Hans Arnholdt

From latent disseminated cells to overt metastasis: Genetic analysis of systemic breast cancer progression

According to the present view, metastasis marks the end in a sequence of genomic changes underlying the progression of an epithelial cell to a lethal cancer. Here, we aimed to find out at what stage of tumor development transformed cells leave the primary tumor and whether a defined genotype corresponds to metastatic disease. To this end, we isolated single disseminated cancer cells from bone marrow of breast cancer patients and performed single-cell comparative genomic hybridization. We analyzed disseminated tumor cells from patients after curative resection of the primary tumor (stage M0), as presumptive progenitors of manifest metastasis, and from patients with manifest metastasis (stage M1). Their genomic data were compared with those from microdissected areas of matched primary tumors. Disseminated cells from M0-stage patients displayed significantly fewer chromosomal aberrations than primary tumors or cells from M1-stage patients (P < 0.008 and P < 0.0001, respectively), and their aberrations appeared to be randomly generated. In contrast, primary tumors and M1 cells harbored different and characteristic chromosomal imbalances. Moreover, applying machine-learning methods for the classification of the genotypes, we could correctly identify the presence or absence of metastatic disease in a patient on the basis of a single-cell genome. We suggest that in breast cancer, tumor cells may disseminate in a far less progressed genomic state than previously thought, and that they acquire genomic aberrations typical of metastatic cells thereafter. Thus, our data challenge the widely held view that the precursors of metastasis are derived from the most advanced clone within the primary tumor.

Perioperative Activation of Disseminated Tumor Cells in Bone Marrow of Patients With Prostate Cancer

PURPOSE The outcome of prostate cancer is highly unpredictable. To assess the dynamics of systemic disease and to identify patients at high risk for early relapse we followed the fate of disseminated tumor cells in bone marrow for up to 10 years and genetically analyzed such cells isolated at various stages of disease. PATIENTS AND METHODS Nine hundred bone marrow aspirates from 384 patients were stained using the monoclonal antibody A45-B/B3 directed against cytokeratins 8, 18, and 19. Log-rank statistics and Cox regression analysis were applied to determine the prognostic impact of positive cells detected before surgery (244 patients) and postoperatively (214 patients). Samples from primary tumors (n = 55) and single disseminated tumor cells (n = 100) were analyzed by comparative genomic hybridization. RESULTS Detection of cytokeratin-positive cells before surgery was the strongest independent risk factor for metastasis within 48 months (P < .001; relative risk [RR], 5.5; 95% CI, 2.4 to 12.9). In contrast, cytokeratin-positive cells detected 6 months to 10 years after radical prostatectomy were consistently present in bone marrow with a prevalence of approximately 20% but had no influence on disease outcome. Characteristic genotypes of cytokeratin-positive cells were selected at manifestation of metastasis. CONCLUSION Cytokeratin-positive cells in the bone marrow of prostate cancer patients are only prognostically relevant when detected before surgery. Because we could not identify significant genetic differences between pre- and postoperatively isolated tumor cells before manifestation of metastasis, we postulate the existence of perioperative stimuli that activate disseminated tumor cells. Patients with cytokeratin-positive cells in bone marrow before surgery may therefore benefit from adjuvant therapies.

Endoscopic Submucosal Dissection of Early Cancers, Flat Adenomas, and Submucosal Tumors in the Gastrointestinal Tract

BACKGROUND & AIMS Endoscopic submucosal dissection (ESD) is a promising technique in the treatment of large premalignant and early malignant gastrointestinal lesions. In contrast to Japan and Asian countries, few data are available from Western countries. The objective of this study was to assess the feasibility of ESD in a European center, with special regard for the success rate and learning curve. METHODS Over a 4-year-period, 82 epithelial or submucosal lesions were referred for ESD. Seventy-one ESDs were performed (51 gastric, 17 rectal, 2 esophageal, and 1 duodenal). Resection rates, procedure times, specimen sizes, complications, and recurrences were noted. The mean follow-up period was 15 months. RESULTS Specimen size increased significantly (P < .05) and procedural duration decreased significantly (P < .005) over time. En bloc resection rates and R0 en bloc resection rates were 77.1% and 65.7%, respectively, in the first half of the study and increased to 86.1% and 72.2%, respectively, in the second half (P = NS). No recurrence was observed after R0 en bloc resection whereas the recurrence rate was 38.5% after piecemeal resections (P < .001). Two perforations in the first series were treated by surgery; 2 other perforations, 8 minor bleedings, and 2 pyloric stenoses were treated endoscopically. CONCLUSIONS ESD is technically feasible and shows promising results in this German single-center-study. ESD is time consuming and difficult but shows a learning curve resulting in a decrease of the procedural duration over time. R0 en bloc resection is mostly possible and can avoid the risk of local recurrence.

Peripheral nerve sheath tumors of the gastrointestinal tract: a multicenter study of 58 patients including NF1-associated gastric schwannoma and unusual morphologic variants

The frequency and morphological spectrum of gastrointestinal peripheral nerve sheath tumors (PNSTs) from consecutive case material has not been studied in the c-KIT era. We reviewed all mesenchymal gastrointestinal (GI) lesions at our departments according to current diagnostic criteria. PNSTs formed the third commonest group of mesenchymal GI tumors with a lower frequency (≤5%) compared to gastrointestinal stromal tumors (GISTs; ∼50%) and smooth muscle neoplasms (∼30%). Granular cell tumors (GCTs; n = 31) and schwannomas (n = 22) were the most common types of PNSTs encountered. Rare tumors included neurofibromatosis 1 (NF1)-associated PNSTs (n = 5) and gastric perineurioma (n = 1). Thirteen schwannomas (including also some recent cases) were initially diagnosed as GIST, leiomyoma, or neurofibroma. Unusual histological variants included sigmoid GCT with prominent lipomatous component (n = 1), reticular–microcystic schwannoma of small (n = 1) and large (n = 1) bowel, NF1-associated gastric schwannoma (the first case to date), and psammomatous melanotic colonic schwannoma unrelated to Carney complex (n = 1). PNSTs coexisted with GIST in four patients (three had definite NF1). In conclusion, PNSTs of the GI tract are rare uniformly benign neoplasms that may show schwannian, perineurial, fibroblastic, or mixed differentiation. Most of them (92%) occurred sporadically unassociated with NF1 or NF2. Gastrointestinal PNSTs are still underrecognized by general pathologists. Awareness of their diverse morphology will help to avoid confusing them with smooth muscle neoplasms and GIST that they may closely mimic.

Methylene blue injection into the rectal artery as a simple method to improve lymph node harvest in rectal cancer.

Adequate lymph node assessment in colorectal cancer is crucial for prognosis estimation and further therapy stratification. However, there is still an ongoing debate on required minimum lymph node numbers and the necessity of advanced techniques such as immunohistochemistry or PCR. It has been proven in several studies that lymph node harvest is often inadequate under routine analysis. Lymph nodes smaller than 5 mm are especially concerning as they can carry the majority of metastases. These small, but affected lymph nodes may escape detection in routine analysis. Therefore, fat-clearing protocols and sentinel techniques have been developed to improve accuracy of lymph node staging. We describe a novel and simple method of ex vivo methylene blue injection into the superior rectal artery of rectal cancer specimens, which highlights lymph nodes and makes them easy to detect during manual dissection. Initially, this method was developed for proving accuracy of total mesorectal excision. We performed a retrospective study comparing lymph node recovery of 12 methylene blue stained and an equal number of unstained cases. Lymph node recovery differed significantly with average lymph node numbers of 27±7 and 14±4 (P<0.001) for the methylene blue and the unstained group, respectively. The largest difference was found in size groups between 1 and 4 mm causing a shift in size distribution toward smaller nodes. Metastases were confirmed in 21 and 19 lymph nodes occurring in five and four cases, respectively. Hence, we conclude that methylene blue injection technique improves accuracy of lymph node staging by heightening the lymph node harvest in rectal resections. In our experience, it is a very simple time and cost effective method that can be easily established under routine circumstances.

Methylene Blue-Assisted Lymph Node Dissection in Colon Specimens A Prospective, Randomized Study

Recently, we introduced ex vivo intra-arterial methylene blue injection into the inferior mesenteric artery as a novel method to improve lymph node (LN) harvest in rectal cancer. We have now adapted this method to the other segments of the colon. A total of 60 cases were enrolled. Primary LN dissection was followed by fat clearance and a secondary dissection. The mean +/- SD primary LN harvest differed highly significantly with 35 +/- 18 and 17 +/- 10 LNs in the methylene blue-stained and unstained groups, respectively. Primary insufficient LN harvest occurred in 8 cases of the unstained group and in only 1 case of the methylene blue-stained group (P = .0226). After secondary dissection, upstaging was seen exclusively in the unstained group. The time/LN ratio differed significantly with 0.9 and 0.6 min/LN in the unstained and methylene blue-stained groups, respectively. Intraarterial methylene blue injection is recommended as a routine technique in the histopathologic study of colon cancer.

Impaired wound-healing, local eczema, and chronic inflammation following titanium osteosynthesis in a nickel and cobalt-allergic patient: a case report and review of the literature.

Patients known to develop allergic reactions to nickel (Ni), cobalt (Co), or chromium (Cr) often develop eczema in association with items of daily use such as jewelry, earrings, or watchbands. The overall sensitization rates to these metals may range between 1.1% (chromium) and 13% (nickel) in the general population, with further differences based on age and sex1. Chromium-cobalt alloys and stainless steel are widely used as orthopaedic implants and may release nickel, chromium, or cobalt into the surrounding tissues as a consequence of either wear or corrosion2. Some patients with a metal allergy may develop dermatitis in association with orthopaedic implants, and the prevalence of dermal sensitivity in patients with a joint replacement, particularly a failed implant, is higher than that in the general population3. Metal sensitivity rates to nickel, cobalt, or chromium may be as high as 43% in orthopaedic patients with well-functioning implants and as high as 71% in patients with poorly functioning implants3. In contrast, because of their excellent biocompatibility, titanium (Ti)-based materials are not considered to provoke allergic reactions. Our patient developed eczema and impaired wound-healing following the fixation of an ankle fracture with titanium-based implants. Histological analysis of the tissue around the implant demonstrated inflammation primarily with lymphocytes, and a contact allergy to nickel and cobalt was found in the absence of titanium hyperreactivity, raising the question of a prior unknown nickel exposure as the source of the complications. The patient was informed that data concerning this case would be submitted for publication, and she consented. A fifty-nine-year-old woman with a trimalleolar ankle fracture with rupture of the syndesmosis (Fig. 1) underwent open reduction and internal fixation of the fractures with a titanium plate (catalogue number 441.370; Synthes, STRATEC Medical, Oberdorf, Switzerland) and screws (catalogue numbers 404.812, …

The clinical significance of lymph node size in colon cancer

To date, the clinical value of lymph node size in colon cancer has been investigated only in a few studies. Only in radiological diagnosis is lymph node size routinely recognized, and nodes ≥10 mm in diameter are considered pathologic. However, the few studies regarding this topic suggest that lymph node size is not a reliable indicator of metastatic disease. Moreover, we hypothesized that increasing lymph node size is associated with favorable outcome. By performing a morphometric study, we investigated the clinical significance of lymph node size in colon cancer in terms of metastatic disease and prognosis. A cohort of 237 cases with excellent lymph node harvest (mean lymph node count: 33±17) was used. The size distribution in node-positive and -negative cases was almost identical. In all, 151 out of the 305 metastases detected (49.5%) were found in lymph nodes with diameters ≤5 mm. Only 25% of lymph nodes >10 mm showed metastases. Minute lymph nodes ≤1 mm were involved only very rarely (2 of 81 cases). In 67% of the cases, the largest positive lymph node was <10 mm. The prognostic relevance of lymph node size was investigated in a subset of 115 stage I/II cases. The occurrence of ≥7 lymph nodes that were >5 mm in diameter was significantly associated with better overall survival. Our data show that lymph node size is not a suitable factor for preoperative lymph node staging. Minute lymph nodes have virtually no role in correct histopathological lymph node staging. Finally, large lymph nodes in stage I/II disease might indicate a favorable outcome.

Injecting methylene blue into the inferior mesenteric artery assures an adequate lymph node harvest and eliminates pathologist variability in nodal staging for rectal cancer.

PURPOSE: The American Joint Committee on Cancer recommends examination of a minimum of 12 lymph nodes in rectal cancer for accurate staging. Despite this, several studies have demonstrated that nodal harvest is highly variable and often inadequate. This study was designed to determine if staining the nodes with methylene blue dye produced a better and more accurate harvest in comparison with standard pathologic lymph node dissection. METHODS: Fifty patients with primary resectable rectal cancer were randomly assigned to undergo a standard nodal harvest or a harvest after ex vivo injection of the inferior mesenteric artery with methylene blue. A fat clearance technique was subsequently used to identify the maximum possible number of lymph nodes and metastasis. RESULTS: The average lymph node harvest was 30 ± 13.5 in the stained group and 17 ± 11 in the unstained group (P < 0.001). At least 12 nodes were identified in every case in the stained group. In the unstained group, 7 of 25 cases (28 percent) did not meet the minimum criteria of 12 nodes (P < 0.01). Among the pathologists for the stained group, no difference was found in the harvest (P < 0.05), but variability was detected between the pathologists in the unstained group (P = 0.6). After fat clearance, one case in the unstained group was upstaged, whereas no cases in the stained group were upstaged. CONCLUSIONS: Staining the lymph nodes with methylene blue dye is an accurate staging technique and reliably produces an adequate harvest.

Tumour budding, uPA and PAI‐1 are associated with aggressive behaviour in colon cancer

The proteases PAI‐1 and uPA play a major role in extracellular matrix degradation, which facilitates tumour progression. Tumour budding is a histomorphological expression of enhanced tumour cell migration.