Mini-laparoscopy as a diagnostic tool for abdominal tuberculosis: a retrospective series of 29 cases.

OBJECTIVES: Abdominal tuberculosis (TB) is a "great mimic," and diagnosis remains challenging even for experienced clinicians. While mini-laparoscopy has already been demonstrated to be an efficient diagnostic tool for a variety of diseases, we aimed to demonstrate the feasibility of this technique in diagnosing abdominal TB. METHODS: We retrospectively included patients who underwent mini-laparoscopy at the University Medical Center Hamburg-Eppendorf between April 2010 and January 2022 for suspected abdominal TB. Demographic, clinical, and laboratory data, radiological findings as well as macroscopic, histopathologic, and microbiologic results were analyzed by chart review. RESULTS: Out of 49 consecutive patients who underwent mini-laparoscopy for suspected abdominal TB, the diagnosis was subsequently confirmed in 29 patients (59%). Among those, the median age was 30 years (range 18-86 years) and the majority were male (n = 22, 76%). Microbiological diagnosis was established in a total of 16 patients. The remaining patients were diagnosed with abdominal TB either by histopathological detection of caseating granulomas (n = 3), or clinically by a combination of typical presentation, mini-laparoscopic findings, and good response to anti-tuberculous treatment (n = 10). Bleeding from the respective puncture site occurred in 19 patients (66%) and either resolved spontaneously or was arrested with argon plasma coagulation alone (n = 10) or in combination with fibrin glue (n = 1). Minor intestinal perforation occurred in 2 patients and was treated conservatively. CONCLUSIONS: Mini-laparoscopy is a useful and safe modality for the diagnosis of abdominal TB.

Outcomes following oropharyngeal squamous cell carcinoma resection and bilateral neck dissection with or without contralateral postoperative radiotherapy of the pathologically node-negative neck.

PURPOSE: There are no consensus guidelines regarding the postoperative treatment of the contralateral pathologically node-negative neck in oropharyngeal squamous cell carcinoma. This study aimed to determine if omission of postoperative irradiation of the contralateral pathologically node-negative neck affects oncological outcomes. METHODS: We retrospectively identified 84 patients with primary surgical treatment including bilateral neck dissection and postoperative (chemo-)radiotherapy (PO(C)RT). Survival was analyzed using the log-rank test and the Kaplan-Meier method. RESULTS: Patients showed no decrease in tumor-free, cause-specific (CSS), or overall survival (OS) when PO(C)RT of the contralateral pathologically node-negative neck was omitted. Increased OS was found in patients with unilateral PO(C)RT and especially an increased OS and CSS was found in unilateral PO(C)RT and in tumors arising from lymphoepithelial tissue. CONCLUSIONS: Omitting the contralateral pathologically node-negative neck appears to be safe in terms of survival and our retrospective study advocates further prospective randomized control de-escalation trials.

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

Gastric NET Subtypes: Do We Need An Additional One?

Depending on etiology, prognosis and malignant potential, recent S2k guideline differentiates gastric neuroendocrine tumors (gNET) in 4 types with different treatment implications.We report on a 55-year-old patient with the accidental finding of a 15 mm gNET. Apart from a prolonged use of proton pump inhibitors (PPI) for 20 years as a treatment for gastroesophageal reflux disease, there were no other associations or risk factors for gNETs. Formally, this patient would have been classified as a type III gNET, implicating gastric surgery. From a pathophysiological point of view, however, the assumed prolonged gastrin hypersecretion would have justified an assignment as a type I gNET. The gNET was resected by ESD, but histology showed an R1 situation. After cessation of PPIs, there is no recurrence so far. Besides, the initially documented numerous and large gland polyps showed an impressive regression only a few weeks after cessation of PPI.This case points to a probably underestimated gap in the present gNET classification. On the basis of present literature, the therapeutic dilemma of PPI-associated gNETs is discussed. A new assignment of PPI associated gNETs as type Ib could help to overcome this dilemma.

[Unusual cause of hypercalcemia in pregnancy]. / Ungewöhnliche Ursache einer Hyperkalzämie in der Schwangerschaft.

BACKGROUND: Neuroendocrine tumors (NET) diagnosed during pregnancy are extremely rare. This case report describes diagnosis and treatment of a metastasized pancreas NET that became symptomatic in the second trimester. CASE DESCRIPTION: A 33-year-old patient presented to the emergency department in the 19th week of pregnancy (WOP) with persistent diarrhea. Laboratory tests showed a pronounced hypercalcemia (3.53 mmol/l). Imaging revealed a mass in the pancreatic corpus/tail with extensive liver metastasis. Histologically, a NET (G2, SSTR-positive) with paraneoplastic parathormone-related-peptide secretion was found to be the cause of hypercalcemia. Under a treatment with octreotide, calcium values normalized and diarrhea stopped. After delivery of a healthy child (32.WOP via cesarean section) tumor progress was found. The pancreatic mass was resected completely, the liver metastases as far as possible. Postoperatively, in a CT scan, residual suspicious liver lesions could be found, and a palliative therapy with lanreotide was initiated. With this treatment, the patient has been asymptomatic for one year, and serum calcium remained normal. The child developed normally. DISCUSSION: This unusual case shows that even in extensively metastasized symptomatic NETs during pregnancy, there may be sufficient diagnostic and therapeutic options that allow for a continuation of pregnancy in close interdisciplinary cooperation under careful risk-benefit assessment for mother and child.

High homogeneity of MMR deficiency in ovarian cancer.

OBJECTIVE: Mismatch repair (MMR) deficiency and Bethesda panel microsatellite instability (MSI) are increasingly analyzed to identify tumors that might benefit from immune checkpoint inhibitors, but tumor heterogeneity is a potential obstacle for such analyses. In ovarian cancer, data on intratumoral heterogeneity of MMR deficiency/MSI are lacking. METHODS: N = 582 ovarian cancers were screened for MMR deficiency by immunohistochemistry (IHC) on a tissue microarray. 10 cases suspect for MMR deficiency were identified among 478 interpretable cancers and repeated IHC on large sections combined with polymerase chain reaction (PCR)-based MSI analysis validated MMR deficiency/MSI in 9 of these tumors. RESULTS: MMR deficiency/MSI was predominantly seen in endmetrioid cancers (8 of 35, 23%) and also in 1 of 358 serous carcinomas (0.3%), but was absent in 34 mucinous carcinomas, 23 clear cell carcinomas, 17 malignant mixed Mullerian tumors (carcinosarcomas), and 11 mixed carcinomas. MMR deficiency involed protein loss of PMS2/MLH1 in 6 cases and of MSH2 and/or MSH6 in 3 cases. 7 MMR deficient cancers were MSI-high (all endometrioid), one was MSI-low (endometrioid) and one cancer with unequivocal MMR protein loss exhibited microsatellite stability (serous). MLH1 promotor methylation was observed in 4 of 5 endometrioid cancers with MLH1 protein loss. Immunostaining of all available cancer-containing tissue blocks (n = 114) of tumors with confirmed MMR deficiency/MSI revealed uniform MMR status throughout the entire tumor mass. CONCLUSIONS: Our data show that MSI is present in a substantial proportion of endometrioid ovarian cancers but can also occur in other tumor subtypes. MMR deficiency/MSI typically involves the entire tumor mass, suggesting that MMR inactivation occurs early in tumorigenesis in a subset of ovarian cancers.

Using Nonexpert Online Reports to Enhance Expert Knowledge About Causes of Death in Dental Offices Reported in Scientific Publications: Qualitative and Quantitative Content Analysis and Search Engine Analysis.

BACKGROUND: Fatalities rarely occur in dental offices. Implications for clinicians may be deduced from scientific publications and internet reports about deaths in dental offices. OBJECTIVE: Data involving deaths in dental facilities were analyzed using Google as well as the PubMed database. By comparing both sources, we examined how internet data may enhance knowledge about deaths in dental offices obtained from scientific medical publications, which causes of death are published online, and how associated life-threatening emergencies may be prevented. METHODS: To retrieve relevant information, we searched Google for country-specific incidents of death in dental practices using the following keywords: "death at the dentist," "death in dental practice," and "dying at the dentist." For PubMed searches, the following keywords were used: "dentistry and mortality," "death and dental treatment," "dentistry and fatal outcome," and "death and dentistry." Deaths associated with dental treatment in a dental facility, attributable causes of death, and documented ages of the deceased were included in our analysis. Deaths occurring in maxillofacial surgery or pre-existing diseases involved in the death (eg, cancer and abscesses) were excluded. A total of 128 cases from online publications and 71 cases from PubMed publications that met the inclusion criteria were analyzed using chi-square statistics after exclusion of duplicates. RESULTS: The comparison between the fatalities from internet (n=117) and PubMed (n=71) publications revealed that more casualties affecting minors appeared online than in PubMed literature (online 68/117, 58.1%; PubMed 20/71, 28%; P<.001). In PubMed articles, 10 fatalities in patients older than 70 years of age were described, while online sources published 5 fatalities (P=.02). Most deaths, both from internet publications and PubMed literature, could be assigned to the category anesthesia, medication, or sedation (online 80/117, 68.4%; PubMed 25/71, 35%; P<.001). Deaths assigned to the categories infection and cardiovascular system appeared more often in the PubMed literature (infection: online 10/117, 8.5%; PubMed 15/71, 21%; P=.01; cardiovascular system: online 5/117, 4.3%; PubMed 15/71, 21%; P<.001). Furthermore, sedative drugs were involved in a larger proportion of fatal incidents listed online compared to in PubMed (online 41/117, 35.0%; PubMed: 14/71, 20%, P=.03). In the United States, more deaths occurred under sedation (44/96, 46%) compared to those in the other countries (Germany and Austria 1/17, 6%, P=.002; United Kingdom 1/14, 7%, P=.006). CONCLUSIONS: Online and PubMed databases may increase awareness of life-threatening risks for patients during dental treatment. Negative aspects of anesthesia and sedation, as well as the number of deaths of young patients, were underestimated when reviewing PubMed literature only. Medical history of patients, medication dosages, and vital function monitoring are significant issues for practitioners. A high-impact finding from online reports was the underestimation of risks when performing sedation and even general anesthesia. Detailed knowledge of the definition and understanding of deep sedation and general anesthesia by dentists is of major concern. By avoiding potentially hazardous procedures, such as sedation-aided treatments performed solely by dentists, the risk of treatment-induced life-threatening emergencies may be reduced.

Survivin expression in head and neck squamous cell carcinomas is frequent and correlates with clinical parameters and treatment outcomes.

OBJECTIVES: Strong expression of survivin is associated with worse survival in many different tumours, and in cell culture, a correlation between radiation resistance and survivin expression can be seen. The potential of survivin expression as a prognostic/predictive marker or therapeutic target has not been examined in head and neck squamous cell carcinomas (HNSCC) yet. MATERIAL AND METHODS: Retrospective study of 452 tissue samples and clinical data from patients with squamous cell carcinomas of the larynx/hypopharynx (LSCC), oral cavity (OSCC) and oropharynx (OPSCC) treated in the University Medical Centre Hamburg-Eppendorf between 2002 and 2006. The expression patterns were detected by tissue microarray technique and correlated with clinical parameters (sex, age, tumour location, TNM 7th edition, grading, recurrence-free and overall survival). RESULTS: 222 OSCC, 126 OPSCC and 105 LSCC tumours of 118 females and 335 males with a mean follow-up of 41.3 months were examined. Survivin expression correlates with pN, cM, pT and overall survival. CONCLUSION AND CLINICAL RELEVANCE: The potential of survivin as a prognostic/predictive marker is very high. The findings have to be confirmed in a larger cohort of HNSCC esp. in those tumours treated primarily with radio/radiochemotherapy.

Immunohistochemically detected IDH1R132H mutation is rare and mostly heterogeneous in prostate cancer.

BACKGROUND: IDH1 mutations are oncogenic through induction of DNA damage and genome instability. They are of therapeutic interest because they confer increased sensitivity to radiation and cytotoxic therapy and hold potential for vaccination therapy. METHODS: In this study, we analyzed more than 17,000 primary prostate cancer tissues with a mutation-specific antibody for the IDH1R132H mutation. RESULTS: IDH1 mutation-specific staining was found in 42 of 15,531 (0.3%) interpretable cancers. IDH1 mutation was associated with higher preoperative PSA and Gleason grade (p < 0.05, each) but was unrelated to PSA recurrence. A comparison with other molecular tumor features available from earlier studies revealed that TMPRSS2-ERG fusion as well as deletion of PTEN, 5q21, 6q15, and 3p13 was less frequent in IDH1-mutated than in non-mutated cancer. Increased lethality of genetically instable, "aberration-rich" cancer cells in the presence of IDH1 mutations could possibly explain this observation. Heterogeneity analysis revealed a homogeneous mutation in only 1 of 16 IDH1-mutated cancers. This high degree of heterogeneity may profoundly limit therapeutic targeting of IDH1 mutations in prostate cancer. CONCLUSIONS: The data show that 0.3% of prostate cancers have an IDH1R132H mutation and that these are mostly heterogeneous. Once specific anti-IDH1 therapy becomes reality, only a very small group of prostate cancer patients may benefit from such a treatment.

Up-regulation of mismatch repair genes MSH6, PMS2 and MLH1 parallels development of genetic instability and is linked to tumor aggressiveness and early PSA recurrence in prostate cancer.

DNA mismatch repair (MMR) is integral to the maintenance of genetic stability. We aimed to evaluate the clinical impact of MMR gene expression in prostate cancer. The MMR genes MSH6, MLH1 and PMS2 were analyzed by immunohistochemistry on a tissue microarray containing 11152 prostate cancer specimens. Results were compared with ETS-related gene status and deletions of PTEN, 3p13, 5q21 and 6q15. MSH6, MLH1 and PMS2 expression was detectable in 89.5%, 85.4% and 85.0% of cancers and was particularly strong in cancers with advanced pathological tumor stage (P < 0.0001 each), high Gleason grade (P < 0.0001 each), nodal metastasis (P ≤ 0.0083) and early biochemical recurrence (P < 0.0001). High levels of MMR gene expression paralleled features of genetic instability, such as the number of genomic deletions per cancer; strong expression of all three MMR genes was found in 24%, 29%, 30%, 33% and 42% of cancers with no, one, two, three or four to five deletions (P < 0.0001). The prognostic value of the analyzed MMR genes was largely driven by the subset of cancers lacking ERG fusion (P < 0.0001), while the prognostic impact of MMR gene overexpression was only marginal in ERG-positive cancers. Multivariate analyses suggested an independent prognostic relevance of MMR genes in ERG-negative prostate cancers when compared with prognostic parameters available at the time of initial biopsy. In conclusion, MMR overexpression is common in prostate cancer and is linked to poor outcome as well as features indicating genetic instability. ERG fusion should be analyzed along with MMR gene expression in potential clinical tests.

Abundant expression of mTOR kinase in salivary gland tumors - potentials as therapy target?

BACKGROUND: Salivary gland tumors constitute 3-6% of all head and neck neoplasms in adults. Because of limited advances made in the treatment of metastatic disease, the more important is the role of new therapeutic strategies, including molecular therapy. The mammalian target of rapamycin (mTOR) has recently been established as a therapeutic target for several drugs. MATERIAL: Evaluation of phospho-mTOR as a possible therapy target by patients with salivary gland tumors. Immunohistochemical semi-quantitative analyses of the expression of phospho-mTOR(Ser2448) were processed on a tissue microarray containing samples from more than 900 patients. For statistical analysis, contingency table and chi-squared test (likelihood) were used. RESULTS: We observed at least weak phospho-mTOR expression in 25.6-41.2% of all 4 histological adenoma and in 36.8-61.6% of all 11 histological carcinoma subtypes analyzed. No association was seen between phospho-mTOR expression and tumor grade in mucoepidermoid carcinomas. CONCLUSION: In conjunction with literature data providing the evidence for a functional role of mTOR in salivary gland tumors, we conclude that treatment with mTOR-antagonists might potentially also be efficient in wide variety of salivary gland carcinomas.

Reduction of gastric inflation by use of a pressure relief valve connected to an acoustic expiration control device during mouth-to-mask ventilation of a manikin model of an unprotected airway.

BACKGROUND: Gastric inflation is a significant issue when ventilation of the unprotected airway is performed. The purpose of this study was to evaluate a prototype pressure relief valve with an acoustic expiration control mechanism connected to two different masks. METHODS: 12 non-physician healthcare professionals (group 1) and 10 newly certified dentists (group 2) performed 10 cycles of cardiopulmonary resuscitation on a manikin using this device compared with mouth-to-mouth technique. Dentists also employed a mask without the valve. Lower oesophageal sphincter pressures had been adjusted to 1.5 kPa (group 1) and 0.3 kPa (group 2); the valve relief pressure to 1.5 kPa (group 1) and 2.0 kPa (group 2). RESULTS: In group 1 tidal volumes by valve mask techniques (medians 350 and 400 ml) differed minimally from mouth-to-mouth ventilation (medians 475 and 600 ml). Almost no gastric inflation was observed. Gastric inflation only occurred using the safety valve connected to the mask (median 122 ml) and the mask alone (median 260 ml) (p=0.004). Only in group 1 the acoustic device delivered adequate signals. CONCLUSION: Gastric inflation occurred less frequently and to a lesser extent when the valve was connected, going ahead with a trend towards lower tidal volumes. The protective effect of the safety valve may be of benefit even if it leads to smaller tidal volumes.

The prognostic impact of B7-H3 and B7-H4 in head and neck squamous cell carcinoma.

PURPOSE: Immune checkpoint inhibition is a therapeutic option in many cancer entities. In head and neck squamous cell carcinoma (HNSCC) targeting of the PD-1/PD-L1 (B7-H1) axis is approved in recurrent/metastatic disease and is being explored in the curative setting. Here, we evaluated two related members of the B7 family, B7-H3 & B7-H4, for their prognostic impact under standard treatment. METHODS: A tissue microarray (TMA) of a single center HNSCC cohort was stained for B7-H3 and B7-H4. Staining intensity and the number of tumor cells stained were assessed, and the expression was scored according to an established algorithm. Staining scores were correlated with clinicopathological parameters and associated with patient survival. mRNA levels of both proteins were associated with patient outcome using the TCGA dataset. RESULTS: mRNA levels of B7-H3 and B7-H4 were not significantly associated with patient survival. TMA analysis revealed interpretable protein staining in 408 samples. Strong staining was the most frequent category for B7-H3 and no staining for B7-H4. In patients with p16-negative oropharyngeal SCC (OPSCC) and in a pooled cohort consisting of p16-negative OPSCC, laryngeal, hypopharyngeal and oral cavity SCC, strong B7-H3 expression was associated with better overall survival. For the latter cohort, this was in part due to reduced lymph node involvement. B7-H3 expression in p16-positive OPSCC and B7-H4 expression were not associated with outcome. CONCLUSION: Despite a possible role in tumor immune escape, B7-H3 was associated with favorable prognosis in HPV-negative HNSCC in our cohort. The underlying mechanisms and a potential impact for B7-H3 targeting remain to be elucidated.

Mismatch repair deficiency occurs very rarely in seminomas.

BACKGROUND: Dense tumor-associated lymphocyte infiltration is linked to mismatch repair (MMR) deficiency in colorectal and endometrial cancer. MMR deficiency is of high clinical importance as MMR deficient cancers tend to react favorably to treatment with immune checkpoint inhibitors. Strong lymphocytic infiltration is a morphological hallmark of seminomas. We thus asked whether seminomas may exhibit MMR deficiency at relevant frequency. METHODS: To screen for tumors with MMR deficiency, protein expression of MLH1, PMS2, MSH2, and MSH6 was analyzed by immunohistochemistry (IHC) on a tissue microarray (TMA) containing 574 seminomas. RESULTS: In total, 536 cases were evaluable resulting in 481 seminomas with unequivocally intact MMR protein expression. In 55 cancers, one or several IHC stains were equivocal and lacked detectable MMR protein in both tumor and stromal cells. Large section IHC analysis of all 55 equivocal cases demonstrated substantial staining issues due to improper fixation in 54 cases and identified one tumor with clear-cut MLH1 and PMS2 protein loss. This seminoma showed homogeneous loss of MLH1 and PMS2 in the entire tumor mass whereas minor adjacent foci of associated germ cell neoplasia in situ (GCNIS) were MMR intact. Polymerase chain reaction (PCR) analysis using the 5 microsatellite loci of the "Bethesda Panel" revealed instability in 1 of 4 interpretable loci ("MSI-low") and additional instability of the complex tetra-penta repeat locus MYCL1 in this tumor. CONCLUSIONS: In summary, one single seminoma with MMR deficiency, characterized by protein loss of MLH1 and PMS2, was identified among 536 interpretable seminomas (0.19%). MMR deficiency is not a relevant determinant of lymphocyte influx in seminoma.

E-Cadherin expression in human tumors: a tissue microarray study on 10,851 tumors.

BACKGROUND: The E-Cadherin gene (CDH1, Cadherin 1), located at 16q22.1 encodes for a calcium-dependent membranous glycoprotein with an important role in cellular adhesion and polarity maintenance. METHODS: To systematically determine E-Cadherin protein expression in normal and cancerous tissues, 14,637 tumor samples from 112 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. RESULTS: E-Cadherin was strongly expressed in normal epithelial cells of most organs. From 77 tumor entities derived from cell types normally positive for E-Cadherin, 35 (45.5%) retained at least a weak E-Cadherin immunostaining in ≥99% of cases and 61 (79.2%) in ≥90% of cases. Tumors with the highest rates of E-Cadherin loss included Merkel cell carcinoma, anaplastic thyroid carcinoma, lobular carcinoma of the breast, and sarcomatoid and small cell neuroendocrine carcinomas of the urinary bladder. Reduced E-Cadherin expression was linked to higher grade (p = 0.0009), triple negative receptor status (p = 0.0336), and poor prognosis (p = 0.0466) in invasive breast carcinoma of no special type, triple negative receptor status in lobular carcinoma of the breast (p = 0.0454), advanced pT stage (p = 0.0047) and lymph node metastasis in colorectal cancer (p < 0.0001), and was more common in recurrent than in primary prostate cancer (p < 0.0001). Of 29 tumor entities derived from E-Cadherin negative normal tissues, a weak to strong E-Cadherin staining could be detected in at least 10% of cases in 15 different tumor entities (51.7%). Tumors with the highest frequency of E-Cadherin upregulation included various subtypes of testicular germ cell tumors and renal cell carcinomas (RCC). E-Cadherin upregulation was more commonly seen in malignant than in benign soft tissue tumors (p = 0.0104) and was associated with advanced tumor stage (p = 0.0276) and higher grade (p = 0.0035) in clear cell RCC, and linked to advanced tumor stage (p = 0.0424) and poor prognosis in papillary RCC (p ≤ 0.05). CONCLUSION: E-Cadherin is consistently expressed in various epithelial cancers. Down-regulation or loss of E-Cadherin expression in cancers arising from E-Cadherin positive tissues as well as E-Cadherin neo-expression in cancers arising from E-Cadherin negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.

Protein Sequence Analysis Using the MPI Bioinformatics Toolkit.

The MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) provides interactive access to a wide range of the best-performing bioinformatics tools and databases, including the state-of-the-art protein sequence comparison methods HHblits and HHpred. The Toolkit currently includes 35 external and in-house tools, covering functionalities such as sequence similarity searching, prediction of sequence features, and sequence classification. Due to this breadth of functionality, the tight interconnection of its constituent tools, and its ease of use, the Toolkit has become an important resource for biomedical research and for teaching protein sequence analysis to students in the life sciences. In this article, we provide detailed information on utilizing the three most widely accessed tools within the Toolkit: HHpred for the detection of homologs, HHpred in conjunction with MODELLER for structure prediction and homology modeling, and CLANS for the visualization of relationships in large sequence datasets. © 2020 The Authors. Basic Protocol 1: Sequence similarity searching using HHpred Alternate Protocol: Pairwise sequence comparison using HHpred Support Protocol: Building a custom multiple sequence alignment using PSI-BLAST and forwarding it as input to HHpred Basic Protocol 2: Calculation of homology models using HHpred and MODELLER Basic Protocol 3: Cluster analysis using CLANS.

High homogeneity of mismatch repair deficiency in advanced prostate cancer.

BACKGROUND: Recent reports have described favorable response rates for immune checkpoint inhibitors in prostate cancers with microsatellite instability (MSI). However, it is unclear whether MSI affects the entire tumor mass or is distributed heterogeneously, the latter potentially impairing treatment efficiency. METHODS: To identify prostate cancers with MSI, 316 advanced prostate cancers were analyzed by immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6 on a TMA format. RESULTS: Out of 200 interpretable cancers, IHC findings were consistent with MSI in 10 tumors. In 9 of these 10 cancers, tissue blocks were available for subsequent large section IHC, confirming MSI in 6 cases, each with combined protein loss of MSH2 and MSH6. One additional tumor with unequivocal loss of MLH1 and PMS2 on the TMA, for which further analyses could not be carried out due to lack of tissue, was also considered to exhibit MSI. In total, 7 of 200 interpretable advanced prostate cancers were found to exhibit MMR deficiency/MSI (3.5%). Subsequent analysis of all available cancer-containing archived tissue blocks (n=114) revealed consistent and homogeneous MMR protein loss in each case. Polymerase chain reaction (PCR)-based analysis using the "Bethesda panel" could be executed in 6 MMR deficient tumors of which 4 were MSI-high and 2 were MSI-low. CONCLUSIONS: The absence of intratumoral heterogeneity for the MMR status suggests that MSI occurs early in prostate cancer. It is concluded that MMR analysis on limited biopsy material by IHC is sufficient to estimate the MMR status of the entire cancer mass.

Homogeneous MMR Deficiency Throughout the Entire Tumor Mass Occurs in a Subset of Colorectal Neuroendocrine Carcinomas.

Neuroendocrine neoplasms comprise a heterogeneous group of tumors, categorized into neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) depending on tumor differentiation. NECs and high-grade NETs (G3) confer a poor prognosis, demanding novel treatment strategies such as immune checkpoint inhibition in tumors with microsatellite instability (MSI). To study any possible intratumoral heterogeneity of MSI, a tissue microarray (TMA) containing 199 NETs and 40 NECs was constructed to screen for MSI using immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. Four cases suspicious for MSI were identified. Validation of MSI by repeated IHC on large sections and polymerase chain reaction (PCR)-based analysis using the "Bethesda Panel" confirmed MSI in 3 cecal NECs. One pancreatic NET G3 with MSI-compatible TMA results was MMR intact on large section IHC and microsatellite stable (MSS). The remaining 235 tumors exhibited intact MMR. Protein loss of MLH1/PMS2 was found in two and MSH6 loss in one cancer with MSI. Large section IHC on all available tumor-containing tissue blocks in NECs with MSI did not identify aberrant tumor areas with intact MMR. Our data indicate that MSI is common in colorectal NECs (3 out of 10) but highly infrequent in neuroendocrine neoplasms from many other sites. The lack of intratumoral heterogeneity of MMR deficiency suggests early development of MSI during tumorigenesis in a subset of colorectal NECs and indicates that microsatellite status obtained from small biopsies may be representative for the entire cancer mass.

MMR deficiency in urothelial carcinoma of the bladder presents with temporal and spatial homogeneity throughout the tumor mass.

BACKGROUND: Microsatellite instability (MSI), a hypermutator phenotype described in many cancers, has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in bladder cancer, but data on the possible extent of intratumoral heterogeneity are lacking. METHODS: To study MSI heterogeneity in bladder cancer, a tissue microarray (TMA) comprising 598 muscle-invasive urothelial carcinomas of the bladder was utilized to screen for MSI by immunhistochemistry with antibodies for MLH1, PMS2, MSH2, and MSH6. RESULTS: In 9 cases suspicious for MSI, MMR status was further evaluated by large section examination and polymerase chain reaction (PCR)-based analysis of microsatellites ("Bethesda panel") resulting in the identification of 5 validated MSI cases from 448 interpretable cancers (prevalence 1.1%). MMR deficiency always involved PMS2 loss, in 3 cases with additional loss or reduction of MLH1 expression. Four cancers were MSI-high and 1 was MSI-low in the PCR analysis. Parallel sequencing revealed an inactivating MLH1 mutation in 1 tumor but no further known pathogenic MMR gene mutations were found. Immunostaining of all available 72 cancer-containing tissue blocks of the 5 confirmed bladder cancer with MSI including prior and subsequent biopsies showed complete homogeneity of the MMR protein defects and the status of the 4 MMR proteins did not markedly change in sequential resections. In all 4 cases with noninvasive precursor lesions, MSI was also detectable. CONCLUSION: These data suggest that MSI occurs early in invasive bladder cancer and immunohistochemical MMR analysis on limited biopsy material is sufficient to estimate MMR status of the entire cancer mass.

Mass Spectrometric Comparison of HPV-Positive and HPV-Negative Oropharyngeal Cancer.

Squamous cell carcinoma of the head and neck (HNSCC) consist of two distinct biological entities. While the numbers of classical, tobacco-induced HNSCC are declining, tumors caused by human papillomavirus (HPV) infection are increasing in many countries. HPV-positive HNSCC mostly arise in the oropharynx and are characterized by an enhanced sensitivity towards radiotherapy and a favorable prognosis. To identify molecular differences between both entities on the protein level, we conducted a mass spectrometric comparison of eight HPV-positive and nine HPV-negative oropharyngeal tumors (OPSCC). Overall, we identified 2051 proteins, of which 31 were found to be differentially expressed. Seventeen of these can be assorted to three functional groups, namely DNA replication, nuclear architecture and cytoskeleton regulation, with the differences in the last group potentially reflecting an enhanced migratory and invasive capacity. Furthermore, a number of identified proteins have been described to directly impact on DNA double-strand break repair or radiation sensitivity (e.g., SLC3A2, cortactin, RBBP4, Numa1), offering explanations for the differential prognosis. The unequal expression of three proteins (SLC3A2, MCM2 and lamin B1) was confirmed by immunohistochemical staining using a tissue microarray containing 205 OPSCC samples. The expression levels of SLC3A2 and lamin B1 were found be of prognostic relevance in patients with HPV-positive and HPV-negative OPSCC, respectively.