Kiyotaka Okuno

Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression‐based stratification. These parameters rely on tumour‐cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM‐I (TNM‐Immune).

Activation of ERBB2 Signaling Causes Resistance to the EGFR-Directed Therapeutic Antibody Cetuximab

Several cancers become resistant to cetuximab by activating a bypass signaling pathway and preventing cetuximab inhibition of ERK1/2-stimulated growth. Combating Resistance to an EGF Receptor Inhibitor Many promising anticancer drugs are effective only for a limited time, because the tumor cells develop resistance. Cetuximab, directed against the epidermal growth factor receptor (EGFR), is no exception, and patients with colorectal, head and neck, or non–small cell lung cancer eventually cease to respond to the drug. Yonesaka and colleagues have determined that cetuximab-resistant cancer cells—both in culture and in patients—can up-regulate signaling through the ERBB2 growth factor receptor in several ways, permanently turning on extracellular signal–regulated kinase 1/2 (ERK1/2)–mediated growth, differentiation, and survival. They further show that interference with the ERBB2 pathway restores the ability of cetuximab to control these cancers, pointing to a promising resistance-fighting approach. The authors generated clones of cetuximab-resistant non–small cell lung and colorectal cancer cell lines by exposing the cells to increasing concentration of the drug. In some of these resistant clones, the ERBB2 receptor oncogene was genetically amplified, resulting in activated ERK1/2 signaling. Down-regulation of ERBB2 with a small interfering RNA or antibody restored sensitivity. Other clones did not have amplified ERBB2 genes but did make excess heregulin, an activating ligand for the ERBB2 receptor. Heregulin depletion or ERBB2 inhibition restored cetuximab sensitivity. After replicating these studies in xenografts in mice, the authors also looked for evidence that these resistance-associated alterations pertain to human tumors. In several groups of patients with colorectal cancer, they saw decreased survival or decreased sensitivity to cetuximab in those who exhibited amplified ERBB2 gene or higher heregulin concentrations. The concordance of their cellular data with patient experience improves confidence that concomitant treatment of certain lung, head and neck, or colorectal cancers with cetuximab and an anti-ERBB2 drug may prevent or delay the development of drug resistance. These studies add to other successes for this approach, which has also been used for analysis of other molecular targeted therapies, including EGFR kinase inhibitors. Cetuximab, an antibody directed against the epidermal growth factor receptor, is an effective clinical therapy for patients with colorectal, head and neck, and non–small cell lung cancer, particularly for those with KRAS and BRAF wild-type cancers. Treatment in all patients is limited eventually by the development of acquired resistance, but little is known about the underlying mechanism. Here, we show that activation of ERBB2 signaling in cell lines, either through ERBB2 amplification or through heregulin up-regulation, leads to persistent extracellular signal–regulated kinase 1/2 signaling and consequently to cetuximab resistance. Inhibition of ERBB2 or disruption of ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vitro and in vivo. A subset of colorectal cancer patients who exhibit either de novo or acquired resistance to cetuximab-based therapy has ERBB2 amplification or high levels of circulating heregulin. Collectively, these findings identify two distinct resistance mechanisms, both of which promote aberrant ERBB2 signaling, that mediate cetuximab resistance. Moreover, these results suggest that ERBB2 inhibitors, in combination with cetuximab, represent a rational therapeutic strategy that should be assessed in patients with cetuximab-resistant cancers.

Cancer classification using the Immunoscore: a worldwide task force

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the ‘Immunoscore’ into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Functional outcome after low anterior resection with low anastomosis for rectal cancer using the colonic J-pouch. Prospective randomized study for determination of optimum pouch size.

PURPOSE: Functional outcome after low anterior resection with ultralow coloanal anastomosis for rectal cancer is improved by construction of a colonic J-pouchvs.straight anastomosis. Optimum size of this pouch has yet to be determined. Therefore, we initiated a prospective, randomized trial using 5-cm and 10-cm pouches to determine this size. METHODS: Patients with tumors 5 to 10 cm from the anal verge were included in the study. Before a low anterior resection anastomosis was performed, patients were randomized to either a 5-cm J-pouch group (5-J group) or a 10-cm J-pouch group (10-J group). Functional assessments were performed one year postoperatively. Clinical functions were evaluated using a functional scoring system. Physiologic functions, such as sphincter and reservoir function, were evaluated by anorectal manometry and evacuation function by the balloon expulsion and saline evacuation tests. RESULTS: Forty patients among 43 randomized patients were assessed for functional outcome one year postoperatively (5-J group, n=20; 10-J group, n=20). The functional score was similar for the two groups, although reservoir function in the 5-J group was significantly less than in the 10-J group. Sphincter function was similar between the two groups. Evacuation function in the 5-J group was significantly superior to that in the 10-J group. CONCLUSIONS: The 5-cm J-pouch conferred adequate reservoir function without compromising evacuation.

Comparison of Long-Term Functional Results of Colonic J-Pouch and Straight Anastomosis After Low Anterior Resection for Rectal Cancer: A Five-Year Follow-Up

PURPOSEFew reports on the long-term functional outcome of colonic J-pouch reconstruction have been published, and data comparing J-pouch and straight reconstruction are contradictory. This prospective study compares the functional outcome of colonic J-pouch and straight anastomosis five years after low anterior resection for rectal cancer.METHODSFunctional outcome was compared in 46 patients with J-pouch reconstruction (J-group) and 48 patients with straight anastomosis (S-group). Clinical status was evaluated with a 17-item questionnaire inquiring about different aspects of bowel function. Reservoir function was evaluated by manovolumetry. The Fisher’s exact test and Wilcoxon’s rank-sum test were used to compare categoric and quantitative data, respectively.RESULTSAmong patients with an ultralow anastomosis (≤4 cm from the anal verge), the number of bowel movements during the day (≥5, 4.3 vs. 29.2 percent; P = 0.028) and at night (>1/week, 4.3 vs. 33.3 percent; P = 0.013) and urgency (4.3 vs. 33.3 percent; P = 0.013) and soiling (21.7 vs. 50.0 percent; P = 0.043) were less in the J-group than in the S-group. Among patients with a low anastomosis (5 to 8 cm from the verge), patients in the J-group had fewer bowel movements at night (>1/week, 0 vs. 20.8 percent; P = 0.028) and less urgency (0 vs. 20.8 percent; P = 0.028). Reservoir function was better in the J-group than in the S-group in both the ultralow (maximum tolerable volume (mean), 101.7 vs. 76.3 ml; P = 0.004; threshold volume (mean), 46.5 vs. 30.4 ml; P < 0.001; compliance (mean), 4.9 vs. 2.5 ml/cm H2O; P < 0.001) and low-anastomosis (maximum tolerable volume, 120.4 vs. 97.9 ml; P < 0.001; threshold volume, 58.3 vs. 40.8 ml; P < 0.001; compliance, 5.2 vs. 3.1 ml/cm H2O; P < 0.001) groups.CONCLUSIONSJ-pouch reconstruction increased reservoir function and provided better functional outcome than straight anastomosis, even five years after surgery, especially in patients whose anastomosis is less than 4 cm from the anal verge.

Surgical Treatment for Digestive Cancer

Background: Due to the westernization of the diet in Japan, the incidence of colorectal cancer has increased 4.5 times in the last 25 years. In this review, the recent results of surgical treatment for colonic cancer and the future perspectives in Japan are described. Materials and Methods: A multi-institutional registry of large bowel cancer in Japan of 10,809 patients with colonic cancer treated from 1991 to 1994 was investigated. The data have been published in the Guidelines of the Japanese Society for Cancer of the Colon and Rectum (2005). Regarding laparoscopic surgery, 1,495 patients with colon cancer were examined in a multicenter study between April 1993 and August 2002. Results: Radical resection with a curative intent is appropriate for 83–99% of the patients with stage I–III localized colon carcinoma. Adequate lymphadenectomy, including paracolic, intermediate and principal node dissection (D3 lymphadenectomy), is of critical importance for both the accurate staging and local control of the disease. This treatment protocol has now been accepted as a ‘standard’ operation by Japanese colorectal surgeons. For patients undergoing a curative resection for colon cancer, the 5-year survival rates vary between 62 (stage III) and 91% (stage I). Adjuvant chemotherapy using 5-FU/leucovorin or oral compounds is commonly administered to patients with stage III disease. Laparoscopic surgery for colonic cancer yielded a comparable oncological outcome to that reported for conventional open surgery in the Japanese registry for all disease stages. Conclusion: Radical resection with a D3 lymphadenectomy provided satisfactory 5-year survival for patients with stage I–III colon cancer in Japan. However, the survival of patients with stage IV disease is still unsatisfactory (only a 14% 5-year survival). Any further improvements depend on both identifying such patients at an earlier stage as well as developing new and effective treatment modalities.

Treatment for unresectable hepatoma via selective hepatic arterial infusion of lymphokine‐activated killer cells generated from autologous spleen cells

Nonspecific tumoricidal effectors, called lymphokine‐activated killer (LAK) cells, can be induced from the tumor‐bearers spleen in vitro. The adoptive transfer of such LAK cells to a patient with unresectable hepatoma was performed in this study. About 2.4 × 109 LAK cells generated from the autologous spleen were adoptively transferred to the patient via hepatic arterial catheter. Signs of toxicity encountered with LAK cell infusions comprised chills and fever only. Chemical studies of hepatic, renal, and hematologic parameters were normal; pulmonary function studies revealed no changes after infusion. With the transfer of LAK cells, serum alpha‐fetoprotein (AFP) levels were markedly decreased and ascitic fluid retention was transiently reduced. Though the therapeutic effect was transient, these trials offered hope for a new therapeutic approach to unresectable hepatoma. Further, the availability of large amounts of recombinant interleukin‐2 (rIL‐2) may now make widespread use of this approach possible. Cancer 58:1001–1006, 1986.

Gene amplification of EGFR, HER2, FGFR2 and MET in esophageal squamous cell carcinoma

Molecular targeted therapy is expected to be a promising therapeutic approach for the treatment of esophageal squamous cell carcinoma (ESCC); however, the gene amplification status of molecular targeted genes in ESCC remains largely unclear. The gene amplification of EGFR, HER2, FGFR2 and MET was examined using a real-time PCR-based copy number assay of 245 ESCC surgical specimens of formalin-fixed, paraffin-embedded samples. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization analyses verified the results of the copy number assay. EGFR mutation was detected using the Scorpions-ARMS method. The EGFR status and drug sensitivity to an EGFR tyrosine kinase inhibitor was then evaluated in vitro. Gene amplification of EGFR and HER2 was observed in 7% (16/244) and 11% (27/245) of the ESCC specimens. A multivariate analysis revealed that HER2 amplification was a significant predictor of a poor prognosis in patients with stage III post-operative ESCC. The L861Q type of EGFR mutation with hypersensitivity to EGFR tyrosine kinase inhibitor was found in one of the eight ESCC cell lines and one del745 type of EGFR mutation was identified in 107 clinical samples. In addition, we demonstrated for the first time that FGFR2 amplification was observed in 4% (8/196) of the ESCC specimens. MET amplification was observed in 1% (2/196). In conclusion, the frequent gene amplification of EGFR, HER2 and FGFR2 and the presence of active EGFR mutations were observed in ESCC specimens. Our results strongly encourage the development of molecular targeted therapy for ESCC.

Optimal ligation level of the primary feeding artery and bowel resection margin in colon cancer surgery: the influence of the site of the primary feeding artery.

PURPOSEIn colon cancer surgery, it is recommended that en bloc resection involving extended lymphadenectomy, characterized as a hemicolectomy, be performed by ligating the primary feeding artery at a high position and resecting proximal and distal with 5-cm to 10-cm bowel margins. However, there is little evidence to unequivocally support such extensive lymphovascular resection.METHODSThe distribution of nodal metastases was obtained by the clearing method in 164 patients with colon cancer.RESULTSFor pericolic spread, for pT1 tumors, the distance from the primary tumor to a diseased node was 2.5 cm; for pT2, the distance was less than 5 cm; for 97.0 percent of pT3 tumors and 93.3 percent of pT4 tumors with nodes involved, the distance was less than 7 cm. For central spread, for pT1 tumors, the rate of spread to central nodes was 0 percent; for pT2, the rate of spread was 20.0 percent to intermediate nodes (for tumors more than 5 cm from the feeding artery, the rate for central nodes was 0 percent); for pT3, the rate was 30.6 percent to intermediate nodes and 15.3 percent to main nodes; for pT4, the rate was 44.4 percent to intermediate nodes and 22.2 percent to main nodes. For curative resection cases with pT3 tumors more than 7 cm from the feeding artery, the rate to central nodes was 0 percent.CONCLUSIONSIn T1 tumors, central node dissection is not required, but resection with proximal and distal 3-cm margins are required; in T2, central node dissection that includes the intermediate node should be performed in addition to resection with proximal and distal 5-cm margins. In T3 and T4, central node dissection that includes the main node should be performed in addition to resection with proximal and distal 7-cm margins. However, for T2 more than 5 cm from the primary feeding artery, and for T3 more than 7 cm from the primary feeding artery, proximal and distal resection alone may be adequate.

Assessment of immunological biomarkers in patients with advanced cancer treated by personalized peptide vaccination

To investigate immunological biomarkers to predict overall survival of advanced cancer patients under treatment with personalized peptide vaccination, correlations between overall survival and biomarkers, including cytotoxic T lymphocyte (CTL) and immunoglobulin G (IgG) responses to the vaccinated peptides, were investigated in 500 advanced cancer patients who received personalized peptide vaccination from October 2000 to October 2008. The best clinical response was assessed for in 436 patients, 43 patients (10%) had partial response, 144 patients (33%) had stable disease and 249 patients (57%) had progressive, with a median overall survival of 9.9 months. Both lymphocyte counts prior to the vaccination (P = 0.0095) and increased IgG response (P = 0.0116) to the vaccinated peptides, along with performance status (P < 0.0001), well correlated with overall survival. To confirm the superiority of IgG response to CTL response, the samples from advanced castration-resistant prostate cancer patients who survived more than 900 days (n=20) and those who died within 300 days (n=23) were analyzed further. As a result, both the numbers of peptides, to which increased IgG responses were observed, and the fold increases in IgG levels were significantly higher in long-term survivors (P = 0.000282 and P = 0.00045). In contrast, CTL responses were not statistically different between the two groups. Both lymphocyte numbers and IgG response were thus suggested to be biomarkers of cancer vaccine for advanced cancer patients.