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Adenoid cystic carcinoma (ACC) is a MYB-driven head and neck malignancy with high rates of local recurrence and distant metastasis and poor long-term survival. New effective targeted therapies and clinically useful biomarkers for patient stratification are needed to improve ACC patient survival. Here, we present an integrated copy number and transcriptomic analysis of ACC to identify novel driver genes and prognostic biomarkers. A total of 598 ACCs were studied. Clinical follow-up was available from 366 patients, the largest cohort analyzed to date. Copy number losses of 1p36 (70/492; 14%) and of the tumor suppressor gene PARK2 (6q26) (85/343; 25%) were prognostic biomarkers; patients with concurrent losses (n = 20) had significantly shorter overall survival (OS) than those with one or no deletions (p < 0.0001). Deletion of 1p36 independently predicted short OS in multivariate analysis (p = 0.02). Two pro-apoptotic genes, TP73 and KIF1B, were identified as putative 1p36 tumor suppressor genes whose reduced expression was associated with poor survival and increased resistance to apoptosis. PARK2 expression was markedly reduced in tumors with 6q deletions, and PARK2 knockdown increased spherogenesis and decreased apoptosis, indicating that PARK2 is a tumor suppressor in ACC. Moreover, analysis of the global gene expression pattern in 30 ACCs revealed a transcriptomic signature associated with short OS, multiple copy number alterations including 1p36 deletions, and reduced expression of TP73. Taken together, the results indicate that TP73 and PARK2 are novel putative tumor suppressor genes and potential prognostic biomarkers in ACC. Our studies provide new important insights into the pathogenesis of ACC. The results have important implications for biomarker-driven stratification of patients in clinical trials. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Adenoid cystic carcinoma (ACC) is an aggressive head and neck malignancy characterized by a t (6;9) translocation resulting in an MYB-NFIB gene fusion or, more rarely, an MYBL1 fusion. The true frequency and clinical significance of these alterations are still unclear. Here, we have used tissue microarrays and analyzed 391 ACCs and 647 non-ACC salivary neoplasms to study the prevalence, expression, and clinical significance of MYB/MYBL1 alterations by FISH and immunohistochemistry. Alterations of MYB or MYBL1 were found in 78% of the cases, of which 62% had MYB alterations and 16% had MYBL1 rearrangements. Overexpression of MYB/MYBL1 oncoproteins was detected in 93% of the cases. MYB split signal, seen in 39% of the cases, was specific for ACC and not encountered in non-ACC salivary tumors. Loss of the 3'-part of MYB was enriched in grade 3 tumors and was a significant independent prognostic biomarker for overall survival in multivariate analyses. We hypothesize that loss of the 3'-part of MYB results from an unbalanced t(6;9) leading to an MYB-NFIB fusion with concomitant loss of the segment distal to the MYB breakpoint in 6q23.3. Our study provides new knowledge about the prevalence and clinical significance of MYB/MYBL1 alterations and indicates the presence of genes with tumor suppressive functions in 6q23.3-qter that contribute to poor prognosis and short overall survival in ACC.

Background/aim: Polymorphous adenocarcinoma (PAC) is a low-grade salivary gland malignancy in contrast to variants with papillary (PAP) or cribriform (CASG) architecture and confers the second most common malignancy of minor salivary glands. Our study aimed to identify prognostic factors and to evaluate histomorphological and molecular diagnostic criteria of PACs., Patients and Methods: A series of 155 PACs, including 10 PAPs and 12 CASGs from the population-based Cancer Registry of North Rhine-Westphalia (LKR-NRW) and the Hamburg Salivary Gland Reference Centre (HRC) were analyzed., Results: One fifth of the tumors were located in the major salivary glands and PACS/CASGS invariably lacked p40 expression. Fifty-two percent of PACs showed a PRKD1 E710D mutation. Ordinary PACs had a disease-specific 10-year survival probability of 97% compared to 90% when combining PAPs and CASGs. T-stage at diagnosis was a prognostic factor with 98% for stages T1/T2 versus 75% for T3/T4., Conclusion: Diagnostic algorithms for the PAC/CASG spectrum of tumors need to be improved and should include molecular markers., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Abstract: Extraocular sebaceous carcinoma (ESC) is a rare appendiceal skin tumor. In contrast to ocular sebaceous carcinoma, information about the exact cellular architecture of these lesions is scarce and the histogenesis of ESC is unknown. Here, we extend our previous study and investigate 28 extraocular carcinomas in comparison to 54 benign sebaceous tumors and 8 cases of normal sebaceous glands using a broad spectrum of antibodies against p63, several keratins, adipophilin, EMA, Ki67, androgen receptor, and mismatch repair proteins. This observational study demonstrates that p63- and K5/14-positive basaloid cells are key cells in normal sebaceous gland and in all sebaceous tumors and that these basaloid cells give rise to EMA+, adipophilin+ sebocytes, and K5/14+, K7±, K10± ductal structures. Finally, about half of ESC is associated with superficial in situ neoplasia, which provides evidence that at least part of these carcinomas arises from flat superficial in situ carcinoma. In contrast to the normal sebaceous gland, about half of all sebaceous tumors lack keratin K7. MMR protein IHC-profiles role will be discussed., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

The histogenesis of extraocular sebaceous carcinomas is - in contrast to ocular sebaceous carcinomas - unclear, and information about the exact cellular architecture of these lesions and even of the normal sebaceous gland is still scarce. This study attempts to elucidate the histogenesis of sebaceous tumors, using multicolor immunofluorescence stainings to analyze 21 cases of sebaceous tumors (six each of extraocular sebaceous carcinoma, sebaceous adenoma and sebaceoma, and three cases of steatocystomas) and eight cases of normal sebaceous glands for p63, several keratins, androgen receptor, adipophilin, epithelial membrane antigen (EMA) and Ki-67. The data of this observational study provide evidence for the existence of two subpopulations of progenitors in normal sebaceous glands: (i) p63 + K5 + progenitors which generate the K10 + luminal cells of sebaceous ducts; and (ii) p63 + K5 + K7 + progenitors which finally generate K7 + adipophilin + EMA + sebocytes. Without exception, all types of sebaceous tumors contained p63 + K5 + cells. Furthermore, these tumors showed a cellular hierarchy and differentiation to adipophilin + and/or EMA + mature sebocytes and to K10 + ductal cells through intermediary cells. Notably, a considerable number of sebaceous tumors lack the K7 pathway of cell maintenance in the normal sebaceous lobule. Based on our data, we propose a cellular algorithmic model of the hierarchy of normal sebaceous glands and of sebocytic tumors in which p63 + K5 + cells play a major role., (© 2019 Japanese Dermatological Association.)

Understanding the mechanisms regulating human mammary epithelium requires knowledge of the cellular constituents of this tissue. Different and partially contradictory definitions and concepts describing the cellular hierarchy of mammary epithelium have been proposed, including our studies of keratins K5 and/or K14 as markers of progenitor cells. Furthermore, we and others have suggested that the p53 homolog p63 is a marker of human breast epithelial stem cells. In this investigation, we expand our previous studies by testing whether immunohistochemical staining with monospecific anti-keratin antibodies in combination with an antibody against the stem cell marker p63 might help refine the different morphologic phenotypes in normal breast epithelium. We used in situ multilabel staining for p63, different keratins, the myoepithelial marker smooth muscle actin (SMA), the estrogen receptor (ER), and Ki67 to dissect and quantify the cellular components of 16 normal pre- and postmenopausal human breast epithelial tissue samples at the single-cell level. Importantly, we confirm the existence of K5+ only cells and suggest that they, in contrast to the current view, are key luminal precursor cells from which K8/18+ progeny cells evolve. These cells are further modified by the expression of ER and Ki67. We have also identified a population of p63+K5+ cells that are only found in nipple ducts. Based on our findings, we propose a new concept of the cellular hierarchy of human breast epithelium, including K5 luminal lineage progenitors throughout the ductal-lobular axis and p63+K5+ progenitors confined to the nipple ducts.

Background: Uterine leiomyosarcomas are rare tumors with adverse prognosis. Recently, it has been suggested that a possible genetic subgroup of these tumors might be characterized by bi-allelic deletions of the RB1 locus. Here we report another uterine leiomyosarcoma with bi-allelic deletion of RB1 along with other genetic alterations., Case Report: A 52-year-old patient was admitted to the hospital for surgical removal of a polyp-like lesion in the uterine cavity. Histological examination revealed a grade 1 leiomyosarcoma with atypical mitoses and areas corresponding to a leiomyoma with bizarre nuclei., Results and Conclusion: This is the third case of a uterine leiomyosarcoma revealing bi-allelic RB1 deletions. Thus, in the absence of monosomy 14 and/or mutations of MED12, this genetic alteration seems, indeed, to constitute a separate entity of these tumors. Histological analysis of the tumor along with its genetic intratumoral heterogeneity suggests its origin to be from a leiomyoma with bizarre nuclei. Furthermore, of considerable interest in the case presented here, is the identification of a large segment of chromosome 22 showing uniparental disomy. Along with the case presented here, recent data show that a genetic classification of all uterine leiomyosarcomas is recommended to reveal more information about clinical correlations of their different genetic subtypes. Due to array-based methods these analyses can be well-carried out using paraffin-embedded samples., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

We contend that knowledge about the cellular composition of normal breast epithelium is a prerequisite for understanding proliferative breast disease. Against this background, we used multicolor immunofluorescence to study normal breast epithelium and two types of intraepithelial proliferative breast lesion for expression of the p63, basal keratin K5, glandular keratin K8/18, SMA, ER-alpha, and Ki67. We studied eight normal breast epithelium samples, 12 cases of usual ductal hyperplasia, and 33 cases of low-grade intraepithelial neoplasia (9 flat epithelial atypia, 14 low-grade ductal carcinoma in situ and 10 cases of lobular neoplasia). Usual ductal hyperplasia showed striking similarity to normal luminal breast epithelium including p63+ and/or K5+ luminal progenitor cells and the full spectrum of luminal progeny cells. In normal breast epithelium and usual ductal hyperplasia, expression of ER-alpha was associated with lack of expression of the proliferation antigen Ki67. In contrast, we found in both types of low-grade intraepithelial neoplasia robust expression of keratin K8/18 and a positive association between ER-alpha and Ki67 expression. However, these lesions were consistently negative for p63 and/or K5. Our observational study supports the view that usual ductal hyperplasia and low-grade intraepithelial neoplasia are different entities rather than part of a spectrum of the same disease. We propose a new operational model of cell differentiation that may serve to better understand correlations between normal breast epithelium and proliferative breast diseases. From our data we conclude that p63+ and/or K5+ progenitor cells contribute to maintenance of normal epithelium and usual ductal hyperplasia, but not to low-grade intraepithelial neoplasia of the breast.

Background: microRNAs (miRs)-371-3 are suggested to be novel biomarkers of germ cell tumors (GCTs), but their specificity is unresolved. We aimed at clarifying the origin of miR 371a-3p by measuring this miR in peripheral vein blood, and in fluids present in the vicinity of GCTs., Methods: miR-371a-3p levels were measured by quantitative PCR in 9 tumor surrounding hydroceles and in cubital vein blood (CVB) and testicular vein blood (TVB) of 64 GCT patients, 51 with clinical stage (CS) 1, 13 with CS2-3. Thirty three CS1 cases had also postoperative CVB measurement. TVB miR levels were compared with those of CVB. Associations with clinical factors were analyzed statistically., Results: TVB miR levels were 294-fold, 80-fold and 4.6-fold higher than those in CVB of CS1 patients, CS2-3 patients and controls, respectively. Neoplastic hydrocele fluid comprised of very high miR levels. In CS1, miR levels dropped to normal postoperatively. Statistically, CVB miR levels are significantly associated with tumor size (p = 0.0211) and testis length (p = 0.0493). TVB miR levels are associated with testis length (p = 0.0129)., Conclusions: This study provides evidence for the origin of circulating miR 371a-3p molecules from GCT cells. miR-371a-3p represents a specific serum biomarker for germ cell cancer., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

A small group of tumors of breast and salivary glands contains squamous/epidermoid elements as a constitutive feature (e.g., squamous carcinoma, syringomatous tumors, and mucoepidermoid carcinoma). Other tumors (e.g., pleomorphic adenoma, adenomyoepithelial tumors, and adenoid cystic carcinoma) may show occasionally squamous differentiation. Furthermore, squamous metaplasia may be observed in non-neoplastic breast and salivary tissues. However, the histogenesis of these squamous differentiations is far from being understood. Based on our earlier in situ triple immunofluorescence and quantitative reverse transcription (RT)-PCR experiments for basal keratins K5/14 and p63 as well as for glandular keratins (K7/K8/18), squamous keratins (K10 and K13), and myoepithelial lineage markers (smooth muscle actin, SMA), we here traced the squamous/epidermoid differentiation lineage of 60 tumors of the breast and/or salivary glands, cultured tumor cells of 2 tumors, and of 7 squamous metaplasias of non-neoplastic breast and salivary tissues. Our results indicate that both the neoplastic lesions as well as the non-neoplastic squamous metaplasia contain p63/K5/14+ cells that differentiate toward K10/13+ squamous cells. Thus, cells with squamous/epidermoid differentiation undergo a transition from its original p63/K5/14+ precursor state to K10/13+ squamous lineage state, which can be pictured by triple-immunofluorescence experiments. Given the immunophenotypic similarity of p63/K5/14+ tumor cells to their physiological p63/K5/14+ counterparts in normal breast and salivary duct epithelium, we suggest that these cells provide an important histogenetic key to understanding the pathogenesis of squamous differentiation both in normal breast/salivary gland tissues and their corresponding tumors.

Aims: Syringomatous tumour of the nipple and low-grade adenosquamous carcinoma (LGAdSC) of the breast are regarded as distinct entities. To clarify the nature of these two lesions, we compared the expression of different lineage/differentiation markers in 12 syringomatous tumours of the nipple, nine LGAdSCs, and normal breast epithelium., Methods and Results: Using triple immunofluorescence labelling and quantitative RT-PCR for keratins, p63, and smooth muscle actin, we demonstrated that syringomatous tumour and LGAdSC contain p63+/K5/14+ tumour cells, K10+ squamous cells, and K8/18+ glandular cells, with intermediary cells being found in both lineages. Identical p63+/K5/14+ cells were also found in the normal breast duct epithelium., Conclusions: Our data provide evidence that syringomatous tumour of the nipple and LGAdSC are identical or nearly identical lesions. They contain p63+/K5/14+ cells as the key cells from which the K10+ squamous lineage and the K8/18+ glandular lineage arise. On the basis of our findings in normal breast tissue and associated benign lesions, we suggest that p63+/K5/14+ cells of the normal breast duct epithelium or early related cells might play a key role in the neoplastic transformation of both syringomatous tumour and LGAdSC. We propose that the differentiation patterns found in both lesions reflect the early ontogenetic stages of the normal breast epithelium., (© 2014 John Wiley & Sons Ltd.)

Salivary gland-like tumors of the breast show a great variety of architectural patterns and cellular differentiations such as glandular, myoepithelial, squamous, and even mesenchymal phenotypes. However, currently little is known about the evolution and cellular differentiation of these tumors. For that reason, we performed an in situ triple immunofluorescence lineage/differentiation tracing (isTILT) and qRT-PCR study of basal (K5/K14), glandular (K7/K8/18), and epidermal-specific squamous (K10) keratins, p63, and smooth muscle actin (SMA; myoepithelial marker) with the aim to construct and trace different cell lineages and define their cellular hierarchy in tumors with myoepithelial differentiation. isTILT analysis of a series of 28 breast, salivary, and lacrimal gland tumors, including pleomorphic adenomas (n=8), epithelial-myoepithelial tumors (n=9), and adenoid cystic carcinomas (n=11) revealed that all tumor types contained K5/K14-positive progenitor cells in varying frequencies from a few percent up to 15%. These K5/K14-positive tumor cells were found to differentiate to glandular- (K8/18-positive) and myoepithelial-lineage (SMA-positive)-specific cells and were also shown to generate various heterologeous cell differentiations such as squamous and mesenchymal progenies. p63 was co-expressed with K5/K14 in basal-like progenitor cells, myoepithelial, and squamous cells but not in glandular cells. Our results show that the corresponding counterpart tumors of breast and salivary/lacrimal glands have identical cellular compositions. Taken together, our isTILT and RNA-expression data indicate that look-alike tumors of the breast represent a special subgroup of basal-type tumors with benign or usually low malignant potential.

Purpose: As HER2 status is a strong predictor of the response to trastuzumab, clinical guidelines recommend that all breast tumours are first evaluated for HER2 protein expression by immunohistochemistry (IHC) followed by confirmatory testing for HER2 gene amplification using fluorescence in situ hybridisation (FISH) for 2+ cases. Alternatively, chromogenic in situ hybridisation (CISH) offers a simpler, less expensive approach to detect HER2 amplification., Methods: In this prospective, multi-centre study, based on the largest dataset for HER2 testing in Germany to date, we evaluated the concordance between FISH and CISH in 399 samples from adjuvant and metastatic breast cancer patients. Tumour specimens from routine diagnostic practice were analysed by IHC, FISH and CISH in four reference centres., Results: FISH and CISH results were strongly concordant (κ = 0.83), with 95% of cases showing agreement. Despite variable IHC scoring across testing centres, complete consensus among the three methods was observed for 246 cases, representing 91% of all IHC positive (3+) or negative (0/1+) cases. Confirmatory testing of 132 IHC equivocal (2+) cases also yielded highly concordant results between FISH and CISH., Conclusions: These data validate CISH for the assessment of HER2 gene amplification in breast tumours and, confirm CISH as a valid alternative to FISH in HER2 testing.

We present two monoclonal antibodies (mab), Mano 4/4 and 486 P3/12, directed against tumor-associated antigens of different subpopulations of transitional cell bladder carcinoma tumors. A detailed description is given concerning their reactivity patterns in an ELISA assay against different cell lines. Using immunohistochemical staining against normal bladder mucosa, mab Mano 4/4 reacts with distinct cells in the basal layer and mab 486 P 3/12 recognizes single cells in the superficial layer. Analysing different transitional-cell carcinomas, mab Mano 4/4 reacts with 17 of 20 and mab 486 P 3/12 with 17 of 19 bladder tumors. It is emphasized that both mabs may add new information in respect to a better characterization of the heterogeneity of bladder carcinoma. A biochemical characterization of the mabs and their corresponding antigens is given. Mab Mano 4/4 is directed against a 28 kD glycoprotein and mab 486 P 3/12 is reacting with a 200 kD glycoprotein belonging to the family of CEA-like proteins.

Monoclonal antibodies directed against tumor-associated antigens of bladder carcinoma were used to identify tumor cells in bladder washout specimens of 40 patients with bladder carcinoma (group 1), 41 with no bladder disease or with urinary tract infections (group 2), 41 who received long-term mitomycin C instillation therapy after excision of the tumors (group 3) and 39 who received no prophylaxis after excision of the tumors (group 4). In all groups the same bladder washout specimen was used for standard urinary cytological and immunocytological tests. True positive results were obtained in 90 per cent of the patients in group 1 according to our immunocytological criteria and in 43 per cent according to standard cytology studies. No urine specimens in group 2 (controls) were immunocytologically positive, while 16 of 41 in group 3 and 17 of 39 in group 4 were positive immunocytologically but only 4 and 5, respectively, were positive according to standard cytology studies. Further followup of these patients will show whether cells positive for monoclonal antibody 486 P 3/12 will permit early detection of recurrent bladder cancer and whether one can identify patients who require prophylaxis after removal of the superficial bladder tumors.

Previous studies revealed that the filaments of cytokeratin express different antigenic sites in the basal and suprabasal cell compartment of the skin and oral mucosa. It has been demonstrated that the keratin polypeptide subunit of molecular weight 67,000 dalton (67K) is a valuable marker of the suprabasal cells, since the normal basal cells remain unlabeled by antibodies against this large polypeptide. In the present study, the distribution of the 67K polypeptide has been investigated on human and guinea pig keratinocyte suspensions, purified basal cells of guinea pigs, normal human abdominal skin and normal human buccal mucosa, using the indirect immunoperoxidase method. The results of this investigation confirmed ultrastructurally that the 67K-antigen expression is lacking in the epidermal basal cell compartment. However, it could be shown that 67K polypeptide is a component of the tonofilaments in the upper Malpighian cell layers of the skin and oral mucosa. These observations suggest that keratinocyte differentiation is accompanied by modifications of antigenic determinants of the keratin filaments.