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Vulvar lichen sclerosus (LS) is an inflammatory dermatosis that can progress to human papillomavirus (HPV)-independent vulvar intraepithelial neoplasia (HPVi VIN) and vulvar squamous cell carcinoma (VSCC). Although LS has a much lower cancer risk compared with HPVi VIN (5% vs 50%, respectively), its incidence is significantly higher. Therefore, there is a clinical need to identify LS patients with an increased cancer risk. Our objective was to study the value of DNA methylation and p53 immunohistochemistry (IHC) as prognostic biomarkers for progression to cancer in patients with LS. Vulvar tissues from 236 patients were selected, including 75 LS and 68 HPVi VIN, both with and without cancer development, 32 VSCC, and 61 healthy vulvar controls. Samples were subjected to p53 IHC and DNA methylation analysis of a 3-gene marker panel containing ZNF582, SST, and miR124-2. Methylation levels and p53 IHC status (mutant or wild-type) were assessed and compared among all disease categories. Odds ratios were determined to identify whether the biomarkers were associated with progression to cancer in patients with LS. The highest methylation levels were found in HPVi VIN and VSCC, followed by LS and healthy vulvar controls. The largest heterogeneity in methylation levels was observed in LS cases. In fact, the 3-marker panel tested positive in 70% of LS, which progressed to VSCC vs only 17% of LS in patients without cancer development (P = .002). Also, mutant p53 IHC was observed more frequently in LS with progression to VSCC compared with nonprogressive LS cases (42% vs 3%, respectively, P = .001). Multivariable analysis identified a mutant p53 status as the only independent risk factor for cancer development in LS (odds ratio: 34.0, 95% CI: 1.4-807.4). In conclusion, DNA methylation testing and p53 IHC show strong potential as prognostic biomarkers for the identification of LS patients at high risk of progression to cancer., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The concept of a "p53 null phenotype" (complete loss of staining) is well-recognized in the gynecologic pathology literature, implicitly reflecting that this staining pattern represents a TP53 mutation. However, in the genitourinary pathology literature, a p53 null phenotype has only been addressed regarding the prognosis of invasive urothelial carcinoma, and not as a diagnostic biomarker for urothelial carcinoma in situ (CIS). Herein, 25 cases of urothelial carcinoma in situ [diagnoses made on hematoxylin and eosin (H&E) stained sections] showing null pattern p53 staining were retrieved from 22 different patients (16 males and 6 females, age range 52-85 years; average 69.6 years), most commonly showing large cell pleomorphic pattern morphology. One representative tissue block per case was selected for next-generation DNA sequencing (NGS). All 21 cases (100%) passing quality control for NGS showed at least 1 TP53 mutation (majority nonsense or frameshift mutations), including 3 cases with 2 mutations and 3 cases with 3 mutations. Three patients with multiple available samples harbored 1 or more shared TP53 mutations at 2 different time points, indicating clonality of the temporally distinct lesions. Additionally, 2 patients had an additional unique TP53 mutation at a later time point, suggesting intratumoral heterogeneity and/or temporal clonal evolution. While urothelial CIS remains an H&E diagnosis in most cases, a p53 immunostain may be useful in a subset of challenging cases. This study demonstrates that a p53 null phenotype represents an aberrant result in urothelial CIS with supportive molecular analysis showing a previously unknown level of complexity for TP53 mutations among these noninvasive lesions. Adequate recognition of the p53 null phenotype as a "biologically supportive result", similar to strong and diffuse staining with p53, is important and may warrant a formal consensus statement for recommended p53 reporting (i.e., "wild type" versus "aberrant or mutant")., (© 2022. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC) is an aggressive clinical entity. Current diagnostic guidelines for premalignant lesions are ambiguous, and their molecular profile and progression events are still unclear. We selected 75 samples, from 40 patients, including 33 VSCC, 8 verrucous carcinomas (VC), 13 differentiated-type vulvar intraepithelial neoplasia (dVIN), 11 suspicious for dVIN (?dVIN), 6 differentiated exophytic vulvar intraepithelial lesions (DE-VIL), 2 vulvar acanthosis with altered differentiation (VAAD), and 2 usual-type vulvar intraepithelial neoplasia (uVIN/HSIL). Invasive and precursor lesions were matched in 29 cases. Clinical information, p16 immunohistochemistry, and mutation analysis were performed on all lesions. All dVIN, ?dVIN, DE-VIL, and VAAD were p16 negative, all uVIN/HSIL were p16 positive. In the HPV-independent group, mutations were identified in 6 genes: TP53 (n = 40), PIK3CA (n = 20), HRAS (n = 12), MET (n = 5), PTEN (n = 4), and BRAF (n = 1). TP53 mutations occurred in 73% (22/30) VSCC, 85% (11/13) dVIN, 70% (7/10) ?dVIN and no VC (0/8), DE-VIL (0/6) nor VAAD (0/2). Basal atypia was the only reliable feature of TP53 mutations. ?dVIN lesions that were non-acanthotic and atypical but obscured by inflammation, all harbored TP53 mutations. In lesions without TP53 mutations, PIK3CA (50% VC, 33% DE-VIL, 100% VAAD, 40% VSCC) and HRAS (63% VC, 33% DE-VIL, 0% VAAD, 20% VSCC) mutations were found. Mutational progression from in situ to invasive was seen (7/26, 27%) and usually involved TP53 (4/26, 15%). Cases with TP53 and PIK3CA co-mutations had the worse clinical outcomes (p < 0.001). We recommend testing for p53 in all HPV-independent lesions suspicious for dVIN, even in the presence of marked inflammation or non-acanthotic skin, particularly when close to a margin. VC, VAAD, and DE-VIL, were almost never mutated for TP53, but instead often harbored PIK3CA and HRAS mutations. In VSCC, combined TP53 and PIK3CA mutations may inform prognosis.

Although squamous cell carcinomas (SCC) are the most frequent human solid tumor at many anatomic sites, the driving molecular alterations underlying their progression from precursor lesions are poorly understood, especially in the context of photodamage. Therefore, we used high-depth, targeted next-generation sequencing (NGS) of RNA and DNA from routine tissue samples to characterize the progression of both well- (cutaneous) and poorly (ocular) studied SCCs. We assessed 56 formalin-fixed paraffin-embedded (FFPE) cutaneous lesions (n = 8 actinic keratosis, n = 30 carcinoma in situ [CIS], n = 18 invasive) and 43 FFPE ocular surface lesions (n = 2 conjunctival/corneal intraepithelial neoplasia, n = 20 CIS, n = 21 invasive), from institutions in the US and Brazil. An additional seven cases of advanced cutaneous SCC were profiled by hybrid capture-based NGS of >1500 genes. The cutaneous and ocular squamous neoplasms displayed a predominance of UV-signature mutations. Precursor lesions had highly similar somatic genomic landscapes to SCCs, including chromosomal gains of 3q involving SOX2, and highly recurrent mutations and/or loss of heterozygosity events affecting tumor suppressors TP53 and CDKN2A. Additionally, we identify a novel molecular subclass of CIS with RB1 mutations. Among TP53 wild-type tumors, human papillomavirus transcript was detected in one matched pair of cutaneous CIS and SCC. Amplicon-based whole-transcriptome sequencing of select 20 cutaneous lesions demonstrated significant upregulation of pro-invasion genes in cutaneous SCCs relative to precursors, including MMP1, MMP3, MMP9, LAMC2, LGALS1, and TNFRSF12A. Together, ocular and cutaneous squamous neoplasms demonstrate similar alterations, supporting a common model for neoplasia in UV-exposed epithelia. Treatment modalities useful for cutaneous SCC may also be effective in ocular SCC given the genetic similarity between these tumor types. Importantly, in both systems, precursor lesions possess the full complement of major genetic changes seen in SCC, supporting non-genetic drivers of invasiveness.

Verruciform proliferations of the vulva unrelated to HPV infection are rare. The term differentiated exophytic vulvar intraepithelial lesion (DEVIL) was recently proposed for these lesions, which harbor recurrent PIK3CA mutations. It is still unclear whether DEVIL is related to verrucous carcinoma, a neoplasm characterized by persistence and local recurrence but nil risk of distant spread. Specimens identified using the words "verruciform" and "verrucous" were reviewed. Diagnosis of DEVIL required verruciform acanthosis, hyper and/or parakeratosis, hypogranulosis, cytoplasmic pallor, and bland nuclei. Verrucous carcinoma required, in addition, discontinuous, bulbous, puzzle-like nests in the stroma. A targeted next-generation sequencing using a custom 11-gene panel was performed. Eighteen specimens corresponding to ten patients with DEVIL and/or verrucous carcinoma were included. Median age at presentation was 66 years for DEVIL and 70 years for verrucous carcinoma. A similar spectrum of prevalent mutations was found in both lesions involving HRAS, PIK3CA, and BRAF. DEVIL preceded verrucous carcinoma and/or was diagnosed concurrently or in subsequent follow-up in five patients. In four of these, the same mutation was identified in DEVIL and synchronous or metachronous carcinoma. All cases showed wild-type 53 staining and lacked pathogenic TP53 mutations. DEVIL is a rare form of squamous proliferation characterized by prevalent PIK3CA and HRAS mutations. Its temporal relationship with verrucous carcinoma and their shared mutational profile in some patients suggest that DEVIL is a precursor of verrucous carcinoma. Moreover, given their morphologic and molecular overlap and the nil risk of verrucous carcinoma for distant spread, it is conceivable that DEVIL and verrucous carcinoma represent a spectrum of the same entity.

The switch from in situ to invasive tumor growth represents a crucial stage in the evolution of lung adenocarcinoma. However, the biological understanding of this shift is limited, and 'Noguchi Type C' tumors, being early lung adenocarcinomas with mixed in situ and invasive growth, represent those that are highly valuable in advancing our understanding of this process. All Noguchi Type C adenocarcinomas (n = 110) from the LATTICE-A cohort were reviewed and two patterns of in situ tumor growth were identified: those deemed likely to represent a true shift from precursor in situ to invasive disease ('Noguchi C1') and those in which the lepidic component appeared to represent outgrowth of the invasive tumor along existing airspaces ('Noguchi C2'). Overall Ki67 fraction was greater in C2 tumors and only C1 tumors showed significant increasing Ki67 from in situ to invasive disease. P53 positivity was acquired from in situ to invasive disease in C1 tumors but both components were positive in C2 tumors. Likewise, vimentin expression was increased from in situ to invasive tumor in C1 tumors only. Targeted next generation sequencing of 18 C1 tumors identified four mutations private to the invasive regions, including two in TP53, while 6 C2 tumors showed no private mutations. In the full LATTICe-A cohort, Ki67 fraction classified as either less than or greater than 10% within the in situ component of lung adenocarcinoma was identified as a strong predictor of patient outcome. This supports the proposition that tumors of all stages that have 'high grade' in situ components represent those with aggressive lepidic growth of the invasive clone. Overall these data support that the combined growth of Noguchi C tumors can represent two differing biological states and that 'Noguchi C1' tumors represent the genuine biological shift from in situ to invasive disease.

Recently it has been reported that serous tubal intraepithelial carcinoma (STIC), the likely precursor of ovarian/extra-uterine high-grade serous carcinoma, are frequently located in the vicinity of tubal-peritoneal junctions, consistent with the cancer-prone features of many epithelial transitional regions. To test if p53 (aka TP53)-signatures and secretory cell outgrowths (SCOUTs) also localize to tubal-peritoneal junctions, we examined these lesions in the fallopian tubes of patients undergoing salpingo-oophorectomy for sporadic high-grade serous carcinomas or as a prophylactic procedure for carriers of familial BRCA1 or 2 mutations. STICs were located closest to the tubal-peritoneal junctions with an average distance of 1.31 mm, while SCOUTs were not detected in the fimbriated end of the fallopian tube. As many epithelial transitional regions contain stem cells, we also determined the expression of stem cell markers in the normal fallopian tube, tubal intraepithelial lesions and high-grade serous carcinomas. Of those, LEF1 was consistently expressed in the tubal-peritoneal junctions and all lesions, independent of p53 status. All SCOUTs demonstrated strong nuclear expression of β-catenin consistent with the LEF1 participation in the canonical WNT pathway. However, β-catenin was preferentially located in the cytoplasm of cells comprising STICs and p53 signatures, suggesting WNT-independent function of LEF1 in those lesions. Both frequency of LEF1 expression and β-catenin nuclear expression correlated with the worst 5-year patient survival, supporting important role of both proteins in high-grade serous carcinoma. Taken together, our findings suggest the existence of stem cell niche within the tubal-peritoneal junctions. Furthermore, they support the notion that the pathogenesis of SCOUTs is distinct from that of STICs and p53 signatures. The location and discrete patterns of LEF1 and β-catenin expression may serve as highly sensitive and reliable ancillary markers for the detection and differential diagnosis of tubal intraepithelial lesions.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53 mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CA and ARID2, respectively. No TP53 mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53 and CDKN2A mutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CA mutations followed by a keratinizing carcinoma with mutations in both PIK3CA and TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CA mutant/TP53 wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CA mutations are found in <10% of vulvar squamous cell carcinomas, they may be specific for a particular pathway involving atypical verruciform lesions, which could function as either a direct precursor or a risk factor for vulvar squamous cell carcinoma. Given the presence of a molecular signature, we propose the term 'differentiated exophytic vulvar intraepithelial lesion' for this group. Whether they function as direct precursors to a less common form of squamous cell carcinoma will require further study, but carcinomas associated with these lesions might warrant testing for PIK3CA mutations to address this question.

Aberration in chromosomal structure characterizes almost all cancers and has profound biological significance in tumor development. It can be facilitated by various mechanisms including overexpression of cyclin E1 and centrosome amplification. As ovarian high-grade serous carcinoma has pronounced chromosomal instability, in this study we sought to determine whether increased copy number of CCNE1 which encodes cyclin E1 and centrosome amplification (>2 copies) occurs in its putative precursor, serous tubal intraepithelial carcinoma. We found CCNE1 copy number gain/amplification in 8 (22%) of 37 serous tubal intraepithelial carcinomas and 12 (28%) of 43 high-grade serous carcinomas. There was a correlation in CCNE1 copy number between serous tubal intraepithelial carcinoma and high-grade serous carcinoma in the same patients (P<0.001). There was no significant difference in the percentage of CCNE1 gain/amplification between serous tubal intraepithelial carcinoma and high-grade serous carcinoma (P=0.61). Centrosome amplification was recorded in only 5 (14%) of 37 serous tubal intraepithelial carcinomas, and in 10 (40%) of 25 high-grade serous carcinomas. The percentage of cells with centrosome amplification was higher in high-grade serous carcinoma than in serous tubal intraepithelial carcinoma (P<0.001). Induced expression of cyclin E1 increased the percentage of fallopian tube epithelial cells showing centrosome amplification. Our findings suggest that gain/amplification of CCNE1 copy number occurs early in tumor progression and precedes centrosome amplification. The more prevalent centrosome amplification in high-grade serous carcinoma than in serous tubal intraepithelial carcinoma supports the view that serous tubal intraepithelial carcinoma precedes the development of many high-grade serous carcinomas.

Serous endometrial intraepithelial carcinoma is often associated with extrauterine disease. It is currently unclear where does the extrauterine disease come from. This study addressed this issue. A total of 135 samples from 21 serous endometrial intraepithelial carcinoma patients were studied. Cellular lineage relationships between intrauterine and extrauterine serous carcinomas were determined by TP53-mutation analysis and correlated to the clinicopathologic features. There were three conditions contributing the extrauterine disease: metastasis from serous endometrial intraepithelial carcinoma (n=10) showed identical TP53 mutation between intrauterine lesions and extrauterine disease, cases of adnexal origin (n=5) had discordant TP53 mutations, and the mixed cellular origin cases (n=6) with both identical and discordant mutation status. Patients with extrauterine disease from serous endometrial intraepithelial carcinoma metastasis typically had small tumor masses (<2 cm) in extrauterine sites and without finding of serous tubal intraepithelial carcinoma, while extrauterine disease with adnexal or tubal origin commonly had larger tumor masses in extrauterine sites including ovary and omentum and serous tubal intraepithelial carcinoma. The majority of extrauterine diseases associated with serous endometrial intraepithelial carcinoma are metastasized from the endometrium. Serous endometrial intraepithelial carcinoma is frequently associated with serous cancers of adnexal or tubal origin, indicating that endometrial and adnexal or tubal serous cancers may share similar etiologies. TP53-mutation analysis provides a strong linkage for cellular lineage analysis. Tumor size in extrauterine disease and presence of serous tubal intraepithelial carcinoma or not are useful clinicopathologic features to determine primary cancer site, which helps in clinical management.

Intraductal papillary neoplasms of the bile duct are still poorly characterized regarding (1) their molecular alterations during the development to invasive carcinomas, (2) their subtype stratification and (3) their biological behavior. We performed a multicenter study that analyzed these issues in a large European cohort. Intraductal papillary neoplasms of the bile duct from 45 patients were graded and subtyped using mucin markers and CDX2. In addition, tumors were analyzed for common oncogenic pathways, and the findings were correlated with subtype and grade. Data were compared with those from 22 extra- and intrahepatic cholangiocarcinomas. Intraductal papillary neoplasms showed a development from preinvasive low- to high-grade intraepithelial neoplasia to invasive carcinoma. Molecular and immunohistochemical analysis revealed mutated KRAS, overexpression of TP53 and loss of p16 in low-grade intraepithelial neoplasia, whereas loss of SMAD4 was found in late phases of tumor development. Alterations of HER2, EGFR, β-catenin and GNAS were rare events. Among the subtypes, pancreato-biliary (36%) and intestinal (29%) were the most common, followed by gastric (18%) and oncocytic (13%) subtypes. Patients with intraductal papillary neoplasm of the bile duct showed a slightly better overall survival than patients with cholangiocarcinoma (hazard ratio (cholangiocarcinoma versus intraductal papillary neoplasm of the bile duct): 1.40; 95% confidence interval: 0.46-4.30; P=0.552). The development of biliary intraductal papillary neoplasms of the bile duct follows an adenoma-carcinoma sequence that correlates with the stepwise activation of common oncogenic pathways. Further large trials are needed to investigate and verify the finding of a better prognosis of intraductal papillary neoplasms compared with conventional cholangiocarcinoma.

Immunohistochemical loss of keratin (K)13 is one of the most valuable diagnostic criteria for discriminating carcinoma in situ (CIS) from non-malignancies in the oral mucosa while K13 is stably immunolocalized in the prickle cells of normal oral epithelium. To elucidate the molecular mechanism for the loss of K13, we compared the immunohistochemical profiles for K13 and K16 which is not expressed in normal epithelia, but instead enhanced in CIS, with their mRNA levels by in-situ hybridization in formalin-fixed paraffin sections prepared from 23 CIS cases of the tongue, which were surgically removed. Reverse transcriptase-PCR was also performed using RNA samples extracted from laser-microdissected epithelial fragments of the serial paraffin sections in seven of the cases. Although more enhanced expression levels for K16 were confirmed at both the protein and gene levels in CIS in these seven cases, the loss of K13 was associated with repressed mRNA levels in four cases, but not in the other three cases. The results suggest that the loss of K13 is partly due to its gene repression, but may also be due to some unknown post-translational events.

Aldo-keto reductase family 1B10 (AKR1B10) exhibits more restricted lipid substrate specificity (including farnesal, geranylgeranial, retinal and carbonyls), and metabolizing these lipid substrates has a crucial role in promoting carcinogenesis. Overexpression of AKR1B10 has been identified in smoking-related carcinomas such as lung cancer. As development of pancreatic cancer is firmly linked to smoking, the aim of the present study was to examine the expression and oncogenic role of AKR1B10 in pancreatic adenocarcinoma. AKR1B10 expression was analyzed in 50 paraffin-embedded clinical pancreatic cancer samples using immunohistochemistry. Oncogenic function of AKR1B10 was examined in pancreatic carcinoma cells in vitro using western blotting and siRNA approaches, mainly on cell apoptosis and protein prenylation including KRAS protein and its downstream signals. Immunohistochemistry analysis revealed that AKR1B10 overexpressed in 70% (35/50) of pancreatic adenocarcinomas and majority of pancreatic intraepithelial neoplasia, but not in adjacent morphologically normal pancreatic tissue. Compared with a normal pancreatic ductal epithelial cell (HPDE6E7), all of the six cultured pancreatic adenocarcinoma cell lines had an overexpression of AKR1B10 using immunoblotting, which correlated with increase of enzyme activity. siRNA-mediated silencing of AKR1B10 expression in pancreatic cancer cells resulted in (1) increased cell apoptosis, (2) increased non-farnesyled HDJ2 protein and (3) decreased membrane-bound prenylated KRAS protein and its downstream signaling molecules including phosphorylated ERK and MEK and membrane-bound E-cadherin. Our findings provide first time evidence that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis possibly via modulation of cell apoptosis and protein prenylation.

Telomeres protect against chromosomal breakage, fusion, and interchromosome bridges during cell division. Shortened telomeres have been observed in the lowest grade of pancreatic intraepithelial neoplasia (PanIN). Genetically engineered mouse models of pancreatic neoplasia develop acinar-to-ductal metaplasia prior to the development of PanIN, suggesting that acinar-to-ductal metaplasias can be an early precursor lesion to pancreatic cancer. Some human PanINs are associated with acinar-to-ductal metaplasias, and it has been suggested that these acinar-to-ductal metaplasias arise as a consequence of growth of adjacent PanINs. As the earliest known genetic lesions of PanINs is shortened telomeres, we compared the telomere lengths of acinar-to-ductal metaplasia lesions, PanINs, and adjacent normal cells of human pancreata to determine whether acinar-to-ductal metaplasias could be precursors to PanIN. We used quantitative fluorescent in situ hybridization to measure the telomere length of cells from pancreatic lesions and adjacent normal pancreata from 22 patients, including 20 isolated acinar-to-ductal metaplasias, 13 PanINs associated with acinar-to-ductal metaplasias, and 12 PanINs. Normalized mean telomere fluorescence was significantly different among the cell types analyzed; 12.6 ± 10.2 units in normal acinar cells, 10.2 ± 6.4 in ductal cells, 8.4 ± 5.9 in fibroblasts, 9.4 ± 7.3 in isolated acinar-to-ductal metaplasias, 4.1 ± 2.9 in PanIN-associated acinar-to-ductal metaplasias, and 1.6 ± 1.9 in PanINs, respectively (P<0.001, ANOVA with randomized block design). Telomeres were significantly shorter in PanIN-associated acinar-to-ductal metaplasias (P<0.05, post hoc Duncan test) and in PanINs (P<0.05), than in normal cells, or isolated acinar-to-ductal metaplasias. Thus, shortened telomeres are found in PanIN-associated acinar-to-ductal metaplasias, but not in isolated acinar-to-ductal metaplasia lesions. These results indicate that isolated acinar-to-ductal metaplasias are not a precursor to PanIN, and support the hypothesis that PanIN-associated acinar-to-ductal metaplasias arise secondary to PanIN lesions.

High-grade endometrioid and serous carcinomas of the ovary and fallopian tube are responsible for the majority of cancer deaths and comprise a spectrum that includes early or localized (tubal intraepithelial carcinoma) and advanced (invasive or metastatic) disease. We subdivided a series of these tumors into three groups, (1) classic serous, (2) mixed serous and endometrioid and (3) endometrioid carcinomas and determined: (1) the frequencies of coexisting tubal intraepithelial carcinoma, (2) frequency of a dominant ovarian mass suggesting an ovarian origin and (3) immuno-localization of WT-1, p53, PTEN, PAX2 and p16(ink4). All tumors were analyzed for p53 mutations. Thirty six, 25 and 8% of groups 1-3 were associated with tubal intraepithelial carcinoma (P=0.09) and 34, 45 and 62% predominated in one ovary (P=0.028), respectively. Differences in frequencies of diffuse p53 immunostaining (85-93%), WT-1 (70-98%) and p16(ink4) positivity (69-75%) were not significant for all groups. Greater than 95% reduction in PAX2 and PTEN occurred in 67-75 and 5-12%, respectively; however, PAX2 and PTEN staining intensity, when present, was often heterogeneous, highlighting different tumor populations. PAX2 and PTEN expression were markedly reduced or absent in 12 of 12 and 4 of 12 tubal intraepithelial carcinomas. In summary, high-grade müllerian carcinomas share identical frequencies of altered or reduced expression of p53, PTEN and PAX2, all of which can be appreciated in tubal intraepithelial carcinomas. Because only a subset of these tumors appears to arise in the fallopian tube, attention to expression of these biomarkers in the ovary and other müllerian sites might facilitate the identification of other carcinogenic pathways. PAX2 and PTEN, in addition to p53 and p16(ink4), comprise a potentially important gene combination in high-grade pelvic carcinogenesis.

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.

The main objectives of this review are (1) to review the histological criteria and terminology for usual ductal hyperplasia, atypical ductal hyperplasia, flat epithelial atypia and ductal carcinoma in situ; (2) to provide an overview of the risk of breast cancer associated with these various lesions; (3) to discuss established and emerging data on the molecular biology and pathogenesis of these lesions and (4) to discuss management when detected in diagnostic needle core and surgical diagnostic specimens.

Differentiated vulvar intraepithelial neoplasia is a unique precursor to vulvar squamous cell carcinoma that is typically HPV-negative and frequently associated with nuclear p53 staining. These features imply a mode of pathogenesis involving somatic mutations. However, the genetic relationship of differentiated vulvar intraepithelial neoplasm and vulvar squamous cell carcinoma and the role of Tp53 mutations in this process have not been resolved. We analyzed 11 differentiated vulvar intraepithelial neoplasms and 6 associated vulvar squamous cell carcinomas. Sections were stained for p53 and p63 and DNA from multiple epithelial sites, representing normal control tissues (n=10), differentiated vulvar intraepithelial neoplasias (n=18), and vulvar squamous cell carcinomas (n=6), were obtained by laser capture microdissection, and sequenced for exons 2-11 of Tp53. Six of 10 cases contained at least one Tp53 mutation-positive differentiated vulvar intraepithelial neoplasia focus; 4 strongly p53 immuno-positive and 2 negative. Staining for p53 and p63 co-localized, targeting the immature epithelium, but surface epithelium was Tp53 mutation-positive. Four of five vulvar squamous cell carcinomas were Tp53 mutation-positive; two shared identical Tp53 mutation with adjacent differentiated vulvar intraepithelial neoplasia. Disparate foci of differentiated vulvar intraepithelial neoplasia often showed different mutations consistent with multiple neoplastic clones. Differentiated vulvar intraepithelial neoplasia is, with few exceptions, associated with Tp53 mutations and will be p53 immunopositive when missense mutations (versus some nonsense and all deletion mutations) are present. Multiple Tp53 mutations in different sites supports the presence of multiple independent genetic events, but shared Tp53 mutations in both differentiated vulvar intraepithelial neoplasia and vulvar squamous cell carcinoma support a genetic relationship between the two. The confinement of p53 staining to immature cell nuclei is consistent with maturation-dependent degradation of mutant p53 protein.

Squamous morules are a common component of premalignant glandular lesions that are followed by glandular, rather than squamous, carcinomas. We tested the hypothesis that the appearance of glands associated with morules predicts cancer risk, and undertook molecular testing to determine the clonal and hormonal response properties of admixed squamous and glandular elements. A total of 66 patients with squamous morules in an index endometrial biopsy had follow-up clinical data (average follow-up: interval 31 months, 2.5 biopsies) showing development of carcinoma in 11% (7/66) of cases. The histological appearance of morule-associated glands in the index biopsy was significantly associated with this clinical outcome, with the majority (71%, 5/7) of cancer occurrences following an overtly premalignant lesion (endometrial intraepithelial neoplasia) with squamous morules. Eight endometrial intraepithelial neoplasias with squamous morules were examined by immunohistochemistry for estrogen and progesterone receptors and mitotic activity (Ki-67 antigen percent stained). Glandular components had abundant estrogen and progesterone receptors, and high levels of mitotic activity in all cases. In sharp contrast, all squamous morules were devoid of sex hormone receptors and had undetectable or extremely low-proliferation rates. When mutated, the same specific PTEN mutation was detected in squamous and glandular elements, indicating that both are of common lineage. The clinical and laboratory data are consistent with a model of morule biology in which squamous morules are a hormonally incompetent subpopulation of endometrial glandular lesions. Isolated morules might result from artifactual displacement from their native glandular context, or selective hormonally induced regression of the glandular but not squamous components over time. Subsequent cancer risk, as promoted by estrogens, is greatest when the glandular component has the appearance of endometrial intraepithelial neoplasia. Even isolated morules should be carefully followed, however, to exclude a coexisting undersampled, or occult, glandular lesion.

Somatic mutations of mitochondrial DNA (mtDNA) are associated with various types of human cancer. To elucidate their role in gastric carcinogenesis, we analyzed mutations in the displacement loop region of mtDNA in 24 paraffin-embedded gastric intraepithelial neoplasias (formerly dysplasia) from a high gastric cancer risk area in northern Italy. Helicobacter pylori infection was assessed by histological examination (Giemsa staining). Gastritis was classified according to the guidelines of the Updated Sydney System. The mtDNA displacement loop region was amplified and sequenced from gastric intraepithelial neoplasia samples and adjacent non-neoplastic gastric mucosa. The gastric intraepithelial neoplasias were divided into two groups by their association with H. pylori gastritis. Group A with lesions arising on a background of H. pylori-positive gastritis contained 7 patients, and group B with lesions associated with H. pylori-negative gastritis contained 17 patients. Group A had a larger proportion of high-grade lesions than group B and showed a foveolar phenotype (type II dysplasia). Group B had a larger proportion of cases with mtDNA displacement loop region mutations than group A (P=0.004, Fisher's exact test) and exhibited an intestinal phenotype. No evidence of heteroplasmic variants in the mtDNA displacement loop, suggestive of mutations, was detected in gastric biopsies from 25 H. pylori-negative subjects and 60 cancer-unaffected H. pylori-positive patients. These results provide further evidence for the morphologic and mtDNA biomolecular differences of gastric intraepithelial neoplasias, and suggest the existence of two distinct pathways to gastric cancer--corpus-dominant H. pylori gastritis and the atrophy-metaplasia pathway.

Biliary tract carcinoma, including carcinoma of the gallbladder, intrahepatic bile ducts (cholangiocarcinoma), and extrahepatic bile ducts, affects 7500 people in the United States annually, and has an overall 32% 5-year survival rate for disease limited to the mucosa, and a dismal 10% 5-year survival for more advanced disease. The identification of factors involved in the pathogenesis and progression of biliary tract carcinoma is critical for devising effective methods of screening and treatment. Recent evidence suggests that reduction of the length of telomeres, which normally help maintain chromosomal stability, may promote the development and progression of a variety of carcinomas. Using a novel, recently validated telomere fluorescence in situ hybridization method, we examined telomere length in normal and inflamed gallbladder epithelium, metaplasia and dysplasia of the gallbladder, and biliary tract carcinoma to determine whether telomere shortening is associated with neoplastic progression in the biliary tract. Although normal and inflamed gallbladder epithelium demonstrated uniform normal telomere lengths, over half of all metaplastic lesions demonstrated shortened telomeres, supporting prior evidence that metaplastic lesions of the gallbladder are pre-neoplastic. Dysplastic epithelium and invasive carcinomas demonstrated almost universally abnormally short telomeres, indicating that telomere shortening occurs at an early, preinvasive stage of cancer development. In addition, invasive adenocarcinoma of the biliary tract frequently demonstrated intratumoral heterogeneity of telomere lengths. We conclude that telomere shortening is a consistent and early finding in the development of biliary tract carcinoma.

Tumor development from an early lesion through to invasive disease is not a clearly defined progression in the breast. Studies of invasive lobular carcinoma have reported mutations, loss of heterozygosity (LOH) and loss of protein expression in epithelial (E)-cadherin, a protein involved in cell adhesion. Our study examines in situ lobular neoplastic lesions without concurrent invasive carcinoma for E-cadherin gene alterations and protein expression, beta-catenin, alpha-catenin and p120-catenin protein expression, and LOH at the chromosome 16q locus, with the goal of determining the events occurring at the stage of lobular neoplasia. In all, 13 atypical lobular hyperplasia lesions and 13 lobular carcinoma in situ lesions from archived cases were examined. E-cadherin sequence alterations were evaluated using single strand conformation polymorphism and DNA sequencing, and PCR-based LOH analysis was carried out for the 16q locus. Using immunohistochemistry, we assessed protein expression. A total of 23 of 24 lesions evaluated by immunohistochemistry were negative for both E-cadherin and beta-catenin protein expression, and 21 of 23 lesions were negative for alpha-catenin. Cytoplasmic (rather than membrane) localization of p120-catenin was observed in 20 of 21 cases. Lobular carcinoma in situ cases were characterized by mutations; however, atypical lobular hyperplasia cases were not. LOH at 16q was an infrequent event. From our study, we conclude that an altered E-cadherin adhesion complex is an early event affecting atypical lobular hyperplasia as well as lobular carcinoma in situ and occurs prior to progression to invasive disease. However, the loss of protein expression is accompanied by E-cadherin DNA alterations in lobular carcinoma in situ but not in atypical lobular hyperplasia. These cases lacking both protein expression and gene alterations suggest that another mechanism is involved, possibly as early as at the hyperplastic stage, causing silencing of the E-cadherin complex.

We attempted to identify deleted segments in two model tumor suppressor gene loci on chromosomes 13q14 and 17p13 that were associated with clonal expansion of in situ bladder preneoplasia using single nucleotide polymorphisms (SNPs)-based whole-organ histologic and genetic mapping. For mapping with SNPs, the sequence-based maps spanning approximately 27 and 5 Mb centered around RB1 and p53, respectively, were assembled. The integrated gene and SNP maps of the regions were used to select 661 and 960 SNPs, which were genotyped by pyrosequencing. Genotyping of SNPs was performed on DNA samples corresponding to histologic maps of the entire bladder mucosa in human cystectomy specimens with invasive urothelial carcinoma. By using this approach, we have identified deleted regions associated with clonal expansion of intraurothelial neoplasia; which ranged from 0.001 to 4.3 Mb (average 0.67 Mb) and formed clusters of discontinuous deleted segments. The high resolution of such maps is a prerequisite for future positional targeting of genes involved in early phases of bladder neoplasia. This approach also permits analysis of the overall genomic landscape of the involved region and discloses that a unique composition of noncoding DNA characterized by a high concentration of repetitive sequences may predispose to deletions.

Array-based comparative genomic hybridization (CGH) is a technique that allows genome wide screening of gains and losses in DNA copy number. In cases where multiple tumors are encountered, this genetic technique may prove useful in differentiating new primary tumors from recurrences. In this case report, we used array-based CGH to examine the genomic relationships among two leiomyosarcomas and two breast cancers in the same patient, three of which were diagnosed synchronously. Array-based CGH was performed on the four tumor samples using random prime amplified microdissected DNA. Samples were hybridized onto bacterial artificial chromosome arrays composed of approximately 2400 clones. Patterns of alterations within the tumors were compared and genetic alterations among the leiomyosarcomas and breast lesions were found. Overall, three distinct genetic profiles were observed. While the two leiomyosarcomas shared a similar pattern of genetic alterations, the two invasive breast lesions did not. The nearly identical pattern of genetic alterations belonging to the two metachronous leiomyosarcomas confirmed metastatic recurrence while the two different genetic profiles of the invasive ductal carcinomas suggest that the two lesions represented two distinct foci of multifocal disease rather than clonal extension of the primary tumor. We conclude that genetic analysis by array-based CGH can clearly elucidate the relationships between multiple tumors and may potentially serve as an important clinical tool.

Methylation of tumor suppressor genes has been implicated in breast cancer development. However, methylation profiles of different breast lesions, subtypes of carcinoma in particular, have not been examined in detail. In this study, we use methylation-specific PCR (MSP) to generate gene methylation profiles of different breast lesions and to test the clinical utility of such profiles. We examined the methylation status of three genes, RARbeta2, RASSF1A, and cyclin D2, on 102 samples of breast tissue, from benign (n = 36), to in situ carcinoma (n = 21), to invasive carcinoma (n = 45). We found that almost all cases of invasive carcinoma (96%) contained at least one methylated gene from our panel, whereas gene methylation was less common among benign lesions (42%) and in situ carcinoma (76%). Of the three genes, cyclin D2 methylation was most specific for malignancy because only 1 of 35 benign cases was methylated at this gene (1 case was not informative). The major histologic subtypes of invasive carcinoma show similar methylation profiles in the genes examined. We next performed MSP analysis on archival breast fine-needle aspiration (FNA) biopsy samples and corresponding surgical biopsy specimens and found a high concordance between the two types of specimens. We then analyzed 17 breast FNA biopsy samples with an indeterminate diagnosis. In this setting, MSP had a high specificity (100%) and modest sensitivity (67%) for identifying malignancy.

E-cadherin is a calcium-dependent, intercellular adhesion molecule that is specifically expressed in epithelial tissues and plays an important role in maintaining epithelial stability. E-cadherin is widely regarded as a prognostic marker in many types of human cancers. The inactivation of the E-cadherin gene is linked to increased potential for tumor invasiveness and distant metastasis. We previously demonstrated reduced expression of E-cadherin protein immunohistochemically in invasive squamous cell carcinomas of the skin as compared with adjacent normal skin. An epigenetic alteration in association with promoter hypermethylation is one important mechanism of gene silencing. In the present study, we analyze the E-cadherin gene promoter hypermethylation in preneoplastic and neoplastic skin lesions to determine whether epigenetic alteration of the E-cadherin gene also plays an important role in cutaneous squamous carcinogenesis. A total of 33 cases was examined for evidence of E-cadherin promoter hypermethylation, and these consist of nine cases of spongiotic dermatitis as nonneoplastic skin control, nine cases of actinic keratosis, eight cases of squamous cell carcinoma in situ, and seven cases of invasive squamous cell carcinoma. Promoter hypermethylation of the E-cadherin gene was detected in 6 of 7 cases (85%) of invasive squamous cell carcinoma, 4 of 8 cases (50%) of squamous cell carcinoma in situ, 4 of 9 cases (44%) of actinic keratosis, and 2 of 9 cases (22%) of nonneoplastic skin. We conclude that E-cadherin promoter hypermethylation occurs frequently and may represent an important mechanism of E-cadherin inactivation in cutaneous preneoplastic and neoplastic lesions. The frequencies of E-cadherin promoter hypermethylation appear to be correlated with more advanced stage of squamous carcinogenesis in skin.

We reviewed 10 cases of pleomorphic lobular (ductal lobular) carcinoma in situ (PL/DLCIS) of the breast and compared them with 14 cases of pleomorphic lobular carcinoma in situ (PLCIS) found in association with invasive pleomorphic lobular carcinoma. The histologic features; immunohistochemical staining for estrogen receptors (ERs), p53, Ki67, E-cadherin, and gross cystic disease fluid protein-15 (GCDFP-15); and results of fluorescence in situ hybridization for HER-2/neu gene amplification were evaluated in all 24 cases. Histologically, PL/DLCIS cells were similar to those of PLCIS with invasion in that they were discohesive and medium to large in size with moderate to marked nuclear pleomorphism, small to prominent nucleoli, and moderate to abundant eosinophilic or vacuolated cytoplasm. In both groups, central necrosis was present in a small number of cases, and classic LCIS coexisted with the in situ lesion in less than half of the cases; in situ carcinomas were positive for ERs in 23 (100%) of 23 cases, p53 in 6 (25%) of 24 cases, and GCDFP-15 in 14 (74%) of 19 cases. The percentage of Ki67-positive tumor nuclei indicated moderate to high (more than 20%) proliferative activity in 8 (47%) of 17 cases. Immunostaining for E-cadherin was negative in all 24 cases. HER-2/neu gene amplification was observed in 1 (4%) of 23 cases. In cases with associated invasion, PLCIS had cytologic features and immunostaining patterns similar to those of the invasive pleomorphic component. Seven of the 10 patients who had PL/DLCIS without invasion underwent lumpectomy or simple mastectomy. Six of these patients had no evidence of disease in follow-up periods ranging from 4 to 32 months; the seventh patient developed recurrent disease 12 months after undergoing lumpectomy. We conclude that the cytologic features and biomarker expression profile of PL/DLCIS are similar to those of PLCIS with invasion but somewhat different from those of classic LCIS and ductal carcinoma in situ. Long-term follow-up studies are needed to further define the natural history of PL/DLCIS and its optimal management.

Amplification of Her-2/neu in breast carcinoma is associated with poor prognosis, short disease-free interval, and short survival time in both node-negative and -positive patients. Little is known about the starting point of amplification of Her-2/neu and how it progresses from benign to malignant breast lesions. We attempted to address these questions by evaluating amplification of Her-2/neu in benign, premalignant, and malignant lesions using fluorescence in situ hybridization (FISH). Twenty-six patients with Her-2/neu-overexpressing invasive ductal carcinomas (as judged by strong immunoreactivity with Her-2/neu antibody) and coexisting lesions of ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) in the vicinity of the invasive tumor (as judged by review of the hematoxylin and eosin-stained sections), as well as metastatic carcinoma in axillary lymph nodes (mets) were selected for this study. In the primary carcinomas, a close relationship was present between overexpression as detected by immunohistochemistry (IHC) and amplification as demonstrated by FISH (85% concordance). Among these patients, amplification of Her-2/neu in ADH was demonstrated in 7 of 13 cases with ADH, and in DCIS, in 21 of 22 cases with DCIS. There was no amplification in DH or normal ductal epithelium. Significantly, in all 12 patients with synchronous positive axillary lymph nodes, there was concordant amplification of Her-2/neu in the primary and metastatic carcinoma. Amplification was consistent in multifocal metastases, despite morphological heterogeneity in some patients. Amplification ratios increased from ADH to DCIS to invasive carcinoma (P <.01, ADH versus DCIS; P <.05, DCIS versus invasive cancer), but there was no difference in amplification ratios between primary cancers and synchronous axillary metastases (P >.05). We also evaluated Her-2/neu amplification in 21 patients without Her-2/neu overexpression in their primary carcinomas (as judged by absent immunoreactivity with Her-2/neu antibody). Three showed amplification in both primary and metastatic lesions, with a low amplification ratio (approximately 2). One patient had amplification in the primary tumor but not in an axillary metastasis. Two patients exhibited slight amplification in the metastatic carcinoma (ratios 1.6 and 2), but not in their primary cancers. This FISH study indicates that amplification of Her-2/neu can emerge de novo in any stage of the disease process, from ADH to metastatic lesions, but most often appears first in ADH or DCIS. The degree of Her-2/neu amplification increases with progression to invasive carcinoma, there being no further increase in synchronous metastasis. Our data suggest that amplification of Her-2/neu appears to be mainly involved in initiation of breast oncogenesis and that its role in progression of breast cancers is uncertain.

In breast cancer amplification of the HER-2/neu oncogene and over-expression of the protein product is associated with poor prognosis, predicts response to some chemotherapeutic regimens and is the target for Herceptin treatment. To date there are several methods to assess the amplification/over-expression of HER-2/neu with each having advantages and disadvantages. We have studied amplification and over-expression of HER-2/neu in 250 consecutive cases of breast cancer (220 invasive and 30 in situ carcinomas) presenting to the Department of Pathology at Women's College Campus of Sunnybrook and Women's College Health Sciences Center. Thirty percent of the invasive carcinomas were node positive. HER-2/neu protein over-expression was assessed by immunohistochemistry (IH) using antibody CB11 and amplification of the gene by differential PCR. The percentage of tumor cells showing CB11 staining was determined and the most significant cut off point for positivity was > or =10% moderate or strong complete membranous staining. The gene was considered amplified if the density score of the product was > or =2. There was 94% concordance between the two methods (P value.0001). Both methods were positive in 16% of cases and negative in 78% of cases. Discrepant cases were examined by FISH which confirmed the IH results in 9/11 invasive carcinomas. These results show that there is excellent concordance between IH and PCR. However, immunohistochemistry is easier to perform and cheaper than PCR and could be used in routine assessment of HER-2/neu in breast cancer patients.

Various molecular markers have been used for the detection of circulating breast cancer cells in blood by reverse transcriptase-polymerase chain reaction (RT-PCR). Using nested RT-PCR, we compared the specificity and sensitivity of human mammaglobin (hMAM), epidermal-growth-factor receptor (EGF-R), and cytokeratin 19 (CK-19) expression as markers for circulating carcinoma cells in the peripheral blood of patients with breast cancer. Blood samples from 12 patients with ductal carcinoma in situ, 133 patients with invasive breast cancer, 20 patients with hematological malignancies, 31 healthy volunteers, and tumor tissues from 40 patients with invasive breast cancer were screened for mRNA encoding hMAM, EGF-R, or CK-19 by nested RT-PCR. In all breast cancer tissues, mRNA for hMAM, EGF-R, and CK-19 was detectable. In blood samples from patients with invasive breast cancer, 11 (8%), 13 (10%), and 64 (48%) were positive for mRNA encoding hMAM, EGF-R, or CK-19, respectively. Blood samples from none of the healthy volunteers and patients with hematological disorders were positive for hMAM, while CK-19 mRNA was found in the blood of 12 (39%) healthy volunteers and transcripts for EGF-R and CK-19 were detectable in 5 (25%) and 2 (10%), respectively, of the patients with hematological malignancies. Only hMAM mRNA expression in blood correlated with clinical parameters such as nodal status, metastasis, and CA 15-3 serum levels. In summary, hMAM transcripts detectable in blood by RT-PCR represent the most specific molecular marker for hematogenous spread of breast cancer cells. With the nested RT-PCR method, aberrant EGF-R mRNA expression might occasionally be found in hematological malignancies, whereas CK-19 mRNA expression proved to be rather nonspecific. The prognostic value of hMAM RT-PCR-based tumor cell detection in peripheral blood should be further tested and validated in prospective studies.

P53 allelic polymorphism at codon 72 has been studied as a possible predisposing factor for cervical carcinogenesis with inconsistent results. Storey and colleagues recently published the interesting finding of a 7-fold increased risk for cervical cancer in women homozygous for the arginine allele at codon 72. This stimulated a number of independent investigations, the majority of which found no association of cervical cancer and arginine homozygosity. With the use of a modified Storey method for determining codon 72 allelotypes, DNA was examined from 431 microdissected, formalin-fixed, archival cervical conization specimens ranging from low-grade squamous lesions to invasive cancer. An alternative independent method using restriction fragment length polymorphism analysis was performed on all arginine homozygotes and all indeterminate cases for confirmation and final allelotype assignment. With the use of Storey's method alone, logistic regression suggested an association (odds ratio, 1.42) between arginine homozygosity and invasive disease. However, with the use of the combined method for accurate allelotyping, this trend disappeared (odds ratio, 1.00), the discordance was clearly resolvable as being due to methodologic variables. With the use of two separate methods for codon 72 allelotyping and accounting for a number of the issues raised in previously published reports, there is no increased risk for invasive cervical cancer associated with arginine homozygosity at codon 72 of p53.

Ductal carcinoma in situ (DCIS) of the breast is often an indolent disease, although some cases are reported to recur many years after a limited surgical resection. It is not known whether these recurrences reflect a resurgence of residual disease or an independent development of a second tumor in susceptible individuals. Therefore, we conducted a longitudinal molecular study of four women with reappearance of DCIS 2 to 15 years after an initial conservative resection. Loss of heterozygosity (LOH) was characterized in both tumors in each case, using several polymerase chain reaction-amplified microsatellite markers on five chromosomal arms commonly affected in breast cancer. In three cases with ipsilateral recurrent disease, all of the allelic losses seen in the initial tumors were also seen in the recurrent lesions, suggesting a common genetic pathway for the development of both lesions and continuous proliferation of residual disease. The presence of at least one additional LOH in all of the three recurrent tumors, however, suggests that the recurrent tumors developed after genetic progression. In contrast, in one case of DCIS that was followed by the development of DCIS in the contralateral breast 7 years later (a case of bilateral DCIS), unrelated LOH patterns were present in the two lesions. These findings suggest that the reappearance of DCIS in the same breast is most commonly the result of a tumor derived from (but not identical to) the original lesion, with acquisition of additional genetic changes, even when the recurrent lesion manifested itself many years (15 years, in one case) after the initial presentation. Furthermore, genetic progression could be detected in tumors recurring in as little as 2 years after the initial resection.

The histogenesis of carcinosarcomas is controversial, specifically with respect to clonality and cell of origin. To answer these questions, tumor cells from both epithelial and spindle-cell components were microdissected from three cases each of mammary carcinosarcoma and its postulated precursor, carcinoma with spindle-cell metaplasia. Clonality was assessed using the principle of X chromosome inactivation. Loss of heterozygosity (LOH) was evaluated at seven chromosomal loci to assess shared and distinctive genetic alterations for the two components in each tumor. All of the six cases demonstrated identical clonality of the carcinomatous and spindle-cell components, identical to a focus of ductal carcinoma in situ present in one case. LOH for NM23 was detected in both components in one carcinosarcoma, whereas LOH for INT-2 was detected in both components in one metaplastic carcinoma. Differential LOH for D11S904 was present in only the mesenchymal components of these two cases. We conclude that the two components of carcinosarcoma and its precursor are clonal and that the sarcomatous and spindle-cell components arise from mutation of the carcinoma. Presence of differential LOH at D11S904 in only the spindle-cell components suggests that this mutation might be critical to the development of this second phenotype.

Background: Recently we have shown, by combining in situ hybridization and immunohistochemistry, that carcinoma in situ (CIS) adjacent to seminoma (CIS/SE), like SE, usually contains three copies of the centromeric region of chromosome 15/tumor cell. In contrast, CIS adjacent to nonseminomatous testicular germ cell tumors of adults (CIS/NS), as well as NS itself, have two copies., Experimental Design: In the present study we have used this approach to investigate the clonal origin of the SE and NS components of combined tumors (CTs). We counted the number of copies of chromosome 15 centromeric regions in tumor cell nuclei of the CIS, SE, and NS components of nine CTs., Results: We show that the number of copies of centromeric regions of chromosome 15 in both the SE and the NS component, and the adjacent CIS of the same CT, may be high (SE-pattern) or low (NS-pattern). In two cases, the copy numbers were high in the SE component and its adjacent CIS, and low in the NS component and its adjacent CIS., Conclusions: The data suggest that in most CTs the SE and the NS components have a monoclonal origin, and that karyotype evolution in CIS and the invasive tumor is very similar.

Unlabelled: The diagnosis of ductal carcinoma in situ of the breast (DCIS) has become common with the advent of breast screening programs., Methods: Proliferation indices (S-phase fraction) were studied in 76 cases of pure DCIS. Tumors were classified according to conventional criteria and also according to a novel simplified classification based on cellular necrosis and morphology. This new classification defines three distinct tumor groups: pure comedo in 19 (25.0%) cases, DCIS with necrosis (non-pure comedo) in 21 (27.6%) patients, and DCIS without necrosis in 36 (47.4%) of cases, the latter group comprising largely classical cribriform or micropapillary architectural subtypes., Results: Flow cytometric DNA analysis showed a significantly higher S-phase fraction in comedo DCIS than in the subgroup of DCIS tumors without necrosis (P < 0.01 [anova]). A preliminary analysis of disease recurrence and disease-free survival in a large series of 391 cases of pure DCIS showed that of 181 cases of pure comedo DCIS there were 19 local recurrences at the 7-year stage (82% 7-year disease-free survival), with 5 local recurrences in 51 cases of DCIS with necrosis (non-pure comedo) (85% 7-year disease-free survival) and only 6 local recurrences in the 159 cases of the DCIS-without-necrosis subgroup (94% 7-year disease-free survival). The chi 2 value for the frequency of disease recurrence of all cases of DCIS with necrosis (i.e., combining the groups of comedo DCIS and DCIS with necrosis (non-pure comedo)) as compared to DCIS without histological evidence of necrosis was 5705 (df = 2; P = 0.0001), and the chi 2 for disease-free survival of types of DCIS with necrosis as compared to cases without necrosis was 178 (df = 2; P = 0.0001). This analysis indicates that the histological presence of necrosis appears to be a relatively powerful predictor of increased disease recurrence and poorer disease-free survival after treatment for DCIS., Conclusions: Necrosis in DCIS in the absence of pure classical comedo morphology is a feature of more biologically aggressive in situ breast cancer with an intermediate proliferative fraction as compared with the high proliferative fraction of pure comedo DCIS and the low proliferative fraction of DCIS without necrosis. There was no significant difference in DNA ploidy (diploid or aneuploid) between the subgroups as assessed by chi 2 analysis. Further larger studies are required to establish if DCIS with necrosis (non-pure comedo) also shows a greater tendency to local recurrence after breast conservation treatment than do subtypes of DCIS without necrosis. DCIS with necrosis (non-pure comedo) should be adopted as a distinct histological subgroup of DCIS in future clinical studies of in situ mammary carcinoma.

Oncogenes and oncoproteins have been shown to play an important role in the neoplastic progression of tumors. It is now evident that certain subsets of human tumors show preferential expression of specific oncogenes and oncoproteins that may in turn predict tumor morphology. The phenomenon of preferential oncogene or oncogene protein expression in certain morphologic types of tumor may help to better define the biologic behavior and aggressiveness of a given tumor, so leading to advances in the diagnosis, treatment, and understanding of human neoplasia.

Background: No detailed data are available concerning the possible chromosomal heterogeneity within testicular germ cell tumors (TGCT) of adults. In addition, little is known about the chromosomal constitution of carcinoma in situ (CIS) of the human testis, the precursor of TGCTs, and the possible relation with the different histological tumor types (seminomas (SE), nonseminomatous (TGCTs (NS)., Experimental Design: Interphase cytogenetics in combination with immunohistochemistry was performed on tissue sections of SE, NS, and their adjacent CIS to study the numerical distributions of centromeric regions of chromosomes 1, 12, and 15., Results: No differences in chromosomal constitution were found between CIS adjacent to SE and the invasive tumor itself. NS showed a significant lower number of copies of chromosome 15 than SE, which was also found for the adjacent CIS. In addition a significantly higher number of copies of chromosome 12 was found in CIS adjacent to SE compared with CIS adjacent to NS. Invasive NS showed a significantly higher chromosome 1 and 12 copy number compared with its adjacent CIS., Conclusions: Net loss of chromosomes during tumor evolution can explain the differences between SE and NS and also between their adjacent CIS. Based on these results, we hypothesize that CIS-S may progress not only into SE but also into CIS-NS. It is conceivable, therefore, that NS may evolve from SE or from CIS-NS.

The ploidy of carcinoma in situ and invasive germ cell tumors of the adult testis was compared by DNA flow cytometry. Irrespective of the tumor type with which it was associated, the median DNA index of carcinoma in situ was about the same as that of seminomas and higher than the DNA index of invasive nonseminomatous germ cell tumors. These data indicate that seminoma and carcinoma in situ cells are not only phenotypically similar but also have the same ploidy.