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Purpose: Tumor-infiltrating B lymphocytes (TIL-B) have demonstrated prognostic and predictive significance in solid cancers. In this study, we aimed to distinguish TIL-Bs from malignant B-cells in diffuse large B-cell lymphoma (DLBCL) and determine the clinical and biological significance., Experimental Design: A total of 269 patients with de novo DLBCL from the International DLBCL R-CHOP Consortium Program were studied. Ultra-deep sequencing of the immunoglobulin genes was performed to determine B-cell clonotypes. The frequencies and numbers of TIL-B clonotypes in individual repertoires were correlated with patient survival, gene expression profiling (GEP) data, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex IHC at single-cell resolution., Results: TIL-B abundance, evaluated by frequencies of normal B-cell clonotypes in the immunoglobulin repertoires, remarkably showed positive associations with significantly better survival of patients in our sequenced cohorts. DLBCLs with high versus low TIL-B abundance displayed distinct GEP signatures, increased pre-memory B-cell state and naïve CD4 T-cell state fractions, and higher CD4+ T-cell infiltration. TIL-B frequency, as a new biomarker in DLBCL, outperformed the germinal center (GC) B-cell-like/activated B-cell-like classification and TIL-T frequency. The identified TIL-B-high GEP signature, including genes upregulated during T-dependent B-cell activation and those highly expressed in normal GC B cells and T cells, showed significant favorable prognostic effects in several external validation cohorts., Conclusions: TIL-B frequency is a significant prognostic factor in DLBCL and plays a crucial role in antitumor immune responses. This study provides novel insights into the prognostic determinants in DLBCL and TIL-B functions with important therapeutic implications., (©2023 American Association for Cancer Research.)

Purpose: Diffuse large B-cell lymphoma (DLBCL) is molecularly and clinically heterogeneous, and can be subtyped according to genetic alterations, cell-of-origin, or microenvironmental signatures using high-throughput genomic data at the DNA or RNA level. Although high-throughput proteomic profiling has not been available for DLBCL subtyping, MYC/BCL2 protein double expression (DE) is an established prognostic biomarker in DLBCL. The purpose of this study is to reveal the relative prognostic roles of DLBCL genetic, phenotypic, and microenvironmental biomarkers., Experimental Design: We performed targeted next-generation sequencing; IHC for MYC, BCL2, and FN1; and fluorescent multiplex IHC for microenvironmental markers in a large cohort of DLBCL. We performed correlative and prognostic analyses within and across DLBCL genetic subtypes and MYC/BCL2 double expressors., Results: We found that MYC/BCL2 double-high-expression (DhE) had significant adverse prognostic impact within the EZB genetic subtype and LymphGen-unclassified DLBCL cases but not within MCD and ST2 genetic subtypes. Conversely, KMT2D mutations significantly stratified DhE but not non-DhE DLBCL. T-cell infiltration showed favorable prognostic effects within BN2, MCD, and DhE but unfavorable effects within ST2 and LymphGen-unclassified cases. FN1 and PD-1-high expression had significant adverse prognostic effects within multiple DLBCL genetic/phenotypic subgroups. The prognostic effects of DhE and immune biomarkers within DLBCL genetic subtypes were independent although DhE and high Ki-67 were significantly associated with lower T-cell infiltration in LymphGen-unclassified cases., Conclusions: Together, these results demonstrated independent and additive prognostic effects of phenotypic MYC/BCL2 and microenvironment biomarkers and genetic subtyping in DLBCL prognostication, important for improving DLBCL classification and identifying prognostic determinants and therapeutic targets., (©2022 American Association for Cancer Research.)

Purpose: MYC is a critical driver oncogene in many cancers, and its deregulation in the forms of translocation and overexpression has been implicated in lymphomagenesis and progression of diffuse large B-cell lymphoma (DLBCL). The MYC mutational profile and its roles in DLBCL are unknown. This study aims to determine the spectrum of MYC mutations in a large group of patients with DLBCL, and to evaluate the clinical significance of MYC mutations in patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy., Experimental Design: We identified MYC mutations in 750 patients with DLBCL using Sanger sequencing and evaluated the prognostic significance in 602 R-CHOP-treated patients., Results: The frequency of MYC mutations was 33.3% at the DNA level (mutations in either the coding sequence or the untranslated regions) and 16.1% at the protein level (nonsynonymous mutations). Most of the nonsynonymous mutations correlated with better survival outcomes; in contrast, T58 and F138 mutations (which were associated with MYC rearrangements), as well as several mutations occurred at the 3' untranslated region, correlated with significantly worse survival outcomes. However, these mutations occurred infrequently (only in approximately 2% of DLBCL). A germline SNP encoding the Myc-N11S variant (observed in 6.5% of the study cohort) was associated with significantly better patient survival, and resulted in reduced tumorigenecity in mouse xenografts., Conclusions: Various types of MYC gene mutations are present in DLBCL and show different impact on Myc function and clinical outcomes. Unlike MYC gene translocations and overexpression, most MYC gene mutations may not have a role in driving lymphomagenesis. Clin Cancer Res; 22(14); 3593-605. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: Activated signal transducer and activator of transcription 3 (STAT3) regulates tumor growth, invasion, cell proliferation, angiogenesis, immune response, and survival. Data regarding expression of phosphorylated (activated) STAT3 in diffuse large B-cell lymphoma (DLBCL) and the impact of phosphorylated STAT3 (pSTAT3) on prognosis are limited., Experimental Design: We evaluated expression of pSTAT3 in de novo DLBCL using immunohistochemistry, gene expression profiling (GEP), and gene set enrichment analysis (GSEA). Results were analyzed in correlation with cell-of-origin (COO), critical lymphoma biomarkers, and genetic translocations., Results: pSTAT3 expression was observed in 16% of DLBCL and was associated with advanced stage, multiple extranodal sites of involvement, activated B-cell-like (ABC) subtype, MYC expression, and MYC/BCL2 expression. Expression of pSTAT3 predicted inferior overall survival (OS) and progression-free survival (PFS) in patients with de novo DLBCL. When DLBCL cases were stratified according to COO or MYC expression, pSTAT3 expression did not predict inferior outcome, respectively. Multivariate analysis showed that the prognostic predictability of pSTAT3 expression was due to its association with the ABC subtype, MYC expression, and adverse clinical features. GEP demonstrated upregulation of genes, which can potentiate function of STAT3. GSEA showed the JAK-STAT pathway to be enriched in pSTAT3(+) DLBCL., Conclusions: The results of this study provide a rationale for the ongoing successful clinical trials targeting the JAK-STAT pathway in DLBCL., (©2014 American Association for Cancer Research.)

Purpose: Epstein-Barr virus-positive (EBV(+)) diffuse large B-cell lymphoma (DLBCL) of the elderly is a variant of DLBCL with worse outcome that occurs most often in East-Asian countries and is uncommon in the Western hemisphere. We studied the largest cohort of EBV(+) DLBCL, independent of age, treated with rituximab combined with CHOP (R-CHOP) in developed Western countries., Experimental Design: A large cohort (n = 732) of patients with DLBCL treated with R-CHOP chemotherapy is included from the multicenter consortium. This study group has been studied for expression of different biomarkers by immunohistochemistry, genetic abnormalities by FISH and mutation analysis, genomic information by gene expression profiling (GEP), and gene set enrichment analysis (GSEA)., Results: Twenty-eight patients (4.0%) were positive for EBV with a median age of 60.5 years. No clinical characteristics distinguished patients with EBV(+) DLBCL from patients with EBV-negative (EBV(-)) DLBCL. Genetic aberrations were rarely seen. NF-κB p50, phosphorylated STAT-3, and CD30 were more commonly expressed in EBV(+) DLBCLs (P < 0.05). Significant differences in survival were not observed in patients with EBV(+) DLBCL versus EBV(-) DLBCL. However, CD30 expression combined with EBV conferred an inferior outcome. GEP showed a unique expression signature in EBV(+) DLBCL. GSEA revealed enhanced activity of the NF-κB and JAK/STAT pathways independent of molecular subtype., Conclusions: The clinical characteristics of patients with EBV(+) versus EBV(-) DLBCL are similar and EBV infection does not predict a worse outcome. EBV(+) DLBCL, however, has a unique genetic signature. CD30 expression is more common in EBV(+) DLBCL and, consistent CD30 and EBV is associated with an adverse outcome. Clin Cancer Res; 20(9); 2338-49. ©2014 AACR.

Purpose: Risk assessment for locoregional disease recurrence would be highly valuable in preoperative treatment planning for patients undergoing primary rectal tumor resection. Epigenetic aberrations such as DNA methylation have been shown to be significant prognostic biomarkers of disease outcome. In this study, we evaluated the significance of a quantitative epigenetic multimarker panel analysis of primary tumors to predict local recurrence in rectal cancer patients from a retrospective multicenter clinical trial., Experimental Design: Primary tumors were studied from patients enrolled in the trial who underwent total mesorectal excision for rectal cancer (n=325). Methylation levels of seven methylated-in-tumor (MINT) loci were assessed by absolute quantitative assessment of methylated alleles. Unsupervised random forest clustering of quantitative MINT methylation data was used to show subclassification into groups with matching methylation profiles., Results: Variable importance parameters [Gini-Index (GI)] of the clustering algorithm indicated MINT3 and MINT17 (GI, 20.2 and 20.7, respectively) to be informative for patient grouping compared with the other MINT loci (highest GI, 12.2). When using this two-biomarker panel, four different patient clusters were identified. One cluster containing 73% (184 of 251) of the patients was at significantly increased risk of local recurrence (hazard ratio, 10.23; 95% confidence interval, 1.38-75.91) in multivariate analysis, corrected for standard prognostic factors of rectal cancer. This group showed a significantly higher local recurrence probability than patients receiving preoperative radiation (P<0.0001)., Conclusion: Quantitative epigenetic subclassification of rectal cancers has clinical utility in distinguishing tumors with increased risk for local recurrence and may help tailor treatment regimens for locoregional control., (Copyright (c) 2010 AACR.)

Purpose: The development of local and distant recurrences is a major problem in the treatment of rectal cancer patients. In this study, we investigated whether epithelial human leukocyte antigen-DR (HLA-DR) expression allowed discrimination between high and low tumor recurrence rates, and analyzed the mechanism behind its expression., Experimental Design: The role of IFNgamma in HLA-DR expression was studied in rectal cancer cell lines and tumors by promoter-specific analyses of class II transactivator (CIITA). The predictive value of epithelial HLA-DR expression was investigated by immunohistochemical evaluation of 1,016 rectal tumors, obtained from a large prospective trial. Associations with recurrences and survival were determined by univariate and multivariate log-rank testing., Results: HLA-DR was induced by IFNgamma in rectal cancer cell lines. Activity of the IFNgamma-inducible pIV-CIITA promoter correlated with epithelial HLA-DR expression in rectal tumors. Patients with HLA-DR-positive tumors developed less frequent local and distant recurrences [1.6% versus 9.1% (P = 0.0015) and 15.3% versus 29.9% (P < 0.0001), respectively, after 5 years of follow-up] and had better survival (78.6% versus 61.3%; P < 0.0001) than patients with HLA-DR-negative tumors. Epithelial HLA-DR was more often found in lower tumor-node-metastasis (TNM) stages. Next to TNM and circumferential resection margin, HLA-DR expression was independently associated with lower distant recurrence rates and prolonged survival., Conclusions: Epithelial HLA-DR expression can be used as a marker to discriminate patients with high or low risk of developing recurrences. The possible involvement of IFNgamma, the relationship with lower TNM stages, and the independent effect on recurrence development together suggest that the host immune response plays an important role in controlling tumor cells.

Purpose: After preoperative (radio)chemotherapy, histologic determinants for prognostication have changed. It is unclear which variables, including assessment of tumor regression, are the best indicators for local recurrence and survival., Experimental Design: A series of 201 patients with locally advanced rectal cancer (cT3/T4, M0) presenting with an involved or at least threatened circumferential margin (CRM) on preoperative imaging (<2 mm) were evaluated using standard histopathologic variables and four different histologic regression systems. All patients received neoadjuvant radiochemotherapy or radiotherapy. The prognostic value of all factors was tested with univariate survival analysis of time to local recurrence and overall survival., Results: Local recurrence occurred in only 8% of the patients with a free CRM compared with 43% in case of CRM involvement (P < 0.0001). None of the four regression systems were associated with prognosis, not even when corrected for CRM status. However, we did observe a higher degree of tumor regression after radiochemotherapy compared with radiotherapy (P < 0.001). Absence of tumor regression was associated with increasing invasion depth and a positive CRM (P = 0.02 and 0.03, respectively)., Conclusions: Assessment of CRM involvement is the most important pathologic variable after radiochemotherapy. Although tumor regression increases the chance on a free CRM, in cases with positive resection margins prognosis is poor irrespective of the degree of therapy-induced regression.

Purpose: Radiotherapy followed by total mesorectal excision surgery has been shown to significantly reduce local recurrence rates in rectal cancer patients. Radiotherapy, however, is associated with considerable morbidity. The present study evaluated the use of biochemical detection of enzymatic caspase-3 activity as preoperative marker for apoptosis to preselect patients that are unlikely to develop a local recurrence to spare these patients from overtreatment and the negative side effects of radiotherapy., Experimental Design: Nonirradiated freshly frozen tissue samples from 117 stage III rectal cancer patients were collected from a randomized clinical trial that evaluated preoperative radiotherapy in total mesorectal excision surgery. Additional frozen archival tissues from 47 preoperative biopsies and corresponding resected colorectal tumors were collected. Level of apoptosis was determined by measuring the enzymatic activity of caspase-3 in a biochemical assay., Results: In tumor tissue, caspase-3 activity lower than the median was predictive of 5-year local recurrence (hazard ratio, 7.4; 95% confidence interval, 1.7-32.8; P = 0.008), which was unaffected by adjustment for type of resection, tumor location, and T status (adjusted hazard ratio, 7.5; 95% confidence interval, 1.7-34.1; P = 0.009). Caspase-3 activity in preoperative biopsies was significantly correlated with caspase-3 activity in corresponding resected tumors (r = 0.56; P < 0.0001)., Conclusion: Detection of tumor apoptosis levels by measuring caspase-3 activity, for which a preoperative biopsy can be used, accurately predicted local recurrence in rectal cancer patients. These findings indicate that caspase-3 activity is an important denominator of local recurrence and should be evaluated prospectively to be added to the criteria to select rectal cancer patients in which radiotherapy is redundant.

Purpose: To determine the effect of cyclooxygenase (COX)-2 expression on clinical behavior in irradiated and nonirradiated rectal carcinomas., Experimental Design: Tumor samples were collected from 1,231 patients of the Dutch TME trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term (5 x 5 Gy) radiotherapy or no preoperative radiotherapy. Tissue microarrays were constructed from primary tumor material, and COX-2 expression was assessed by immunohistochemistry. Tumor cell apoptosis was determined by M30 immunostaining., Results: A high level of COX-2 expression after radiotherapy was associated with low levels of tumor cell apoptosis (P=0.001). COX-2 expression had no significant effect on patient survival or tumor recurrence in nonirradiated tumors. However, in patients receiving preoperative radiotherapy, high level of COX-2 expression was associated with higher incidence of distant recurrences [P=0.003; hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.2-2.5] and shorter disease-free survival (P=0.002; HR, 1.8; 95% CI, 1.2-2.5) and overall survival (P=0.009; HR, 1.5; 95% CI, 1.1-2.0), independent of patient age, tumor stage, tumor location, or the presence of tumor cells in the circumferential resection margin., Conclusions: A high level of COX-2 expression after preoperative radiotherapy in resection specimens is associated with apoptosis resistance, high distant recurrence rates, and a poor prognosis in rectal cancer.

Purpose: The combination of radiotherapy and good quality surgery reduces local recurrence rate for rectal cancer patients. This study assesses the prognostic value of both intrinsic and radiotherapy-induced apoptosis and evaluates the relevance of radiotherapy for outcome of rectal cancer patients., Experimental Design: Tumor samples (1,198) were available from the Dutch Total Mesorectal Excision trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term radiotherapy or not. Tumor samples were obtained at time of surgery. Tissue microarrays were constructed and stained with the active caspase-specific M30 antibody to determine the amount of apoptotic epithelial tumor cells., Results: Nonirradiated patients with a negative circumferential margin displaying lower than median levels of apoptosis developed more local recurrences (10.5% versus 6.1%; P=0.06) and more rapidly after surgery than patients with high intrinsic apoptosis in their tumors (median time to recurrence, 13.0 versus 21.3 months; P=0.04). In multivariate analysis, intrinsic apoptosis was an independent predictor for the development of local recurrences (hazard ratio, 2.0; P=0.05). Radiotherapy increased apoptosis level (11 versus 23 apoptotic cells/mm2 tumor epithelium; P<0.001), but this apoptosis did not influence patients' prognosis., Conclusions: Rectal cancer patients with low intrinsic apoptosis will benefit from radiotherapy with respect to the development of local recurrences. Because apoptosis is an inherent characteristic of tumors, patients who do not need radiotherapy may be selected based on the apoptotic index of the primary tumor.

The objective of this study was to evaluate the histologic and immunohistopathologic effects of intratumorally given recombinant human interleukin-12 on the immune cells in the primary tumors and regional lymph nodes. Ten previously untreated patients with head and neck squamous cell carcinoma (HNSCC) were injected in the primary tumor twice to thrice, once weekly, at two dose levels of 100 or 300 ng/kg, before surgery. These patients were compared with 20 non-IL-12-treated control HNSCC patients. In the primary tumor, the number of CD56+ natural killer (NK) cells was increased in IL-12-treated patients compared with control patients. In some IL-12-treated patients, an impressive peritumoral invasion of CD20+ B cells was noticed. No differences were seen in the CD8+ or CD4+ T lymphocytes. Interestingly, major differences were apparent in the architecture of the enlarged lymph nodes of IL-12-treated patients; in particular, the distribution of B cells differed and fewer primary and secondary follicles with smaller germinal centers were observed. In addition, a decrease of dendritic cell lysosyme-associated membrane glycoprotein-positive cells in the paracortex was noted, resulting in a reduction of paracortical hyperplasia. In the lymph nodes, especially the CD56+ NK cells but also the CD8+ and CD4+ T lymphocytes, produced a high amount of IFN-gamma. Patients, irrespectively of IL-12 treatment, with a high number of CD56+ cells in the primary tumor had a better overall survival than those with a low number. In conclusion, after i.t. IL-12 treatment in HNSCC patients, the largest effect was seen on the NK cells, with a higher number in the primary tumor and a high IFN-gamma mRNA expression in the lymph nodes. Significant effects were noted on B cells, with altered lymph node architecture in every IL-12-treated patient and excessive peritumoral infiltration in some patients.

Purpose: In malignant lymphoma, angiogenesis has been associated with adverse outcome or more aggressive clinical behavior. This correlation has been established in groups of patients with a large heterogeneity regarding lymphoma subtypes and treatment regimens. The aim of this study is to investigate the significance of vascularization in patients with follicular lymphoma receiving uniform first-line treatment., Experimental Design: We assessed microvessel density (MVD) in pretreatment lymph node biopsies of 46 previously untreated patients with follicular lymphoma using anti-CD34 immunohistochemical staining and interactive quantification. In a selection of cases, vascular endothelial growth factor (VEGF)-RNA in situ hybridization was done. Patients were treated with cyclophosphamide-vincristine-prednisone induction chemotherapy combined with IFN-alpha2b. Thirty-six patients responded and received IFN-alpha as maintenance therapy., Results: MVD ranged from 10 to 70 per measurement field of 0.19 mm2 (median, 38). Median progression-free survival was 47 months in patients with MVD in the highest tertile and only 13 months in patients with lower MVD. Overall survival in patients with low vessel density was 59 months. In patients with high vessel density, median overall survival was not reached. Multivariate analysis indicated that MVD was independently associated with overall survival. There was a lack of correlation between VEGF-RNA expression and vessel density., Conclusion: This study shows that in follicular lymphoma increased vascularization is associated with improved clinical outcome. Furthermore, VEGF-A expression seems not to be involved in follicular lymphoma angiogenesis.

Purpose: Autosomal dominant hereditary diffuse gastric cancer (HDGC) is caused by germ-line E-cadherin (CDH1) gene mutations. Early detection of cancer in carriers is difficult because HDGC escapes endoscopic detection. We hypothesized that the glucose metabolism is enhanced in HDGC and that this can be detected with [18F]fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET)., Experimental Design and Results: An asymptomatic twenty-eight year-old female was seen at our outpatient clinic because of a request for screening on HDGC. Her father and younger sister died of diffuse gastric cancer, at the ages of 52 and 27, respectively. Mutational analysis of the CDH1 gene in this patient demonstrated a novel heterozygous splice-site mutation in exon 8 (1135delACGGTAATinsTTAGA). Upper gastrointestinal endoscopies revealed no macroscopic abnormalities, but one of the 40 random biopsy specimens showed well-differentiated signet-cell carcinoma. A FDG-PET scan demonstrated two spots of FDG accumulation, one located in the proximal part of the stomach and the second in the region of the pylorus. A total gastrectomy was performed and microscopic examination showed focal localization of intramucosal adenocarcinoma of the signet-cell type in the cardiac and antrum area. Most notably, the localization of the FDG accumulation matched the localization of the carcinoma., Conclusions: We present an asymptomatic patient from a HDGC family carrying a novel CDH1 mutation in whom FDG-PET scanning facilitated early detection of HDGC. This calls for further investigation of the role of FDG-PET scan as a screening modality in HDGC.

Purpose: Metallothionein (MT) is a small protein with a high affinity for divalent heavy metal ions. This metalloproteinis involved in many (patho)physiological processes, like metal homeostasis and detoxification, cell proliferation, apoptosis, therapy resistance, and protection against oxidative damage. Alterations in the immunohistochemical expression of MT have been reported for various human tumors, and a high expression has been found to be associated with a poor clinical outcome. We showed previously that gastrointestinal cancer is accompanied by a decrease in MT expression, but the most malignant phenotypes had the highest MT levels. The purpose of the present study was to assess the clinical relevance of MT in gastrointestinal cancer., Experimental Design: In this study, we determined the MT levels, by radioimmunoassay, in intestinal tissue of 251 patients with colorectal cancer and 81 patients with gastric cancer and assessed the relation with the overall survival of these patients., Results: More than 74% of the carcinomas were found to have a lower MT level than their corresponding normal mucosa. In colorectal cancer patients, but not in gastric cancer patients, a high MT level in both the carcinomas and normal mucosa was, however, significantly associated with a poor overall survival, independently from clinicopathological features., Conclusions: Overexpression of MT in intestinal tissue of colorectal cancer patients is a prognostic marker for a poor overall survival. In gastric cancer, however, MT expression in the gastric mucosa is not of prognostic significance. This observation emphasizes the clinical relevance of this multifunctional metalloprotein in colorectal carcinogenesis and therapy.

The oxidant-antioxidant balance is thought to be important in the initiation, promotion, and therapy resistance of cancer. In the present study, we assessed the expression of the antioxidants manganese superoxide dismutase (Mn-SOD) and copper/zinc superoxide dismutase in gastric and esophageal carcinomas and their relation with clinical outcome. Adenocarcinomas of the stomach (n = 81) as well as squamous cell carcinomas of the esophagus (n = 10) showed an enhanced immunohistochemical expression of Mn-SOD, which was accompanied by a significantly higher tissue level (P < or = 0.007) compared with their corresponding normal mucosa. In contrast, copper/zinc superoxide dismutase was found to be marginally lower in these malignant tissues in comparison with the normal tissues. The superoxide dismutase levels were not found to be associated with major clinicopathological features of the gastric cancer patients. Univariate analysis revealed, however, that a high Mn-SOD level in gastric carcinomas, a low level in the normal gastric mucosa, and a high ratio of these two levels in gastric cancer patients are indicative of a poor overall survival. Multivariate analysis, including all clinicopathological parameters, revealed that the Mn-SOD ratio in particular is an independent prognostic parameter in gastric cancer patients.