Role of the KRT7 Biomarker in Immune Infiltration and Paclitaxel Resistance in Ovarian.

Context: Paclitaxel (PTX) resistance is often associated with poor outcomes for patients with ovarian cancer (OC), but its mechanism is unknown. Clinicians are increasingly using immunotherapy in the management of OC, and the ability to assess tumor-immune interactions and identify effective, predictive, prognostic molecular biomarkers for OC is an urgent need. Objective: The study intended to explore the potential tumorigenesis mechanisms to identify promising biomarkers and improve survival in OC patients. Design: The research team performed a genetic analysis. Setting: The study took place at First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China. Outcome Measures: The research team: (1) obtained GSE66957 and GSE81778 gene expression profiles from the Gene Expression Omnibus (GEO) database and identified 468 differentially expressed genes (DEGs); (2) conducted functional enrichment analysis and constructed a protein-to-protein interaction (PPI) network; (3) identified the OC survival-related genes using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) webserver and compared those genes with upregulated DEGs to identify the core genes; (4) used GEPIA2 and the Kaplan-Meier plotter to explore the expression profiles and the prognostic values of the core genes in OC; (5) used the LinkOmics, Oncomine, and GEPIA2 web servers to perform co-expression analysis and explore functional networks correlated with keratin 7 (KRT7); (6) performed correlation analyses between KRT7, the six main types of tumor-infiltrating lymphocytes (TILs), and immune signatures, using the TIMER tool; and (7) subsequently detected the KRT7 expression in the cell lines IOSE80, A2780, A2780/PTX, ho8910, skov3, and ovcar3 using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) technology. Results: High expression levels of KRT7 were significantly correlated with progression-free survival (PFS) and poor overall survival (OS) for OC patients, with logrank P = .0074 and logrank P = .014, respectively. The expression levels of KRT7 were also significantly correlated with the infiltrated neutrophil levels (r = 0.169, P = .0077). The study identified neutrophils as potential predictors of survival in OC. Moreover, the expression levels of KRT7 in OC were positively correlated with 51 (31.68%) of the 161 immune gene markers. The RT-qPCR analyses revealed a high expression of KRT7 in the paclitaxel-resistant OC cell line. Conclusions: KRT7 is correlated with immune infiltration and paclitaxel resistance in OC patients. Therefore, clinicians could use KRT7 as a prognostic marker and a target in the development of new drugs.

Robotic low anterior resection of rectal metastasis from small bowel adenocarcinoma: A case report.

A 69-year-old female underwent laparoscopic ileal partial resection for ileal adenocarcinoma. Pathological diagnosis was moderately differentiated tubular adenocarcinoma (UICC 8th; T4N0M0 StageIIB). The patient received adjuvant chemotherapy with modified 5-fluorouracil/leucovorin/oxaliplatin. Fourteen months after surgery, computed tomography revealed a mass in the upper rectum. Colonoscopy detected a submucosal protruding mass and a biopsy specimen showed moderately differentiated tubular adenocarcinoma. Robotic low anterior resection was performed. The tumor was located in the upper rectum and there was no macroscopic invasion or peritoneal dissemination. Pathologically, the tumor was moderately differentiated tubular adenocarcinoma located within the rectal wall with little evidence of a carcinoma component in the mucosal lining. Immunohistochemistry showed the same pattern as the previous ileal adenocarcinoma: negativity for cytokeratin 7 and positivity for cytokeratin 20 and caudal-type homeobox 2. In combination with the rectum showing no abnormalities in colonoscopy performed 15 mo previously, the mass was considered hematogenous metastasis from small bowel adenocarcinoma.

Treatment Effects Can Mimic Recurrent Extramammary Paget Disease in Perianal Skin.

The histologic differential diagnosis of perianal Paget disease includes malignant melanoma, pagetoid spread of squamous cell carcinoma, and secondary involvement by colorectal carcinoma. While consideration of these entities is useful when establishing a diagnosis, it does not apply when patients with Paget disease undergo surveillance for recurrent disease. Treatment of perianal Paget disease consists of a combination of surgical excision with skin grafts and topical chemotherapeutic agents that induce cytologic alterations in benign cells and simulate recurrent malignancy. To evaluate the therapy-related changes and possible diagnostic pitfalls in patients with Paget disease, we reviewed 412 posttreatment tissue samples from 3 women with primary perianal Paget disease who underwent wide excision, skin grafting, and topical 5-fluorouracil therapy. Biopsy samples from engrafted skin often displayed single and clustered cells with hyperchromatic nuclei dispersed in the deep epidermis. Similar cells were scattered throughout all levels of the epidermis in biopsy samples following topical chemotherapy. The abnormal cells were negative for cytokeratin 7 (CK7) and mucicarmine in both situations. Disease ultimately recurred in all patients; some Paget cells showed classic features with eosinophilic or mucinous cytoplasm and eccentric nuclei, whereas others were smaller with less conspicuous atypia. All Paget cells showed strong, membranous CK7 staining. In short, treatment of perianal Paget disease can elicit cytologic abnormalities in benign epithelial cells that simulate the cytologic features of recurrent disease, and can diminish the atypia of Paget cells. Immunohistochemical stains for CK7 can be helpful when evaluating surveillance samples from these patients.

Immunohistochemical Profiling of Corneas With Fuchs Endothelial Corneal Dystrophy.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is the leading indication for endothelial keratoplasty. Further insight into its pathophysiology is needed to develop alternative therapies. METHODS: Sixteen genes from a previous microarray expression experiment (FECD vs. normal) were validated using immunohistochemistry on paraffin-embedded corneas (n = 6 FECD, n = 6 normal). The results were quantified manually and semiautomatically. RESULTS: A higher percentage of corneal endothelial cells stained for alpha-smooth muscle actin (αSMA), cytokeratin 7, and superoxide dismutase 3 in FECD versus normal [odds ratios (ORs) of 60.90, 41.70, and 15.16, respectively, P < 0.001]. Dot-like staining for major histocompatibility complex, class II, DR alpha was present in FECD, but not in normal. Higher percentages of stromal cells in FECD versus normal stained for αSMA (OR = 864.26, P < 0.001), brain-derived neurotrophic factor (BDNF, OR = 6.34, P = 0.005), fibroblast growth factor 7 (FGF-7, OR = 2.76, P = 0.011), FGF-9 (OR = 5.97, P < 0.001), receptor FGFR-3 (OR = 13.90, P = < 0.001), and serum amyloid A1 (OR = 3.45, P = 0.023). Higher percentages of corneal epithelial cells stained for αSMA (OR = 2.20, P = 0.006) and BDNF (OR = 3.94, P < 0.001) in FECD versus normal. CONCLUSIONS: These results support a role for epithelial-mesenchymal transition (αSMA), oxidative stress (superoxide dismutase 3), and major histocompatibility complex, class II, DR alpha cells with dendritic morphology in the pathophysiology of FECD. Furthermore, corneal stromal cells express trophic molecules (BDNF and FGFs) and markers of chronic inflammation (serum amyloid A1) in FECD.

Pseudomyxoma Peritonei of Extra-Appendiceal Origin: A Comparative Study.

BACKGROUND: Pseudomyxoma peritonei (PMP) usually originates from appendiceal neoplasms and, less commonly, from extra-appendiceal lesions. To date, the clinical and therapeutic implications of extra-appendiceal origin are largely unknown. METHODS: A prospective database of 225 PMP patients uniformly treated by cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) was reviewed to identify cases with extra-appendiceal primaries. Histologically, negative appendix defined extra-appendiceal origin. Clinical, pathological, and immunohistochemical features (cytokeratin [CK]-20, CK-7, CDX-2, MUC-2, MUC-5A) were correlated with the site of origin. PMP was categorized into low or high grade, according to the 2010 World Health Organization (WHO) classification. The main independent variable for survival analysis was appendiceal versus extra-appendiceal primary. RESULTS: In 19 patients (8.4 %), PMP origin was the ovary (n = 9), uterine cervix (n = 1), mature cystic teratomas (n = 4), and unknown (n = 5). Appendiceal and extra-appendiceal PMP groups were comparable for all characteristics, except for a prevalence of females in the latter. Median follow-up was 64.1 months (95 % confidence interval [CI] 53.9-80.1), and 10-year overall survival was 63.4 % (median 148.2 months; 95 % CI 131.2-165.2) for appendiceal PMP, and 62.0 % (median not reached) for extra-appendiceal PMP. The difference was not significant at univariate ( p = 0.297) and multivariate analysis (hazard ratio 1.51, 95 % CI 0.78-3.14; p = 0.278). High-grade peritoneal histology (p = 0.007), prior systemic chemotherapy (p = 0.003), more than four visceral resections (p = 0.011), and incomplete cytoreduction (p = 0.021) independently correlated with poorer survival. CONCLUSIONS: Clinical-pathological features of PMP, and outcome after CRS/HIPEC, did not differ according to the primary site, thus suggesting that PMP is a relatively homogeneous disease that can be produced by a range of histopathologic entities. Extra-appendiceal origin does not contraindicate CRS/HIPEC.

Cytoreductive Surgery Plus Hyperthermic Intraperitoneal Chemotherapy for Pseudomyxoma Peritonei Arising from Urachus.

BACKGROUND: Pseudomyxoma peritonei (PMP) is a rare locoregional disease characterized by disseminated intraperitoneal mucinous tumors. However, little is known about PMP from urachal neoplasm as a result of its rarity. METHODS: A total of 9 patients with PMP of urachal origin were treated by cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC) in our institution. All specimens of surgeries were submitted for pathologic examination. Representative slides of tumors and normal urachus were submitted for immunohistochemical staining. RESULTS: Four patients were men; the median age was 48 years (range 27-65 years). Initial radiologic examination of all patients showed a cystic tumor located between posterior aspect of umbilicus and the dome of urinary bladder, with or without leaking mucin. Complete CRS and HIPEC were performed in all patients. Until the latest follow-up, local recurrence occurred in 1 patient. Other 8 patients had a median disease-free survival of 27.5 months. Primary urachal tumors of 9 cases were all mucinous adenocarcinoma. Six patients had low-grade mucinous carcinoma peritonei, and 3 patients had high-grade mucinous carcinoma peritonei. Signet ring cells were noted in 4 patients. All tumor specimens of 9 patients were diffuse positive for CK-20, CDX-2, MUC-2, and MUC-5AC, and were variant positive for CK-7. CONCLUSIONS: PMP arising from urachus comes from neoplastic cells with development of intestinal-type mucinous neoplasm. It shares a similar pathophysiology as PMP from appendix. CRS including total urethrectomy, partial cystectomy, and peritonectomy plus HIPEC can be considered as a new option of treatment for PMP originating from urachus.

Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy: an emerging treatment option for advanced goblet cell tumors of the appendix.

BACKGROUND: The debate remains whether appendiceal goblet cell cancers behave as classical carcinoid or adenocarcinoma. Treatment options are unclear and reports of outcomes are scarce. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS+HIPEC) is considered optimal treatment for peritoneal involvement of other epithelial appendiceal tumors. METHODS: Prospective cohorts of patients treated for advanced appendiceal tumors from three peritoneal malignancy centres were collected (1994-2011). All patients underwent complete CRS+HIPEC, when possible, or tumor debulking. Demographic and outcome data for patients with goblet cell cancers were compared to patients with low- or high-grade epithelial appendiceal tumors treated during the same time period. RESULTS: Details on 45 goblet cell cancer patients were compared to 708 patients with epithelial appendix lesions. In the goblet cell group, 57.8 % were female, median age was 53 years, median peritoneal cancer index (PCI) was 24, and CRS+HIPEC was achieved in 71.1 %. These details were similar in patients with low- or high-grade epithelial tumors. Lymph nodes were involved in 52 % of goblet cell patients, similar to rates in high-grade cancers, but significantly higher than in low-grade lesions (6.4 %; p < 0.001). At 3 years, overall survival (OS) was 63.4 % for goblet cell patients, intermediate between that for high-grade (40.4-52.2 %) and low-grade (80.6 %) tumors. On multivariate analysis, tumor histology, PCI, and achievement of CRS+HIPEC were independently associated with OS. CONCLUSIONS: This data supports the concept that appendiceal goblet cell cancers behave more as high-grade adenocarcinomas than as low-grade lesions. These patients have reasonable long-term survival when treated using CRS+HIPEC, and this strategy should be considered.

The role of CK7, Ki-67, CD34 and vimentin in the differentiation between biliary atresia and idiopathic neonatal hepatitis in Egyptian cholestatic neonates.

The differentiation between biliary atresia (BA) and idiopathic neonatal hepatitis (INH) is challenging with many histological overlaps especially in the first weeks of life. This study aimed to investigate the role of immunohistochemical staining of CK7, Ki67, CD34, and vimentin in addition to other clinicopathological features in the differentiation between BA and INH. Cases included 30 infants with BA and 30 infants with INH. The diagnosis was based on clinical, laboratory, and liver biopsy examination. Female gender and elevated serum gamma glutamyle transferase were in favor of BA. Portal tract changes, such as bile ductular proliferation documented by CK7, Ki67 immunostaining and angiogenesis documented by CD34 immunostaining, favored the diagnosis of BA. Copper associated protein was positive in 70% of BA cases, but not detected in INH cases. Parenchymatous changes, such as giant cell transformation and positive iron deposition and Kupffer cell proliferation documented by vimentin immunostaining, favored the diagnosis of INH.CK7, Ki67, CD34, and vimentin are helpful adjuvant immunostaining in the differentiation between BA and INH.

[Huangqi decoction inhibits cholangiocyte proliferation and transdifferentiation in cholestatic liver fibrosis induced by BDL in rats].

OBJECTIVE: To elucidate the antifibrotic mechanism of Huangqi decoction in rats with BDL-induced cholestatic liver fibrosis. METHODS: Liver fibrosis model was induced by ligating the common bile duct (BDL) in rats. Sham-operation was performed in control rats. The BDL rats were randomly divided into two groups: the BDL group and the Huangqi decoction group. Huanqi decoction was given intragastrically for 4 weeks. At the end of the fifth week after BDL, animals were sacrificed. RESULTS: Compared with the sham control group, mortality rate in BDL group was 33.3% and incidence rate of ascites was 90%, and hepatic function was abnormal in most of the rats in BDL group. The number of Hepatocytes was decreased and the number of cholangiocytes significantly increased in BDL group. In addition, Hyp content of liver tissue and protein expression of CK 7 and a-SMA were significantly increased. Immunostaining indicated that CK 7 and a-SMA were co-localized in BDL group. These changes were markedly suppressed by the Huangqi decoction. CONCLUSIONS: These observations suggest that Huangqi decoction can inhibit cholangiocyte proliferation and cholangiocyte transdifferentiation.

Toxicity of smoke extracts towards A549 lung cells: role of acrolein and suppression by carbonyl scavengers.

The noxious 3-carbon electrophile acrolein forms on combustion of diverse organic matter including synthetic polymers such as polyethylene. While known to play a key role in smoke inhalation injury (SII), the molecular basis for the pulmonary toxicity of high dose acrolein-containing smoke is unclear. As a result, drug interventions in SII are poorly directed against pathogenetic smoke toxicants such as acrolein. The first aim of this study was to confirm a role for acrolein in the acute toxicity of smoke extracts towards A549 lung cells by monitoring adduction of known acrolein targets and the expression of acrolein-inducible genes. A second aim was to evaluate carbonyl scavengers for their abilities to protect cell targets and block smoke extract toxicity. Extracts were prepared by bubbling smoke released by smouldering polyethylene through a buffered saline-trap. Acrolein levels in the extracts were estimated via HPLC after derivatisation with 2,4-dinitrophenylhydrazine. Extracts were highly toxic towards A549 cells, eliciting greater ATP depletion than an equivalent concentration of acrolein alone. The toxicity was accompanied by pronounced carbonylation of several cytoskeletal targets, namely vimentin and keratins-7, -8 and -18. Western blotting revealed that polyethylene combustion products also upregulated several acrolein-responsive protein markers, including GADD45beta, NQO1, HMOX, Hsp70, Nur77 and Egr1. Several carbonyl scavengers (bisulfite, d-penicillamine, hydralazine and 1-hydrazinoisoquinoline) strongly attenuated smoke extract toxicity, with bisulfite suppressing both the adduction and cross-linking of intermediate filament targets. Bisulfite also suppressed the cytotoxicity of smoke extracts when detected using real-time monitoring of cellular impedance. These findings confirm a key role for acrolein in smoke cytotoxicity and suggest drugs that block acrolein toxicity deserve further investigation as possible interventions against SII.

Double primary adenocarcinomas of the jejunum and descending colon with lung metastases presenting rare immunohistochemical phenotypes: a case report.

We report a male patient with double advanced tumors in the jejunum and descending colon and multiple lung tumors. The intestinal cancers were surgically resected. Immunoprofiling of the specimens revealed a rare phenotype: the jejunal cancer was positive for cytokeratin (CK) 7, partially positive for CK20, and Cdx-2-negative, whereas the colon cancer was CK7(+), CK20(-), and Cdx-2(-). Biopsied lung tumor was diagnosed as tubular adenocarcinoma, and CK7(+)/CK20(+)/Cdx-2(-). Together with clinical information, we deduced that the jejunal adenocarcinoma had presumably metastasized to the lung. Moreover, postoperative oxaliplatin, including chemotherapy, significantly reduced the lung metastases, suggesting that this regimen is a promising treatment option for advanced small bowel adenocarcinoma.

Huangqi decoction inhibits cholangiocyte proliferation and transdifferentiation in cholestatic liver fibrosis induced by BDL in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To elucidate the antifibrotic mechanism of Huangqi decoction in rats with BDL-induced cholestatic liver fibrosis.</p><p><b>METHODS</b>Liver fibrosis model was induced by ligating the common bile duct (BDL) in rats. Sham-operation was performed in control rats. The BDL rats were randomly divided into two groups: the BDL group and the Huangqi decoction group. Huanqi decoction was given intragastrically for 4 weeks. At the end of the fifth week after BDL, animals were sacrificed.</p><p><b>RESULTS</b>Compared with the sham control group, mortality rate in BDL group was 33.3% and incidence rate of ascites was 90%, and hepatic function was abnormal in most of the rats in BDL group. The number of Hepatocytes was decreased and the number of cholangiocytes significantly increased in BDL group. In addition, Hyp content of liver tissue and protein expression of CK 7 and a-SMA were significantly increased. Immunostaining indicated that CK 7 and a-SMA were co-localized in BDL group. These changes were markedly suppressed by the Huangqi decoction.</p><p><b>CONCLUSIONS</b>These observations suggest that Huangqi decoction can inhibit cholangiocyte proliferation and cholangiocyte transdifferentiation.</p>

High-grade foamy gland prostatic adenocarcinoma on biopsy or transurethral resection: a morphologic study of 55 cases.

Foamy gland carcinoma is a variant of adenocarcinoma of the prostate that typically is assigned a Gleason score 3+3=6. The morphologic features of high foamy gland carcinoma have not been previously studied. We analyzed 55 cases of high-grade (Gleason score 7 or greater) foamy gland carcinoma of the prostate in needle biopsy (n=49) or transurethral resection (n=6) specimens. The number of cores involved by high-grade foamy gland carcinoma ranged from 1 to 12, with more than 1 core involved in 61% of cases (mean 3.4 cores). On average, 84% of the total tumor volume was foamy gland carcinoma, with high-grade foamy gland cancer averaging 73% of the total foamy gland carcinoma. The following results pertain only to the high-grade foamy gland cancer component. The most common architectural pattern was cribriform (73%), followed by fused/poorly defined glands (55%), cords/single cells (11%), and solid sheets (5%). Nuclear enlargement was observed in 45 of the 55 studied cases (82%). Prominent nucleoli were either absent or infrequent in 38 cases (69%). Frequent to numerous prominent nucleoli were seen more frequently in foamy gland carcinoma with Gleason score 8 or above (52%) than those with Gleason score 7 (16%) (P<0.004). Mitotic figures were observed in 22 cases (40%), and present in 65% of the cases with Gleason score 8 or above, but only in 22% of the cases with Gleason score 7 (P<0.002). In 31 cases (56%), intraluminal dense pink secretions were identified. Perineural invasion and extraprostatic extension identified on the biopsy specimens were noted in 18 cases (33%) and in 5 cases (9%), respectively. In 18 cases (33%), there was at least a moderate stromal reaction. A moderate or greater stromal reaction was seen in 48% (11/23) of the cases with Gleason score 8 or above compared with 22% (7/32) of the cases with Gleason score 7 (P=0.04). In 6 cases, there was a peculiar extensive desmoplastic reaction almost obscuring the carcinoma component, 5 of which were Gleason scores 4+4=8. Concurrent ordinary acinar nonfoamy adenocarcinoma was encountered in 26 of 55 cases (47%) with the following Gleason scores: Gleason 6 (27%); Gleason 7 (27%); and Gleason 8 to 10 (46%). Associated ordinary high-grade prostatic intraepithelial neoplasia and foamy gland variant of high-grade prostatic intraepithelial neoplasia/intraductal adenocarcinoma were seen in 13 cases (24%) and 11 cases (20%), respectively. Of the 19 cases with available immunohistochemical stains for high molecular weight cytokeratin, 7 (37%) showed nonspecific labeling of cancer cells in a nonbasal cell pattern. A similar finding was seen in 1 of the 7 (14%) cases with available stains for p63. Alpha-methyl-CoA racemase positivity was noted in all 9 cases stained. In summary, uncommonly foamy gland carcinoma consists of cribriform, fused/poorly formed glands, cords/single cells, and solid sheets typical of Gleason patterns 4 and 5. High-grade foamy gland cancer shares certain morphologic features with more typical lower-grade foamy gland cancer including relatively bland nuclei with more difficult to identify nucleoli and frequent intraluminal dense pink secretions. However, consistent with their higher architectural grade, high-grade foamy gland cancers had more prominent nucleoli and increased mitotic figures compared with lower-grade foamy gland cancer. A unique subset of high-grade foamy gland carcinoma poses particularly difficult diagnostic challenges, with scattered, scant, relatively bland foamy glands imbedded in an extensive densely sclerotic desmoplastic stroma.

Coexistence of primary squamous cell carcinoma of thyroid with classic papillary thyroid carcinoma.

Primary squamous cell carcinoma of the thyroid gland is very rare and its histogenesis is poorly defined so far. Although there have been some cases of squamous cell carcinoma with variant types of papillary thyroid carcinoma (PTC), the present case is the first primary squamous cell carcinoma with classic PTC to be reported. A 43-year-old woman presented with a 20 year history of neck mass. Neck ultrasound indicated a 6x4x3 cm large mass. The patient underwent total thyroidectomy. Histopathology indicated a well-differentiated squamous cell carcinoma and squamous metaplasia in conjunction with classic PTC. On immunohistochemistry cytokeratin 7 was positive in papillary carcinoma and squamous metaplasia, thyroglobulin was positive only in papillary carcinoma, and p63 was positive in squamous metaplasia and squamous cell carcinoma. Postoperatively, the patient received 59.4 Gy adjuvant radiotherapy, hormonal therapy and radioactive iodine therapy. At 8 months after surgery the patient remained disease free.

[A 42-month disease free survival case of combined hepatocellular-cholangiocarcinoma with lymph node metastases treated with multimodal therapy].

Combined hepatocellular and cholangiocarcinoma (HCC-CC) is a rare type of liver cancer. We herein report a case of HCC-CC with lymph node metastases treated by multimodality therapy. The patient has been alive for more than 42 months. A 52-year-old man with a 9 cm diameter mass lesion in the liver was admitted to our hospital. The tumor was diagnosed as peripheral type of cholangiocarcinoma. Preoperative transhepatic arterial chemoenbolization (TACE) was performed. An accumulation pattern of lipiodol after TACE and an increase of serum alpha-fetoprotein led us to diagnosis of combined HCC-CC. A three segmentectomies of the liver and dissection of the local lymph nodes were performed. A histological examination of the resected specimen showed combined HCC-CC with lymph node metastases. Alpha fetoprotein, cytokeratins 7 and 19 were partially positive with immunohistochemical staining. The final diagnosis was a mixed type of combined HCC-CC. To improve a poor prognosis of combined HCC-CC, adjuvant chemotherapy with CDDP, 5 FU and radiation therapy were achieved. Fortunately, the patient is alive without any recurrence for 42 months after the operation.