Monitoring of cancer patients via next-generation sequencing of patient-derived circulating tumor cells and tumor DNA.

Liquid biopsy of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) is gaining attention as a method for real-time monitoring in cancer patients. Conventional methods based upon epithelial cell adhesion molecule (EpCAM) expression have a risk of missing the most aggressive CTC subpopulations due to epithelial-mesenchymal transition and may, thus, underestimate the total number of actual CTC present in the bloodstream. Techniques utilizing a label-free inertial microfluidics approach (LFIMA) enable efficient capture of CTC without the need for EpCAM expression. In this study, we optimized a method for analyzing genetic alterations using next-generation sequencing (NGS) of extracted ctDNA and CTC enriched using an LFIMA as a first-phase examination of 30 patients with head and neck cancer, esophageal cancer, gastric cancer and colorectal cancer (CRC). Seven patients with advanced CRC were enrolled in the second-phase examination to monitor the emergence of alterations occurring during treatment with epidermal growth factor receptor (EGFR)-specific antibodies. Using LFIMA, we effectively captured CTC (median number of CTC, 14.5 cells/mL) from several types of cancer and detected missense mutations via NGS of CTC and ctDNA. We also detected time-dependent genetic alterations that appeared during anti-EGFR therapy in CTC and ctDNA from CRC patients. The results of NGS analyses indicated that alterations in the genomic profile revealed by the liquid biopsy could be expanded by using a combination of assays with CTC and ctDNA. The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (ID: UMIN000014095).

Mucinous micropapillary pattern in lung adenocarcinomas: a unique histology with genetic correlates.

AIMS: In lung adenocarcinoma (ADC), micropapillary carcinomas (MPCs) are associated with poor prognosis because these tumours exhibit higher metastatic potential. Despite this, there are no studies investigating the differences between mucinous and non-mucinous MPC. METHODS AND RESULTS: We evaluated the proportion of micropapillary components in lung ADCs, and compared the differences with respect to the presence or absence of associated mucin. Tumour specimens from 694 patients with consecutively resected primary lung ADC were reviewed, and 37 cases of invasive mucinous ADCs were excluded. A significant (≥5%) micropapillary component was noted in 320 (48.7%) of 657 evaluable cases. When the cases with micropapillary component were divided into 67 (20.9%) mucinous and 253 (79.1%) non-mucinous subtypes, tumours with mucinous micropapillary component exhibited significantly more aggressive pathological features, a higher proportion of HER2 mutations (P = 0.002) and ALK rearrangements (P < 0.001), and a lower proportion of EGFR mutations (P = 0.038) compared to those with a non-mucinous micropapillary component. In survival analyses, mucinous MPC tended to be more aggressive compared with non-mucinous MPC, but its prognostic value was not statistically significant (P = 0.076). CONCLUSIONS: Mucinous micropapillary pattern is an under-recognized unique growth associated significantly with HER2 mutation and ALK rearrangement.

[Liquid Biopsy: Challenges and Possibilities Regarding Its Clinical Application.]

Cell-free DNA is "Fragmented DNA found in circulation in the Cell-free component of whole blood". Cell-free DNA derived from tumors is expressed as circulating tumor DNA. Examination of circulating tu- mor DNA for genetic alterations present in the tumor tissue is defined as liquid biopsy. Currently in the cancer field, Cell-free DNA or CTC (Circulating tumor cells) is the main target of "Liquid Biopsy". Acquir- ing Cell-free DNA or CTC presents little challenge because of the recent technological developments. However, we need to improve the efficiency of CTC retrieval, and we also need to establish how to culture the retrieved CTCs. For clinical applications, PGx (Pharmacogenomics) and PGt (Pharmacogenetics) fol- lowing NGS (Next Generation Sequencing) are attractive areas for new and future applications. The intro- duction of "Liquid Biopsy" to the area of clinical trials is already in progress. As an expert group, members of the JSLM (Japanese Society of Laboratory Medicine) need to indicate the presence of quality control or quality management in the area of "Liquid Biopsy". Besides these quality issues, we, as clinical pathologists, need to think about harmonizing our expertise with surgical pathologists, who have historically handled clas- sical biopsies of solid samples. The field of "Liquid Biopsy" has marked potential; however, we need to overcome various obstacles to realize this.

[Breakthrough Technologies: Liquid Biopsy -Chairmen's Introductory Remarks.]

To date, liquid biopsies have generated much interest as they involve minimally invasive blood tests, pro- vide an ongoing picture of a patient's cancer, and offer valuable insight into the best treatment. Liquid biop- sies detect circulating tumor cells (CTCs), fragments of tumor DNA and exosomes that are shed into the blood from the primary tumor and from metastatic sites. Liquid biopsies offer what tissue biopsies cannot due to risks to the patients and costs. Liquid biopsies allow the monitoring of genomic changes in tumors for the diagnosis of early and recurrent cancer and drug effects. In the future, instead of extensive imaging and invasive tissue biopsies, liquid biopsies could be used to guide cancer treatment decisions and even screen for tumors that are not vet visible on imaging.

[Harmonization of TSH Measurements.]

The measured concentration of thyroid stimulating hormone (TSH) differs depending on the reagents used. Harmonization of TSH is crucial because the decision limits are described in current clinical practice guide- lines as absolute values, e.g. 2.5 mIU/L in early pregnancy. In this study, we tried to harmonize the report- ed concentrations of TSH using the all-procedure trimmed mean. TSH was measured in 146 serum samples, with values ranging from 0.01 to 18.8 mIU/L, using 4 immunoassays. The concentration of TSH was highest with E test TOSOH and lowest with LUMIPULSE. The concentrations with each reagent were recalculated with the following formulas: E test TOSOH 0.855x-0.014; ECLusys 0.993x+0.079; ARCHITECT 1.041x- 0.010; and LUMIPULSE 1.096x-0.015. Recalculation eliminated the between-assay discrepancy. These formulas may be used until harmonization of TSH is achieved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC).

Association between serum ligands and the skin toxicity of anti-epidermal growth factor receptor antibody in metastatic colorectal cancer.

Skin toxicity is a known clinical signature used to predict the prognosis of anti-epidermal growth factor receptor (EGFR) antibody treatment in metastatic colorectal cancer (mCRC). There are no biological markers to predict skin toxicity before anti-EGFR antibody treatment in mCRC patients. Between August 2008 and August 2011, pretreatment serum samples were obtained from KRAS wild-type (WT) patients who received anti-EGFR antibody treatment. Serum levels of ligands were measured by ELISA. A total of 103 KRAS WT patients were enrolled in the study. Progression-free survival and overall survival of patients with a high grade (grade 2-3) of skin toxicity were significantly longer than those with a low grade (grade 0-1) of skin toxicity (median progression-free survival, 6.4 months vs 2.4 months, P < 0.001; median overall survival, 14.6 months vs 7.1 months, P = 0.006). There were significant differences in distribution of serum levels of epiregulin (EREG), amphiregulin (AREG), and hepatocyte growth factor (HGF) between groups of low/high grade of skin toxicity (P < 0.048, P < 0.012, P < 0.012, respectively). In addition, serum levels of HGF, EREG, and AREG were inversely proportional to grades of skin toxicity as determined by the Cochran-Armitage test (P = 0.019, P = 0.047, P = 0.021, respectively). Our study indicated that serum levels such as HGF, EREG, and AREG may be significant markers to predict the grade of skin toxicity and the prognosis of anti-EGFR antibody treatment, which contribute to improvement of the management of skin toxicity and survival time in mCRC patients.

Impact of KRAS mutation on response and outcome of patients with stage III non-squamous non-small cell lung cancer.

The frequency and clinical profile of patients with stage III non-small cell lung cancer harboring KRAS mutations have not yet been well documented. Here, we analyzed hotspot KRAS mutations using high-resolution melting analyses in tumor specimens from patients who received chemoradiotherapy between January 2001 and December 2010 at the National Cancer Center Hospital. The associations between the presence of KRAS mutations and the response rate, relapse-free survival, first relapse sites, survival post-progression and overall survival were investigated. A total of 274 non-squamous non-small cell lung cancer patients received chemoradiotherapy at our hospital. After excluding 121 patients for whom tumor specimens were not available and 34 patients with EGFR mutations, the remaining 119 patients were included in the analysis. KRAS mutations were found at a frequency of 13%. Patients with KRAS mutations had a shorter median relapse-free survival (6.1 vs 10.9 months) and a lower response rate (63% vs 81%). As for the first relapse site, patients with KRAS mutations had fewer local relapses (8% vs 23%) and more brain metastases (46% vs 12%). After disease progression, patients with KRAS mutations had a significantly shorter median survival post-progression (2.5 vs 7.3 months, P = 0.028) and median overall survival (15.1 vs 29.1 months, P = 0.022). Our results suggested that KRAS mutation could be associated with a reduced efficacy of chemoradiotherapy and a shortened survival time.

Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma is an intractable cancer, with limited therapeutic options, in which the molecular mechanisms underlying tumor development remain poorly understood. Identification of a novel driver oncogene and applying it to targeted therapies for molecularly defined cancers might lead to improvements in the outcome of patients. We performed massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients without KRAS/BRAF/ROS1 alterations and identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1. In reverse-transcriptase polymerase chain reaction (RT-PCR) screening, the FGFR2 fusion was detected in nine patients with cholangiocarcinoma (9/102), exclusively in the intrahepatic subtype (9/66, 13.6%), rarely in colorectal (1/149) and hepatocellular carcinoma (1/96), and none in gastric cancer (0/212). The rearrangements were mutually exclusive with KRAS/BRAF mutations. Expression of the fusion kinases in NIH3T3 cells activated MAPK and conferred anchorage-independent growth and in vivo tumorigenesis of subcutaneous transplanted cells in immune-compromised mice. This transforming ability was attributable to its kinase activity. Treatment with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074 effectively suppressed transformation. CONCLUSION: FGFR2 fusions occur in 13.6% of intrahepatic cholangiocarcinoma. The expression pattern of these fusions in association with sensitivity to FGFR inhibitors warrant a new molecular classification of cholangiocarcinoma and suggest a new therapeutic approach to the disease.

Biomarker expression and druggable gene alterations for development of an appropriate therapeutic protocol for pulmonary adenosquamous carcinoma.

AIMS: Pulmonary adenosquamous carcinoma (ASC) is more aggressive than adenocarcinoma (AC) and squamous cell carcinoma (SCC). The genetic features and biomarkers of ASC are not well known. Here, we attempted to identify potential therapeutic markers for ASC. METHODS AND RESULTS: Surgically resected ASC samples from 65 patients were analysed. We examined the expression of ß III-tubulin, thymidylate synthase, breast cancer susceptibility gene 1 and ribonucleotide reductase M1 (RRM1); identified mutations in epidermal growth factor receptor (EGFR), KRAS, BRAF and HER2; and detected ALK, ROS1 and RET rearrangements. Gene amplification and expression of EGFR, human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor-1 and MET were also examined. ß III-Tubulin showed the highest expression (P = 0.002), and its expression was more frequent in the AC than in the SCC component (P = 0.013). RRM1 expression was more frequent in the SCC component (P = 0.046). EGFR and KRAS mutations were detected in both components (21.5 and 10.9%, respectively). ALK and ROS1 rearrangements and MET amplification were detected in both components in one (1.5%) case. CONCLUSIONS: In ASC, drug response-specific gene alterations could occur in both AC and SCC components, suggesting that patients with confirmed or suspected ASC should undergo further testing for driver gene analyses.

[Liquid Biopsy and Laboratory Medicine].

Recent progress in cancer biology has revealed the fact that molecular profiles of primary and metastatic cancer are not necessarily the same. Furthermore, evidence of intra-tumor heterogeneity has been disclosed repeatedly. In addition to these, acquiring resistances to chemoradiation therapy is far more rapid than typical predictions. Under these circumstances, physicians are realizing that one biopsy is not enough to predict the direction of cancer progression or extension. Repeated biopsy was proposed in this context. For "re-biopsy", acquiring blood is much easier compared to regular biopsies of acquiring body tissues. Therefore, CTC or Cell-free DNA is one of the hot topics in clinical and molecular diagnostic fields. The term "liquid biopsy" is used to include these two materials. We utilized a CTC isolation device based on microfluidic principles. Procedures for the extraction of DNA from plasma (Cell-free DNA) is also available. Based on this background, we performed a feasibility study of NGS (Next Generation Sequencing) by analyzing materials from advanced gastrointestinal cancer patients. We have successfully acquired NGS results using these liquid biopsies. We have also investigated the possibility of storing CTCs by evaluating procedures after cytospin using H1975 cells with various fixation conditions under a DIC microscope examination. Because of the paucity of the number of isolated CTCs, H1975 cells were used for this purpose. After cytospin, 95% ETOH and then -80 degrees C storage provided the best results. Attempts at not only NGS but also storage in this sequence of studies have opened new fields of liquid biopsy in clinical laboratories.

[Quality of Human Liquid Specimens Appropriate for Laboratory Tests and Analyses--Chairmen's Introductory Remarks].

One of the major concerns for laboratory medicine communities is the quality of human specimens. Thanks to previous efforts in laboratory medicine field, routine quality control is almost guaranteed. Evidence is continually accumulating, and this evidence keeps the analytical levels in the clinical laboratory high. There is still, however, insufficient scientific evidence when handling and storing routine specimens, especially liquid specimens. Data based on poor quality specimens affects designs of patients' diagnostic and therapeutic strategies. Furthermore, such poor data also affect the outcomes of clinical trials. The only solution to avoid this situation is to acquire specimens of favorable quality. In this symposium, six experts involved in basic science to the clinical frontline gave unique presentations to share their expertise with us, and their concerns about specimen quality.

[Alternative Splicing Detection as Biomarker Candidates for Cancer Diagnosis and Treatment with Establishment of Clinical Biobank in Chiba University].

Alternative splicing is a fundamental process of gene regulation that contributes to protein diversity, a common phenomenon in the mammalian genome. Alternative splicing events not only happen in the normal gene regulation process, but are also closely related to certain diseases, including cancer. In this review, we briefly demonstrate the proof of concept (POC) of the relationship between alternative splicing and DNA damage, and describe the associations among alternative splicing and cancer pathogenesis, DNA damage, and gastrointestinal cancers. We discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. FUSE-binding protein (FBP) -interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2), upregulates c-myc transcription by inactivating wild-type FIR. FIR+/- mice exhibited marked c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Because the single knockout of TP53 generates thymic lymphoma, FIR+/-TP53-/- mice developed T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion and showed a poor prognosis. After describing the POC of alternative splicing of FIR in DNA damage and carcinogenesis, clinical application for cancer diagnosis and treatment by FIR/FIRΔexon2 was briefly summarized. Chiba University has prepared a biobank to support studies to develop biomarker detection, molecular diagnosis, and "Omics" research. In conclusion, alternative splicing of FIR, generating FIRΔexon2, potentially contributes to not only colorectal carcinogenesis, but also leukemogenesis, and a better understanding of the role and mechanism of alternative splicing in tumorigenesis may reveal new directions for cancer biomarker detection.

[Feasibility Study for Storage and Re-Utilization of Circulating Tumor Cells (CTCs)].

BACKGROUND: In this era of precision medicine, monitoring patients requires not only real time but also longitudinal sequence of samples at various time points. Based on this background, we focused on conditioned circumstances on fixation and storage for re-utilization of CTCs. MATERIALS: Instead of actual CTCs, Cell line (H1975) derived from lung cancer was used because of their scarceness of CTCs. METHODS: These cells were put on a slide by using an auto-smear device. The slides were evaluated under various centrifuge forces, fixations for the following storages. RESULTS AND DISCUSSION: The study indicated that 800 rpm for 1 min centrifuge and fixation by 95% ETOH was excellent. Further at least 5 cells per 1 mL cell solution were required for the following procedures including Fluorescence in situ hybridization (FISH) analysis. This study provides insights of new platform for evaluation of CTCs not only real time but also longitudinal sequence at various time points.

Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.

The recent discovery and characterization of an oncogenic ROS1 gene fusion in a subset of lung cancers has raised significant clinical interest because small molecule inhibitors may be effective to these tumors. As lung cancers with ROS1 rearrangements comprise only 1-3% of lung adenocarcinomas, patients with such tumors must be identified to gain optimal benefit from molecular therapy. Recently, immunohistochemical analyses using a novel anti-ROS1 rabbit monoclonal antibody (D4D6) have shown promise for accurate identification of ROS1-rearranged cancers. To validate this finding, we compared the immunostaining results of tissue microarrays (TMAs) containing 17 ROS1-rearranged and 253 ROS1-non-rearranged lung carcinomas. All 17 ROS1-rearranged cancers showed ROS1 immunoreactivity mostly in a diffuse and moderate-to-strong manner with an H-score range of 5-300 (median, 260). In contrast, 69% of ROS1-non-rearranged cancers lacked detectable immunoreactivity, whereas the remaining 31% showed reactivity mainly in a weak or focal manner. The H-score for the entire ROS1-non-rearranged group ranged from 0 to 240 (median, 0). The difference in H-score between the two cohorts was statistically significant, and the H-score cutoff (≥150) allowed optimal discrimination (94% sensitivity and 98% specificity). Similar but slightly less-specific performance was achieved using the extent of diffuse (≥75%) staining or ≥2+ staining intensity as cutoffs. CD74-ROS1 and EZR-ROS1 fusions were significantly associated with at least focal globular immunoreactivity and plasma membranous accentuation, respectively, and these patterns were specific to ROS1-rearranged cases. Although full-length ROS1 is expressed in some ROS1-non-rearranged cases, we showed that establishment of an optimal set of interpretative criteria makes ROS1 immunohistochemistry a valuable method to rapidly and accurately screen lung cancer patients for appropriate molecular therapy.

Soluble interleukin-6 receptor is a serum biomarker for the response of esophageal carcinoma to neoadjuvant chemoradiotherapy.

Preoperative chemoradiotherapy has been shown to improve the outcome of patients with esophageal cancer, but because response to this therapy varies, it is desirable to identify in advance individuals who would be unlikely to benefit, in order to avoid unnecessary adverse drug effects. The serum profiles of 84 cytokines and related proteins were determined in 37 patients with esophageal squamous cell carcinoma who received identical neoadjuvant preoperative chemoradiotherapy regimens and underwent surgical resection. Histological response to this therapy was assessed in surgically resected specimens. The serum soluble interleukin-6 receptor (sIL6R) level was significantly higher in 30 patients who failed to achieve a histological complete response (P = 0.005). Multivariate analysis revealed that the increased level of sIL6R was one of several significant independent predictors of an unfavorable outcome (hazard ratio, 2.87; P = 0.017). The increased level of this cytokine in patients who did not obtain a complete response was reproducibly observed in an independent cohort of 34 patients. Esophageal squamous cell carcinoma patients with an increased serum level of sIL6R are predicted to respond poorly to preoperative chemoradiotherapy, therefore, their exclusion from this treatment may be considered. Persistent systemic inflammation is implicated as a possible mechanism of resistance to this therapy.

Anaplastic lymphoma kinase status in rhabdomyosarcomas.

Rhabdomyosarcoma is a rare soft tissue sarcoma that typically affects children, adolescents, and young adults. Despite treatment via a multidisciplinary approach, the prognosis of advance-stage rhabdomyosarcomas remains poor, and a new treatment strategy is needed. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is a potential target for specific inhibitors. In this study, we investigated 116 rhabdomyosarcomas using a polymer-based ALK immunostaining method and correlated the results with clinicopathological parameters. In addition, we examined ALK status using dual-color fluorescence in situ hybridization, PCR, and sequencing. In immunohistochemical analysis, ALK was detected in 2 (6%) of 33 embryonal rhabdomyosarcomas, 42 (69%) of 61 alveolar rhabdomyosarcomas, and 0 (0%) of 22 other subtypes, including pleomorphic, adult-spindle-cell/sclerosing, and epithelioid variants. Compared with ALK-negative alveolar rhabdomyosarcomas, ALK-positive ones are presented with metastatic spread more frequently and showed a greater extent of myogenin reactivity. Overall survival was not associated with ALK expression. FOXO1 rearrangement was significantly associated with ALK immunoreactivity. The median ALK copy number was greater in ALK-positive tumors than in ALK-negative tumors. Most (93%) cases tested showed no selective increase in the ALK gene dosage. ALK selective amplification and low-level selective gain were noted in one and three cases, respectively. Further, a high-polysomy pattern (≥4 ALK copies in ≥40% of cells) was observed in seven cases. A significant increase in the ALK copy number was exclusive to the ALK-immunopositive cohort, but it was uncommon, accounting for only 30% of the 37 ALK-positive rhabdomyosarcomas. ALK gene rearrangement was not observed in either cohort, while an ALK somatic mutation (I1277T) was found in one ALK-negative embryonal case. Although it remains controversial whether ALK expression without gene rearrangement is therapeutically relevant, this comprehensive analysis may help future studies on the utility of ALK-targeted therapy for patients with rhabdomyosarcoma.

Ewing sarcoma arising after treatment of diffuse large B-cell lymphoma.

We report the case of a patient in whom the diagnosis of Ewing sarcoma arising from a soft tissue was made after successful treatment of diffuse large B-cell lymphoma. A 65-year-old woman presented with a rapidly growing mass in her left scapular region 8 years after successful chemotherapy with the cyclophosphamide, hydroxydaunomycin hydrochloride, vincristine, prednisolone regimen for diffuse large B-cell lymphoma. Computed tomographic examination and magnetic resonance imaging of the thorax revealed an intramuscular tumour measuring 40 mm in size in the left scapular region. Histopathological examination of an open biopsy specimen revealed a small round cell tumour that showed positive staining for CD99. Fluorescence in situ hybridization showed a split signal by a break-apart probe for the EWS gene in chromosome 22q12. Reverse transcriptase-polymerase chain reaction confirmed the expression of EWS-FLI1 fusion transcripts. Based on these findings, the patient was diagnosed as having secondary Ewing sarcoma. Despite adjuvant chemotherapy, however, she died of pulmonary metastases 2 years after the diagnosis of Ewing sarcoma. Therapy-related haematological malignancies with balanced translocations have been reported previously. A mechanism similar to that underlying the development of secondary malignancy might explain the occurrence of this solid cancer.

Improving Public Trust in Biobanking: Roundtable Discussions from the 2021 ISBER Annual Meeting.

Biobanking is a relatively newly recognized and innovative branch of science, which includes the collection of samples and associated data from hospitals, diagnostic centers, and voluntary donations for biomedical and environmental research. It involves diverse stakeholders at the junction of society, science, ethics, law, and politics. A key element in the success of a biobank is the trust and support of public donors, clinicians, and scientists. To achieve trust, it is important to implement strategies that can increase biobank awareness in common people, and different types of communities. Biobank laws and regulations and transparent governance by the biobanks are also crucial to achieving public trust.

Comparison of outcomes after allogeneic hematopoietic stem cell transplantation in patients with follicular lymphoma, diffuse large B-cell lymphoma associated with follicular lymphoma, or de novo diffuse large B-cell lymphoma.

The outcome after allogeneic hematopoietic stem cell transplantation (allo-HCT) for diffuse large B-cell lymphoma (DLBCL) associated with follicular lymphoma (FL), which includes DLBCL with pre- or co-existing FL, remains controversial, and few previous reports have compared the outcomes after allo-HCT for FL, DLBCL associated with FL, and de novo DLBCL. We retrospectively analyzed 97 consecutive patients with FL (n = 46), DLBCL associated with FL (n = 22), or de novo DLBCL (n = 29) who received allo-HCT at our institute between 2000 and 2010. With a median follow-up of 53 months, the 5-year overall survival (OS) and progression-free survival (PFS) were, respectively, 77% and 70% for FL, 62% and 57% for DLBCL associated with FL, and 26% and 23% for de novo DLBCL. The 5-year cumulative incidences of non-relapse mortality and disease progression/relapse were, respectively, 16% and 15% for FL, 19% and 24% for DLBCL associated with FL, and 36% and 41% for de novo DLBCL. By a multivariate analysis, the OS and PFS for DLBCL associated with FL were significantly better than those for de novo DLBCL, whereas they were not significantly different from those for FL. These results suggest that allo-HCT may be a promising option for patients with not only advanced FL but also DLBCL associated with FL.