Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

Residual embryonic cells as precursors of a Barrett's-like metaplasia.

Barrett's esophagus is an intestine-like metaplasia and precursor of esophageal adenocarcinoma. Triggered by gastroesophageal reflux disease, the origin of this metaplasia remains unknown. p63-deficient mice, which lack squamous epithelia, may model acid-reflux damage. We show here that p63 null embryos rapidly develop intestine-like metaplasia with gene expression profiles similar to Barrett's metaplasia. We track its source to a unique embryonic epithelium that is normally undermined and replaced by p63-expressing cells. Significantly, we show that a discrete population of these embryonic cells persists in adult mice and humans at the squamocolumnar junction, the source of Barrett's metaplasia. We show that upon programmed damage to the squamous epithelium, these embryonic cells migrate toward adjacent, specialized squamous cells in a process that may recapitulate early Barrett's. Our findings suggest that certain precancerous lesions, such as Barrett's, initiate not from genetic alterations but from competitive interactions between cell lineages driven by opportunity.

Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection.

The extent of lung regeneration following catastrophic damage and the potential role of adult stem cells in such a process remains obscure. Sublethal infection of mice with an H1N1 influenza virus related to that of the 1918 pandemic triggers massive airway damage followed by apparent regeneration. We show here that p63-expressing stem cells in the bronchiolar epithelium undergo rapid proliferation after infection and radiate to interbronchiolar regions of alveolar ablation. Once there, these cells assemble into discrete, Krt5+ pods and initiate expression of markers typical of alveoli. Gene expression profiles of these pods suggest that they are intermediates in the reconstitution of the alveolar-capillary network eradicated by viral infection. The dynamics of this p63-expressing stem cell in lung regeneration mirrors our parallel finding that defined pedigrees of human distal airway stem cells assemble alveoli-like structures in vitro and suggests new therapeutic avenues to acute and chronic airway disease.

STK11 (LKB1) immunohistochemistry is a sensitive and specific marker for STK11 adnexal tumours.

AIMS: STK11 adnexal tumour is a rare, recently described malignant neoplasm that is associated with Peutz-Jeghers syndrome. [Correction added on 3 October 2024, after first online publication: 'ST11' in preceding sentence has been corrected to 'STK11' in this version.] It predominantly originates from the para-adnexal soft tissues and often shows secondary involvement of the fallopian tube and ovary. STK11 adnexal tumours have a broad differential diagnosis due to their variable morphology and non-specific immunoprofile, and diagnostic confirmation currently requires sequencing to identify an STK11 mutation. We investigate the diagnostic utility of STK11 (LKB1) immunohistochemistry (IHC) in a cohort of STK11 adnexal tumours and morphological mimics. METHODS AND RESULTS: IHC for STK11 was performed on 122 tumours, including 17 STK11 adnexal tumours and 105 morphological mimics (10 female adnexal tumours of Wolffian origin, 22 adult granulosa cell tumours, 10 juvenile granulosa cell tumours, four Sertoli-Leydig cell tumours, two Leydig cell tumours, one Sertoli cell tumour, one steroid cell tumour, four extra-ovarian sex cord-stromal tumours, 16 ovarian endometrioid carcinomas, eight tubo-ovarian high-grade serous carcinomas, five ovarian mesonephric-like adenocarcinomas, 14 ovarian carcinosarcomas, five peritoneal malignant mesotheliomas, two pelvic plexiform leiomyomata and one ovarian solid pseudopapillary tumour). All STK11 adnexal tumours showed complete loss of cytoplasmic staining for STK11. All other tumour types showed retained cytoplasmic staining, except for one endometrioid carcinoma with mucinous differentiation which showed complete loss of STK11 expression and a high-grade serous carcinoma with subclonal loss. CONCLUSIONS: STK11 is a highly sensitive and specific immunohistochemical marker for distinguishing STK11 adnexal tumour from its histological mimics, and can obviate the need for confirmatory molecular studies in the appropriate morphological context.

Inflammatory Activity of Epithelial Stem Cell Variants from Cystic Fibrosis Lungs Is Not Resolved by CFTR Modulators.

Rationale: CFTR (cystic fibrosis transmembrane conductance regulator) modulator drugs restore function to mutant channels in patients with cystic fibrosis (CF) and lead to improvements in body mass index and lung function. Although it is anticipated that early childhood treatment with CFTR modulators will significantly delay or even prevent the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in patients with CF with established lung disease despite modulator therapy, underscoring the need to identify and ultimately target the sources of this inflammation in CF lungs. Objectives: To determine whether CF lungs, like chronic obstructive pulmonary disease (COPD) lungs, harbor potentially pathogenic stem cell "variants" distinct from the normal p63/Krt5 lung stem cells devoted to alveolar fates, to identify specific variants that might contribute to the inflammatory state of CF lungs, and to assess the impact of CFTR genetic complementation or CFTR modulators on the inflammatory variants identified herein. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in COPD and idiopathic pulmonary fibrosis lungs was applied to end-stage lungs of patients with CF (three homozygous CFTR:F508D, one CFTR F508D/L1254X; FEV1, 14-30%) undergoing therapeutic lung transplantation. Single-cell-derived clones corresponding to the six stem cell clusters resolved by single-cell RNA sequencing of these libraries were assessed by RNA sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants after CFTR gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: End-stage CF lungs display a stem cell heterogeneity marked by five predominant variants in addition to the normal lung stem cell, of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophilic inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of three proinflammatory stem cell variants in CF lungs may contribute to the persistence of lung inflammation in patients with CF with advanced disease undergoing CFTR modulator therapy.

Prevalence and genotype distribution of human papillomavirus in cervical adenocarcinoma (usual type and variants): A systematic review and meta-analysis.

Cervical glandular neoplasms represent a heterogeneous group of tumors for which a comprehensive overview of the involvement of high-risk human papillomaviruses (HPV) in pathogenesis is still lacking. We first searched MEDLINE (PubMed), Embase, and Scopus databases (until October 2022), and systematically reviewed available literature. We then quantitatively estimated both pooled and genotype-specific prevalence of HPV DNA as well as the influence of various factors (e.g., geographical region, histological subtype, tissue/sample type) on computed effect size by means of random effects meta-analysis. In total, 379 studies comprising 17 129 cases of cervical adenocarcinoma were identified. The pooled HPV prevalence was 78.4% (95% confidence interval [95% CI]: 76.2-80.3) with a significant between-study heterogeneity (I2 = 79.4%, Q test p < 0.0001). Subgroup analyses indicated that the effect size differed substantially by geographical region (from 72.5% [95% CI: 68.7-76.1] in Asia to 86.8% [95% CI: 82.2-90.3] in Oceania) (p < 0.0001) and histological subtype of cancer (from 9.8% [95% CI: 5.5-17] in gastric-type to 85% [95% CI: 79.6-89.2] in usual-type cervical adenocarcinoma) (p < 0.0001). HPV16 and HPV18 were by far the most frequently detected viral strains with specific prevalence of 49.8% (95% CI: 46.9-52.6) and 45.3% (95% CI: 42.8-47.8), respectively. When stratified by continent or histologic variant, these genotype-specific results varied in a relatively limited manner. Altogether, these findings support that all histological subtypes of cervical adenocarcinoma are etiologically linked to high-risk HPV but to varying degrees. Therefore, a dual-criteria classification taking into account accurately both morphological and virological aspects could be an interesting evolution of the current binary World Health Organization classification, better reflecting the pathogenic diversity of the disease.

Genomic catastrophe, the peritoneal cavity and ovarian cancer prevention.

The current theory of carcinogenesis for the deadliest of 'ovarian' cancers-high-grade serous carcinoma (HGSC)-holds that the malignancy develops first in the fallopian tube and spreads to the ovaries, peritoneum, and/or regional lymph nodes. This is based primarily on the observation of early forms of serous neoplasia (serous tubal intraepithelial lesions [STILs], and serous tubal intraepithelial carcinomas [STICS]) in the fimbria of women undergoing risk reduction surgery. However, these lesions are uncommon in the general population, confer a low risk (5%) of HGSC following their removal in at-risk women with germ-line BRCA1/2 mutations, and require 4 or more years to recur as intraperitoneal HGSC. These features suggest that isolated STILs and STICs behave as precursors, with uncertain cancer risk rather than carcinomas. Their evolution to HGSC within, or after, escape from the tube could proceed stepwise with multiple biologic events; however, it is unclear whether tubal or ovarian HGSCs encountered in the setting of advanced disease evolved in the same fashion. The latter scenario could also be explained by a 'catastrophic' model in which STICs suddenly develop with invasive and metastatic potential, overwhelming or obscuring the site of origin. Moreover, a similar model might explain the sudden emergence of HGSC in the peritoneal cavity following escape of precursor cells years before. Long-term follow-up data from opportunistic or prophylactic salpingectomy should shed light on where malignant transformation occurs, as well as the timeline from precursor to metastatic HGSC. © 2022 The Pathological Society of Great Britain and Ireland.

Mural nodules in mucinous ovarian tumors represent a morphologic spectrum of clonal neoplasms: a morphologic, immunohistochemical, and molecular analysis of 13 cases.

Mucinous ovarian tumors rarely harbor mural nodules, which have historically been classified as sarcoma-like, anaplastic carcinomatous, or sarcomatous on the basis of predominant morphologic features. The molecular relationship between mural nodules and associated mucinous ovarian tumors remains poorly characterized, as does the molecular pathogenesis of these mural nodules. Thus, we analyzed the morphological, immunohistochemical, and genetic features of 13 mucinous ovarian tumors and associated mural nodule(s). Three harbored sarcoma-like mural nodules and ten contained anaplastic carcinomatous nodules, including 1 tumor with spatially discrete anaplastic carcinomatous and sarcomatous nodules. Twelve of 13 cases showed genetic evidence of clonality between the mural nodule(s) and associated mucinous ovarian tumor, including all three tumors with sarcoma-like morphology. Mural nodules were genetically identical in the five cases in which there were multiple discrete mural nodules that were sequenced separately. MTAP and p53 immunohistochemistry confirmed the distribution of neoplastic cells in a subset of sarcoma-like and anaplastic carcinomatous nodules. No single recurrent genetic alteration was associated with mural nodule development. No recurrent genetic differences were identified between mural nodules with sarcoma-like, anaplastic carcinomatous, and sarcomatous morphology. Of 11 patients with clinical follow-up, three died of disease 3, 8, and 9 months after diagnosis, but no recurrent genetic events were associated with poor outcome. These molecular data suggest that sarcoma-like, anaplastic carcinomatous, and sarcomatous nodules represent a morphologic spectrum of clonal neoplasms arising in mucinous ovarian tumors rather than three discrete biological entities.

Interobserver reproducibility of the diagnosis of differentiated exophytic vulvar intraepithelial lesion (DEVIL) and the distinction from its mimics.

AIMS: Most vulvar squamous cell carcinomas are human papillomavirus (HPV)-associated or TP53-mutant. A third category of HPV-independent TP53-wild-type lesions is uncommon and not fully understood. Differentiated exophytic vulvar intraepithelial lesion (DEVIL) has been characterised as a precursor of this latter category. The reproducibility of the diagnosis of DEVIL and its distinction from lesions with overlapping morphology has not been studied. Our aim was to establish the interobserver agreement in the diagnosis of DEVIL and its distinction from neoplastic and reactive conditions of the vulva on haematoxylin and eosin evaluation. METHODS AND RESULTS: A set of 35 slides was evaluated by eight reviewers (two trainees and six practising gynaecological pathologists). The set included DEVIL, condyloma, established vulvar precursors [high-grade squamous intraepithelial lesion (HSIL) and differentiated vulvar intraepithelial neoplasia (dVIN)] with superimposed acanthosis or verruciform growth, lichen simplex chronicus (LSC), and psoriasis. Kappa (κ) values were calculated. Overall, interobserver agreement was moderate (κ = 0.56), improving to substantial (κ = 0.7) when evaluation was performed by practising pathologists. Agreement was strong for the diagnosis of HSIL (κ = 0.88), and substantial for the diagnosis of DEVIL (κ = 0.61), condyloma (κ = 0.79), and LSC (κ = 0.72). Agreement was moderate for the diagnosis of dVIN (κ = 0.59) and psoriasis (κ = 0.53). Perfect agreement (6/6) among practising pathologists was observed in 43% of cases, and majority agreement (5/6 or 4/6) was observed in 48% of cases. CONCLUSIONS: Reproducibility in the diagnosis of verruciform vulvar lesions, including the novel DEVIL, is acceptable overall. Reproducibility is higher for well-known lesions such as HSIL and condyloma than for more challenging diagnoses such as DEVIL, dVIN, and psoriasis. Agreement is higher among practising gynaecological pathologists, suggesting that training and experience improve reproducibility. Our findings support the inclusion of DEVIL as a diagnostic entity in the classification of vulvar squamous lesions.

A Clinical and Pathologic Exploration of Suspected Peritoneal Endometriotic Lesions.

Endometriosis is generally histopathologically defined as the presence of at least 2 of the following: endometrial stroma, Müllerian epithelium, and/or hemosiderin-laden macrophages (HLM). Despite clinically evident endometriotic lesions, biopsies are frequently nondiagnostic. In this study, we conducted a large-scale review of biopsies of lesions clinically thought to represent endometriosis and correlate the histologic findings with clinical appearance to expand sensitivity of the pathologic definition of endometriosis, particularly in patients on hormonal therapy. In all, 112 biopsies from 78 patients (mean age=25, range 18-39 yr) were reviewed for histopathologic features suggestive of or diagnostic for endometriosis including the presence of endometrial stroma, Müllerian epithelium, dystrophic calcifications, HLM, chronic inflammation, adhesions, and vascular proliferation. Endometriosis was confirmed by pathologic criteria in 37 of 78 patients (47%). Biopsies from patients on hormonal therapy (n=62, 80%) were significantly less likely to meet pathologic criteria for endometriosis (P=0.01). Nondiagnostic biopsies (70/112; 63%) frequently displayed HLM (20%), chronic inflammation (29%), dystrophic calcifications (26%), vascular proliferation (20%), or adhesions (20%) and were significantly more likely to have a vascular clinical appearance (P=0.01). Diagnostic biopsies (42/112; 38%) were more likely to have a blue/black clinical appearance (P=0.03), demonstrate HLM (P=0.004), and display pseudodecidualization (P=0.05). Patients with a high clinical suspicion of endometriosis have a range of histologic findings, with less than half meeting the current histopathologic criteria for diagnosing endometriosis. Given the heterogeneous histopathologic appearance, revision of the histologic criteria may be warranted with further exploration, particularly for lesions with predominantly vascular features.

p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration.

Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis involve the progressive and inexorable destruction of oxygen exchange surfaces and airways, and have emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ transplantation, remain limited and the possibility of regenerative medicine has lacked empirical support. However, it is clinically known that patients who survive sudden, massive loss of lung tissue from necrotizing pneumonia or acute respiratory distress syndrome often recover full pulmonary function within six months. Correspondingly, we recently demonstrated lung regeneration in mice following H1N1 influenza virus infection, and linked distal airway stem cells expressing Trp63 (p63) and keratin 5, called DASC(p63/Krt5), to this process. Here we show that pre-existing, intrinsically committed DASC(p63/Krt5) undergo a proliferative expansion in response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial lung inflammation. We also show that the selective ablation of DASC(p63/Krt5) in vivo prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASC(p63/Krt5)-derived pedigrees differentiate to type I and type II pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and also minimize the structural consequences of endogenous stem cell loss on this process. The ability to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests their potential in stem cell-based therapies for acute and chronic lung diseases.

Cloning and variation of ground state intestinal stem cells.

Stem cells of the gastrointestinal tract, pancreas, liver and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, 'ground state' stem cells of the human intestine and colon. We show that derived stem-cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells is likely to enforce the functional specificity of the adult intestinal tract. Using clonally derived colonic epithelia, we show that toxins A or B of the enteric pathogen Clostridium difficile recapitulate the salient features of pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests certain advantages for their use in disease modelling and regenerative medicine.

Molecular characterization of diffuse malignant peritoneal mesothelioma.

Malignant peritoneal mesothelioma is a rare aggressive tumor that arises from the peritoneal lining. While recurrent BAP1 mutations have been identified in a subset of mesotheliomas, molecular characteristics of peritoneal mesotheliomas, including those lacking BAP1 alterations, remain poorly understood. Using targeted next-generation sequencing, we examined the molecular features of 26 diffuse malignant peritoneal mesotheliomas. As part of an exploratory analysis, we analyzed an additional localized peritoneal mesothelioma and one well-differentiated papillary mesothelioma with invasive foci. Genomic characterization identified categories of diffuse malignant peritoneal mesotheliomas: The first group included 18 (69%) tumors with recurrent BAP1 alterations, with eight (31%) having more than one BAP1 alterations, and concomitant alterations in PBRM1 (46%) and SETD2 (35%). All tumors with complete loss of BAP1 expression by immunohistochemistry harbored BAP1 molecular alterations. PBRM1 alterations were significantly enriched in the BAP1-altered cohort. Frequent copy number loss of BAP1, ARID1B, PRDM1, PBRM1, SETD2, NF2, and CDKN2A was noted. The second group included eight (31%) BAP1-wild-type tumors: two with TP53 mutations, one with a TRAF7 activating mutation, one with a SUZ12 inactivating mutation, and three with ALK rearrangements that we previously published. One TP53-mutant biphasic mesothelioma showed evidence of genomic near-haploidization showing loss of heterozygosity of all chromosomes except 5, 7, 16, and 20. The localized peritoneal mesothelioma harbored a nonsense CHEK2 mutation, and the well-differentiated papillary mesothelioma with invasive foci harbored no reportable variants. In conclusion, we described the genetic categories of diffuse malignant peritoneal mesotheliomas, with BAP1-mutant and BAP1-wild-type groups. Our findings implicated DNA repair, epigenetics, and cell cycle regulation in the pathogenesis of peritoneal mesotheliomas, with identification of potential therapeutic targets.

Evidence for a Novel Endometrioid Carcinogenic Sequence in the Fallopian Tube With Unique Beta-Catenin Expression.

Epithelial proliferations in the fallopian tube have been characterized by some as stem cell outgrowths (SCOUTs) and divided into type I and type II. Type II SCOUTs exhibit diffuse cellular beta-catenin nuclear staining (ß-catenin), implying a CTNNB1 mutation. SCOUTs are more common in perimenopausal and postmenopausal women and are associated with ovarian cancer but have not been linked directly to malignancy. We analyzed type II SCOUTs in various gynecologic conditions, and searched for endometrioid atypical hyperplasias (tubal endometrioid intraepithelial neoplasia) or adenocarcinomas in the tube. ß-catenin SCOUT frequency in cases of neoplasia was 66.7% per case and 30.7% per nonfimbrial cross-section for uterine endometrioid carcinomas versus 25% and 13.3% for controls, respectively (P=0.02 and 0.09). Multiple (3 or more) ß-catenin SCOUTs in a single section were uncommon; 6 of 9 were associated with a carcinoma or proliferative lesion in the endometrium. Tubal endometrioid intraepithelial neoplasia/atypical hyperplasia displayed complex growth, including focal cribriform growth patterns and squamous morules. Two cases of type II SCOUTs associated with tubal endometrioid intraepithelial neoplasia/atypical hyperplasia and/or adenocarcinomas in the fallopian tube were identified, both of which coexisted with a separate endometrioid adenocarcinoma, one with bilateral ovarian endometrioid adenocarcinomas. Both benign and neoplastic tubal lesions were ß-catenin. This report is the first to link components of a unique ß-catenin endometrioid carcinogenic sequence in the fallopian tube. It further emphasizes the multifocal nature of endometrioid neoplasia in the female genital tract and poses questions regarding the frequency and biologic underpinnings of ß-catenin proliferations in the oviduct.

Landscape of genomic alterations in cervical carcinomas.

Cervical cancer is responsible for 10-15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma-normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour-normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.

The fallopian tube, "precursor escape" and narrowing the knowledge gap to the origins of high-grade serous carcinoma.

Most ovarian carcinomas are high-grade serous carcinomas (HGSC) that contain TP53 mutations, present at advanced stage, and eventually become resistant to chemotherapy. The rapid evolution of this disease has been attributed to an origin in the distal fallopian tube, in the form of serous tubal intraepithelial carcinomas (STICs). This has led to a disease model where malignancy develops first in the tube and spreads to the peritoneum or regional lymph nodes. However, although most early or incidentally discovered HGSCs manifest in the tube with STICs, many advanced HGSCs are not accompanied by a malignancy in the fimbria. To resolve this paradox, the focus has shifted to earlier, premalignant serous proliferations (ESPs) in the tubes, which lack the cytomorphologic features of malignancy but contain TP53 mutations. These have been termed p53 signatures or serous tubal intraepithelial lesions (STILs). Although they have not been presumed to have cancer-causing potential by themselves, some ESPs have recently been shown to share identical TP53 mutations with concurrent HGSCs, indicating a shared lineage between these early mucosal changes and metastatic malignancy. This discovery supports a paradigm by which HGSCs can emerge not only from STICs but also from exfoliated precursor cells (precursor escape) that eventually undergo malignant transformation within the peritoneal cavity. This paradigm unifies both localized and widespread HGSCs to a visible pre-existing cellular alteration in the tubal epithelium, and highlights a consistent and necessary biologic event (TP53 mutation) rarely encountered in the ovary or secondary Mullerian system. This dual pathway to HGSCs underscores the subtle nature of many serous cancer origins in the tube, explains contrasting clinico-pathologic presentations, and explains why, until recently, the fallopian tube was unappreciated as the principal origin of HGSCs. Moreover, it highlights additional challenges faced in preventing or intercepting HGSCs at a curable stage.

Classic Vulvar Intraepithelial Neoplasia With Superimposed Lichen Simplex Chronicus: A Unique Variant Mimicking Differentiated Vulvar Intraepithelial Neoplasia.

High-grade vulvar intraepithelial neoplasia, a precursor lesion to vulvar squamous cell carcinoma, is subdivided into 2 types, classic or usual vulvar intraepithelial neoplasia (CVIN) and differentiated vulvar intraepithelial neoplasia (DVIN). CVIN, which is a human papilloma virus (HPV)-dependent lesion, is typically distinguished from DVIN, a p53 mutation-dependent process, by its distinct histomorphologic and immunohistochemical characteristics. However, distinguishing between the 2 entities becomes challenging in cases of CVIN with superimposed inflammatory changes, especially lichen simplex chronicus (LSC). Twelve cases of DVIN, 9 cases of LSC, and 9 cases of CVIN with superimposed LSC were assessed for a number of morphologic features, including hyperkeratosis, hypergranulosis, acanthosis, hypercellularity, abnormal maturation (i.e. abnormal keratinization close to the base and/or dyskeratosis), hyperchromasia, and basal atypia. Immunohistochemistry for p53, p16, and MIB-1 was performed for all cases. When sufficient tissue was available, HPV genotyping was performed for cases of CVIN with superimposed LSC. DVIN uniformly demonstrated abnormal maturation, and atypia involving the basal cell layer; they were all p16 negative and demonstrated p53 positivity of moderate to strong intensity in a basal and parabasal distribution. CVIN with superimposed LSC frequently displayed hyperchromasia involving the basal 3 to 4 cell layers, basal to full-thickness atypia, and apoptosis. CVIN with superimposed LSC demonstrated intense p16 positivity extending from the basal cells to the mid-epithelium and a reduction or loss of staining in maturing keratinocytes. P53 staining revealed a unique pattern of parabasal and mid-epithelial weak to moderate staining with sparing of the basal layer. Cases of LSC demonstrated heterogenous p53 positivity and were negative for p16. MIB-1 staining showed a similar range of positivity for all diagnoses. HPV genotyping revealed HPV 16 in all 5 cases of CVIN with LSC that underwent testing. We conclude that, although CVIN with superimposed LSC can closely resemble DVIN, morphologic features such as nuclear hyperchromasia uniformly involving the basal 3 to 4 cell layers, apoptosis, and absent or less pronounced cytoplasmic maturation are more suggestive of CVIN with superimposed LSC. In cases where the morphology remains ambiguous, immunohistochemistry for both p16 and p53 can be helpful. In particular, p53 parabasal and mid-epithelial staining without involvement of the basal layer appears to be a characteristic finding in CVIN with superimposed LSC. MIB-1 staining is of little utility in distinguishing between these entities and should not be routinely performed.

HPV 6-associated HSIL/Squamous Carcinoma in the Anogenital Tract.

Human papillomavirus (HPV) type 6 is historically classified as low-risk HPV type and associates with low-grade squamous intraepithelial lesions of the anogenital tract. Rare squamous carcinomas have been reported in association with these HPV types but the mechanism(s) behind this carcinogenic sequence have been unclear. We report 4 cases of low risk anogenital HPV infections-3 cervical (immature low-grade squamous intraepithelial lesion with metaplastic phenotype) and one anal (exophytic condyloma) lesion-that manifested with high-grade squamous intraepithelial lesion/squamous cell carcinoma. Two were associated with invasion one of which metastasized to a regional node. Two cases exhibited strong p53 positivity in the high-grade squamous intraepithelial lesion/squamous cell carcinoma component analogous to that seen in HPV-negative differentiated intraepithelial lesions of the external genitalia. This series of cases adds to the literature on low risk HPV-associated cervical squamous carcinomas. It underscores the similarities between the baseline cyto-morphology and benign mimics (low-grade squamous intraepithelial lesions), the subtle cytologic and immunohistochemical (MIB1) features heralding biologic aggressiveness, and in some potential pathways (p53) not usually involved in HPV-related anogenital neoplasia.

Evidence of a Monoclonal Origin for Bilateral Serous Tubal Intraepithelial Neoplasia.

Serous tubal intraepithelial carcinoma (STIC) is found in 10% to 60% of cases of tuboovarian high-grade serous carcinoma (HGSC) and is presumed to be the site of origin, linking many HGSCs to the fallopian tube. Bilateral STIC is present in ∼20% of cases. Because clonal Tp53 mutations are a defining feature of HGSC, including their associated STICs, we analyzed 4 cases of bilateral serous tubal intraepithelial neoplasia (STIN), including STIC and Tp53-mutated serous tubal intraepithelial lesions (STILs), associated with HGSC to determine whether they contained the same or different p53 mutations. Extracted DNA from STINs, concurrent HGSCs and control tissues was analyzed for mutations in all exons of Tp53. Sequencing was successful in 3 of the 4 cases, and an identical Tp53 mutation was detected in the HGSC and bilateral STINs in 2 of these 3 cases. One STIN was morphologically a STIL. These findings confirm that a subset of bilateral STINs share the same Tp53 mutation, implying that at least one of the STINs is an intraepithelial metastasis from either the contralateral STIN or HGSC. This study complements others addressing the multiple origins of STIN in the setting of existing HGSC. It further underscores the fact that potential overlap in biologic behavior between STILs and STICs as well as timing and direction of metastatic spread has yet to be resolved.

Targeted Genomic Profiling of Female Adnexal Tumors of Probable Wolffian Origin (FATWO).

Female adnexal tumor of probable Wolffian origin (FATWO) is a rare gynecologic neoplasm of low-malignant potential presumed to be derived from mesonephric remnants in the upper female genital tract. Similarly, mesonephric remnants in the lower female genital tract are thought to be the origin for mesonephric carcinoma. Although the molecular alterations in mesonephric carcinoma have been recently reported, the pathogenesis of and molecular alterations in FATWO are not well understood. The aims of this study were to examine the molecular alterations in FATWO and to establish whether these neoplasms are molecularly similar to mesonephric carcinoma. Eight FATWOs underwent massively parallel sequencing to detect single nucleotide variations, copy number variations, and structural variants by surveying exonic DNA sequences of 300 cancer genes and 113 introns across 35 genes. Good quality DNA was isolated from 7 of 8 cases. Novel KMT2D variants (1 frameshift, 3 missense) were identified in 4 of 7 cases (57%), but were variants of uncertain biologic significance. STK11 mutations (both frameshift) were identified in 2 of 7 cases (29%); one of these was in a patient with a known history of Peutz-Jeghers syndrome. A mutation in the chromatin remodeling gene ARID1B was identified in 1 of 7 cases (14%). No cases harbored KRAS, NRAS, TP53, PIK3CA, PTEN, or DICER1 mutations. There were relatively low numbers of copy number variations, and no recurrent copy number variations were identified. One case demonstrated moderate copy gain of CCND1. No structural variants were identified. In summary, FATWO is characterized molecularly by the absence of KRAS/NRAS mutations (characteristic of mesonephric carcinoma), absence of DICER1 mutations (characteristic of Sertoli-Leydig cell tumor) and frequent KMT2D mutations of unknown biologic significance. FATWOs exhibit a limited number of molecular aberrations that are significantly different from those reported in tumors in the differential diagnosis, and our results question the relationship of mesonephric carcinoma with FATWO. Disease-defining molecular alterations for FATWO have yet to be discovered.