Design, Setting, and Participants: A single-center, retrospective, case series was conducted at a comprehensive cancer center. Pretreatment biopsies from 298 patients with rectal cancer who were later treated with neoadjuvant CRT between April 1, 2004, and September 30, 2020, were analyzed by RNA sequencing. Data analysis was performed from July 1, 2021, to May 31, 2022. Exposures: Chemoradiotherapy followed by total mesorectal excision or watch-and-wait management. Main Outcomes and Measures: Transcriptional subtyping was performed by consensus molecular subtype (CMS) classification. Immune cell infiltration was assessed using microenvironment cell populations-counter (MCP-counter) scores and single-sample gene set enrichment analysis (ssGSEA). Patients with surgical specimens of tumor regression grade 3 to 4 or whose care was managed by the watch-and-wait approach for more than 3 years were defined as good responders. Results: Of the 298 patients in the study, 205 patients (68.8%) were men, and the median age was 61 (IQR, 52-67) years. Patients classified as CMS1 (6.4%) had a significantly higher rate of good response, albeit survival was comparable among the 4 subtypes. Good responders exhibited an enrichment in various immune-related pathways, as determined by ssGSEA. Microenvironment cell populations-counter scores for cytotoxic lymphocytes were significantly higher for good responders than nonresponders (median, 0.76 [IQR, 0.53-1.01] vs 0.58 [IQR, 0.43-0.83]; P < .001). Cytotoxic lymphocyte MCP-counter score was independently associated with response to CRT, as determined in the multivariable analysis (odds ratio, 3.81; 95% CI, 1.82-7.97; P < .001). Multivariable Cox proportional hazards regression analysis, including postoperative pathologic factors, revealed the cytotoxic lymphocyte MCP-counter score to be independently associated with recurrence-free survival (hazard ratio [HR], 0.38; 95% CI, 0.16-0.92; P = .03) and overall survival (HR, 0.16; 95% CI, 0.03-0.83; P = .03). Conclusions and Relevance: In this case series of patients with rectal cancer treated with neoadjuvant CRT, the cytotoxic lymphocyte score in pretreatment biopsy samples, as computed by RNA sequencing, was associated with response to CRT and survival. This finding suggests that the cytotoxic lymphocyte score might serve as a biomarker in personalized multimodal rectal cancer treatment.

Evolution of cytochrome p450 genes from the viewpoint of genome informatics.

Cytochrome P450 (CYP) constitutes a large gene superfamily descended from a single common ancestor. CYP genes are widely distributed in all domains of life from bacteria, archaea, and viruses to higher plants and animals. Because of their monophyletic nature, all CYP genes may be hierarchically classified at several distinct levels based on similarity of the protein amino acid sequences. A five-level classification (class, group, clan, family, and subfamily) is reasonably stable and useful for conceptual categorization of CYP genes. With a few exceptions, genes in a clan are specific to a kingdom or phylum, whereas cross-kingdom genes may belong to the same group, indicating an ancient origin of CYP diversification. CYP proteins are often functionally categorized into catalysts of "endogenous," "secondary," and "xenobiotic" compounds according to their substrate specificities. It was once postulated that xenobiotic-metabolizing enzymes were derived from an endogenous substrate-catalyzing enzyme. Although functional flow from endogenous to xenobiotic substrates occurred, recent evidence from a wide range of genomic analyses has indicated that the opposite is the more dominant stream. Expression of most vertebrate CYP genes is regulated by internal and external stimuli through transcription factors in the nuclear receptor family and bHLH-PAS family. Some aspects of cooperative evolution between transcriptional regulators and their target genes are briefly reviewed.