Real-world data in the molecular era-finding the reality in the real world.

Real-world data (RWD) promises to provide a pivotal element to the understanding of personalized medicine. However, without true representation (or the reality) of the patient-disease biosystem and its molecular contributors, RWD may hamper rather than help this advancement. In this review article, we discuss RWD vs. clinical reality and the disconnects that exist currently (emphasizing molecular medicine), and methods of closing the gaps between RWD and reality.

Activation of NFAT signaling establishes a tumorigenic microenvironment through cell autonomous and non-cell autonomous mechanisms.

NFAT (the nuclear factor of activated T cells) upregulation has been linked to cellular transformation intrinsically, but it is unclear whether and how tissue cells with NFAT activation change the local environment for tumor initiation and progression. Direct evidence showing NFAT activation initiates primary tumor formation in vivo is also lacking. Using inducible transgenic mouse systems, we show that tumors form in a subset of, but not all, tissues with NFATc1 activation, indicating that NFAT oncogenic effects depend on cell types and tissue contexts. In NFATc1-induced skin and ovarian tumors, both cells with NFATc1 activation and neighboring cells without NFATc1 activation have significant upregulation of c-Myc and activation of Stat3. Besides known and suspected NFATc1 targets, such as Spp1 and Osm, we have revealed the early upregulation of a number of cytokines and cytokine receptors, as key molecular components of an inflammatory microenvironment that promotes both NFATc1(+) and NFATc1(-) cells to participate in tumor formation. Cultured cells derived from NFATc1-induced tumors were able to establish a tumorigenic microenvironment, similar to that of the primary tumors, in an NFATc1-dependent manner in nude mice with T-cell deficiency, revealing an addiction of these tumors to NFATc1 activation and downplaying a role for T cells in the NFATc1-induced tumorigenic microenvironment. These findings collectively suggest that beyond the cell autonomous effects on the upregulation of oncogenic proteins, NFATc1 activation has non-cell autonomous effects through the establishment of a promitogenic microenvironment for tumor growth. This study provides direct evidence for the ability of NFATc1 in inducing primary tumor formation in vivo and supports targeting NFAT signaling in anti-tumor therapy.

CRTC1 rearrangements in the absence of t(11;19) in primary cutaneous mucoepidermoid carcinoma.

BACKGROUND: Mucoepidermoid carcinoma (MEC) of the skin is an uncommon neoplasm with a remarkable resemblance to MEC of the salivary glands. The latter has been shown to harbour an oncogenic translocation resulting in a fusion gene consisting of exon 1 of CRTC1/MECT1/TORC1 at 19p and exons 2-5 of MAML2 at 11q. OBJECTIVES: While t(11;19) and rearrangements of the involved loci have been demonstrated in MEC of the salivary gland and other sites, it remains to be determined if morphological similarities in cutaneous MEC are reflected at the molecular level. METHODS: Cases of cutaneous MEC were defined by three histopathological features: (i) cystic dermal nodule with (ii) overlying intact epidermis and (iii) presence of three cell types (squamoid, intermediate, mucinous), and characterized by reverse transcription-polymerase chain reaction (RT-PCR), interphase fluorescent in situ hybridization (FISH) and immunohistochemistry. RESULTS: Eight primary cutaneous MECs were analysed. All informative cases showed CRTC1 rearrangements; none of the cases had MAML2 rearrangements or the presence of t(11;19) by RT-PCR. One case of primary MEC of the breast showed amplification of MAML2 in the absence of CRTC1 or t(11;19). Two MECs metastatic to the skin, histologically identical to primary cutaneous MEC, were included, one of which harboured the CRTC1-MAML2 fusion gene by RT-PCR, verified by interphase FISH and sequencing. CONCLUSIONS: MEC of the skin harbours CRTC1 rearrangements, a molecular finding that reflects morphological similarities between glandular and cutaneous MEC. The absence of oncogenic t(11;19) or MAML2 aberrations in our series, which is the largest reported, may explain the innocuous clinical behaviour of this uncommon adnexal tumour.

Visceral primitive peripheral neuroectodermal tumors: a clinicopathologic and molecular study.

Ewing sarcoma-primitive neuroectodermal tumor (EWS/PNET) belongs to the group of pediatric small round blue cell tumors; although EWS/PNET is classically a tumor of the soft tissue or bone in children and young adults, individual cases have been described in patients of all ages. A group of chromosomal translocations involving the EWS gene and a member of the Ets transcription factor family of genes has been detected in EWS/PNET, and heterogeneity in the precise breakpoint of the translocation has been shown to generate a group of related fusion transcripts that may have prognostic significance. Within the last decade, the clinicopathologic spectrum of EWS/PNET has been markedly expanded by recognition that the tumor may also have a visceral origin. To determine whether visceral EWS/PNET has the same pattern of genetic alterations and range of fusion transcripts as EWS/PNET of bone and soft tissue, we performed reverse-transcription polymerase chain reaction-based testing of formalin-fixed, paraffin-embedded tissue from a series of visceral tumors for which the diagnosis of EWS/PNET was well established. Together with additional cases compiled from the literature, EWS-Fli1 (or a related fusion transcript) was present in 18 of 19 visceral EWS/PNET, with a distribution of transcript types not statistically different from EWS/PNET of soft tissue and bone (P >.05, chi(2) test). These results firmly establish the genetic relationship between EWS/PNET of visceral sites, soft tissue, and bone.

Successful in vitro fertilization pregnancy after conservative management of endometrial cancer.

OBJECTIVE: To report a successful IVF pregnancy in an infertile couple after conservative treatment of endometrial cancer. DESIGN: Case report and literature review. SETTING: University teaching hospital. PATIENT(S): A 29-year-old infertile white woman. MAIN OUTCOME MEASURE(S): Successful pregnancy after conservative management of endometrial cancer. INTERVENTION(S): Grade 1 endometrial adenocarcinoma diagnosed at hysteroscopy, followed by dilatation and curettage (D&C). On follow-up D&C, pathologic examination was normal after high-dose progesterone therapy. The patient subsequently underwent an IVF cycle with transfer of three blastocysts. RESULT(S): The patient delivered triplets by cesarean section. Laparoscopic-assisted vaginal hysterectomy and bilateral salpingo-oophorectomy was then done. No residual endometrial cancer was evident in the hysterectomy specimen, but a 1.1-cm cystic mixed endometrioid and clear cell-type adenocarcinoma was discovered in the left ovary. The patient is doing well after 3 cycles of chemotherapy; her CA-125 level is normal. The triplets are also doing well. CONCLUSION(S): In carefully chosen situations, deferring surgery in infertile patients with endometrial cancer may be a viable option permitting subsequent successful pregnancy.

Primitive neuroectodermal tumors of the biliary and gastrointestinal tracts: clinicopathologic and molecular diagnostic study of two cases.

Primitive neuroectodermal tumor (PNET) is a prototypic malignant small round cell tumor of childhood that is characterized in most cases by t(11;22) resulting in an EWS-FLI1 gene fusion. Once thought to be uncommon, PNET now accounts for almost 20% of malignant soft tissue tumors in children. Increased recognition of PNET is partly due to advances in immunohistochemistry and molecular diagnostics, which have led to the identification of the tumor in non-classical sites. We report the clinical, histologic, immunohistochemical, and molecular findings of two visceral PNETs of the digestive system--one involving the small intestine and the other involving the hepatic duct. Histologically, each tumor was composed of malignant small cells growing in sheets, nests, and lobules; the tumor cells of both cases showed characteristic immunoreactivity for vimentin and O13 (CD99). Reverse transcription-polymerase chain reaction (RT-PCR) analysis for t(11;22) using nested primers was performed with RNA extracted from paraffin-embedded, formalin-fixed tissue and demonstrated an EWS exon 7 to FLI1 exon 5 fusion in both cases, confirmed by Southern blot hybridization and DNA sequence analysis. These results illustrate the expanded clinicopathologic profile of PNET, and demonstrate that visceral PNETs, despite their unusual sites of presentation, maintain the characteristic immunohistochemical and genetic features of PNETs at more conventional sites.

Diagnostic gold standard for soft tissue tumours: morphology or molecular genetics?

The recognition of recurrent genetic alterations in specific tumour types has provided the basis for the reclassification of certain soft tissue neoplasms, and molecular analysis of patient material has the potential to provide both diagnostic and prognostic information. In this review, we evaluate the role of molecular genetic testing as the prospective 'gold standard' for sarcoma diagnosis. Molecular genetic testing, as with every new method, promises to improve accuracy and to be more sensitive and less subjective, claims that have been made previously by histochemistry, electron microscopy and immunohistochemistry. Technical limitations in molecular assays, as well as more general specificity issues, decrease the clinical usefulness of molecular pathological testing significantly and suggest that, at present, molecular evaluation is best considered an ancillary technique that neither supersedes other ancillary techniques nor eclipses traditional pathological examination.

Malignant peripheral nerve sheath tumors with t(X;18). A pathologic and molecular genetic study.

Spindle cell sarcomas often present the surgical pathologist with a considerable diagnostic challenge. Malignant peripheral nerve sheath tumor, leiomyosarcoma, fibrosarcoma, and monophasic synovial sarcoma may all appear similar histologically. The application of ancillary diagnostic modalities, such as immunohistochemistry and electron microscopy, may be helpful in the differentiation of these tumors, but in cases in which these adjunctive techniques fail to demonstrate any more definitive evidence of differentiation, tumor categorization may remain difficult. Cytogenetic and molecular genetic characterization of tumors have provided the basis for the application of molecular assays as the newest components of the diagnostic armamentarium. Because the chromosomal translocation t(X;18) has been observed repeatedly in many synovial sarcomas, it has been heralded as a diagnostic hallmark of synovial sarcoma. To formally test the specificity of this translocation for the diagnosis of synovial sarcoma, RNA extracted from formalin-fixed, paraffin-embedded tissue from a variety of soft tissue and spindle cell tumors was evaluated for the presence of t(X;18) by reverse transcriptase-polymerase chain reaction. Although 85% of the synovial sarcomas studied demonstrated t(X;18), 75% of the malignant peripheral nerve sheath tumors in our cohort also demonstrated this translocation. We conclude that the translocation t(X;18) is not specific to synovial sarcoma and discuss the implications of the demonstration of t(X;18) in a majority of malignant peripheral nerve sheath tumors.

Renal cell carcinoma with rhabdoid features.

Neoplasms with rhabdoid features have been reported at many anatomic sites. In the kidney, rhabdoid tumors are typically found in children, whereas only rare examples have been reported in adults. Little is known of renal cell carcinomas (RCCs) that exhibit rhabdoid features. The objective of this study was to determine the incidence of RCC with rhabdoid attributes and characterize the histologic, immunophenotypic, and ultrastructural features by retrospective analysis of 480 consecutively identified cases of RCC in radical nephrectomy specimens. Immunohistochemical evaluation was performed in cases with rhabdoid foci using a panel of antibodies to pancytokeratin (pan-CK), CK7, CK20, epithelial membrane antigen (EMA), S-100 protein, desmin, vimentin, neuron-specific enolase (NSE), muscle-specific actin (MSA), smooth muscle actin (SMA), human melanoma, black-45 (HMB-45), and glial fibrillary acidic protein (GFAP). Electron microscopy was also performed in selected cases. The presence and extent of rhabdoid foci in relation to pathologic stage and grade were assessed. Twenty-three of 480 cases of RCC (4.7%) exhibited rhabdoid features. The 23 patients were all adults with a mean age of 61.8 years (age range, 33-84 yrs). Fifteen of the patients were men and eight were women. Histologically, the rhabdoid foci were typified by sheets and clusters of variably cohesive, large epithelioid cells with vesicular and often eccentric nuclei, prominent nucleoli, and large, paranuclear intracytoplasmic hyaline globules (inclusions). The presence of these rhabdoid features was related to high histologic Fuhrman grade of the nonrhabdoid carcinoma component, with an incidence of 0 of 84 grade I cases, eight of 300 grade 2 cases (2.6%), six of 70 grade 3 cases (8.9%), and nine of 26 grade 4 cases (34.6%; p = 3 x 10(-9)). The rhabdoid foci were all high grade. The presence of rhabdoid foci was also found in higher stage carcinomas. A total of 52% (12 of 23) of RCC cases with rhabdoid features exhibited extrarenal extension compared with 28% (24 of 92) of contemporary RCCs without rhabdoid features (p = 0.03). The size of the rhabdoid component ranged from 1 mm to more than 2 cm and comprised 1% to 50% of the renal mass. Immunoreactivity for vimentin (100%), NSE (79%), and panCK (56%) was present in the majority of cases. Substantial percentages of cases were immunopositive for EMA (47%) and S-100 protein (37%), with minimal to no immunohistochemical reactivity for CK7 (5%), SMA (5%), CK20 (0%), desmin (0%), MSA (0%), HMB-45 (0%), and GFAP (O%). A distinctive globular, paranuclear reaction pattern was found for the cytokeratin, EMA, and vimentin immunostains. Ultrastructurally, the rhabdoid cells had paranuclear intermediate filament aggregates or paranuclear condensation of organelles, often associated with peripheral vacuolization. Adult RCCs may harbor a rhabdoid component, and these neoplasms can be regarded as "composite" tumors. Rhabdoid elements are important to identify because of their high-grade nature, and association with high stage. Adult RCC with rhabdoid elements should be distinguished from pure rhabdoid tumors of kidney, in light of their clinicopathologic differences. Rhabdoid differentiation in adult renal cell carcinoma may represent clonal divergence and/ or evolution, and emergence of a particularly aggressive element.

WT1 staining reliably differentiates desmoplastic small round cell tumor from Ewing sarcoma/primitive neuroectodermal tumor. An immunohistochemical and molecular diagnostic study.

Differentiating desmoplastic small round cell tumor (DSRCT) from another similar small round cell tumor of childhood, the Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET), can be difficult because morphologic and immunohistochemical features overlap. We studied the predictive value of immunohistochemistry with an antibody to the C-terminal region of the Wilms tumor (WT1) protein for differentiating DSRCT from EWS/PNET in 24 malignant small round cell tumors that had been previously diagnosed as DSRCT or EWS/PNET by standard methods. We performed reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in cases with available tissue as a confirmatory measure: 6 of 13 DSRCTs were informative by RT-PCR, and 6 of 6 showed an EWS-WT1 fusion; all 13 DSRCTs showed strong, definitive nuclear staining with the WT1 antibody. All 11 EWS/PNETs were WT1 antibody negative; 7 of 11 cases classified as EWS/PNET were informative by RT-PCR, and 7 of 7 showed an EWS-FLI-1 fusion. For cases in which the morphologic and immunohistochemical features are consistent with a diagnosis of DSRCT, WT1 antibody staining predicts the EWS-WT1 translocation with high sensitivity and specificity and is, therefore, useful for differentiating DSRCT from EWS/PNET when genetic information is unavailable.

Squamous cell carcinoma arising in recurrent respiratory papillomatosis with pulmonary involvement: emerging common pattern of clinical features and human papillomavirus serotype association.

Squamous papillomas of the lung are an uncommon feature of recurrent respiratory papillomatosis, occurring in fewer than 1% of cases. We describe a 23-year-old patient with pulmonary papillomas who developed a fatal squamous cell carcinoma of the lung. PCR-based human papillomavirus (HPV) typing showed the presence of HPV 11 DNA in both benign papillomas and invasive carcinoma. A review of the literature reveals four reports of malignant transformation of juvenile-onset recurrent respiratory papillomatosis in which HPV typing was performed. Similar clinical features are noted in all of the reports; specifically, each case has arisen in a young adult man with a history of papillomatosis since childhood. In each of the cases, HPV 11 was identified in association with the squamous cell carcinoma. Although HPV 11 is uncommonly associated with the development of invasive carcinoma at other sites, these findings suggest that it is correlated with malignant transformation in the setting of juvenile-onset recurrent respiratory papillomatosis.

An IgM lambda antibody to Escherichia coli produces false-positive results in multiple immunometric assays.

BACKGROUND: Interferences in immunometric assays as a result of human anti-immunoglobulin antibodies frequently have been described in the literature. The etiology of these interfering antibodies is usually not known but has been associated with rheumatoid factors in some assays. It is known that microorganisms in experimental settings can induce anti-immunoglobulin antibodies. METHODS: Following Escherichia coli septicemia, a 56-year-old male patient had increased immunoassay results for cardiac troponin I, thyrotropin, human chorionic gonadotropin, alpha-fetoprotein, and CA-125 that were consistent with myocardial infarction, hyperthyroidism, and pregnancy, and suggestive of an occult neoplasm such as hepatic or ovarian cancer. None of these diagnoses were consistent with the rest of his medical exam. In addition, the patient had a restricted IgM lambda paraprotein by immunofixation. Plasma from the patient was incubated with Sepharose-conjugated protein A, irrelevant murine monoclonal antibodies, and formalin-killed E. coli organisms from his infection to determine whether these immunoassay values were falsely increased. RESULTS: Incubation of the patient's plasma with irrelevant murine monoclonal antibodies or the E. coli organism produced normal immunoassay values and removed the IgM lambda paraprotein. CONCLUSIONS: The patient produced a very restricted IgM lambda antibody response to the E. coli infection that had anti-immunoglobulin activity and caused falsely increased values in numerous immunometric assays. Microorganism-induced anti-immunoglobulin antibodies are discussed in the context of this patient.

Meningioma presenting as an intraoral mass in a patient with neurofibromatosis type 1.

A 77-year-old woman with neurofibromatosis type 1 presented with ill-fitting dentures due to intraoral extension of a right temporal fossa mass. Computed tomographic scanning demonstrated that the masticator space mass bowed the zygomatic arch and remodeled the lateral orbit and maxillary sinus walls, findings that were consistent with the clinical diagnosis of a neurofibroma with possible malignant transformation. However, light microscopic, immunohistochemical, and ultrastructural examination of tissue from an incisional biopsy specimen were diagnostic of meningioma. This case illustrates that the clinicopathologic differential diagnosis of an enlarging mass in patient with neurofibromatosis should include sporadic, unrelated neoplasms as well as tumors known to be associated with the syndrome.

Malignant peripheral nerve sheath tumors with t(X;18). A pathologic and molecular genetic study.

Spindle cell sarcomas often present the surgical pathologist with a considerable diagnostic challenge. Malignant peripheral nerve sheath tumor, leiomyosarcoma, fibrosarcoma, and monophasic synovial sarcoma may all appear similar histologically. The application of ancillary diagnostic modalities, such as immunohistochemistry and electron microscopy, may be helpful in the differentiation of these tumors, but in cases in which these adjunctive techniques fail to demonstrate any more definitive evidence of differentiation, tumor categorization may remain difficult. Cytogenetic and molecular genetic characterization of tumors have provided the basis for the application of molecular assays as the newest components of the diagnostic armamentarium. Because the chromosomal translocation t(X;18) has been observed repeatedly in many synovial sarcomas, it has been heralded as a diagnostic hallmark of synovial sarcoma. To formally test the specificity of this translocation for the diagnosis of synovial sarcoma, RNA extracted from formalin-fixed, paraffin-embedded tissue from a variety of soft tissue and spindle cell tumors was evaluated for the presence of t(X;18) by reverse transcriptase-polymerase chain reaction. Although 85% of the synovial sarcomas studied demonstrated t(X;18), 75% of the malignant peripheral nerve sheath tumors in our cohort also demonstrated this translocation. We conclude that the translocation t(X;18) is not specific to synovial sarcoma and discuss the implications of the demonstration of t(X;18) in a majority of malignant peripheral nerve sheath tumors.

Multiple insertions of fimbrial operons correlate with the evolution of Salmonella serovars responsible for human disease.

On centisome 7, Salmonella spp. contain a large region not present in the corresponding region of Escherichia coli. This region is flanked by sequences with significant homology to the E. coli tRNA gene aspV and the hypothetical E. coli open reading frame yafV. The locus consists of a mosaic of differentially acquired inserts forming a dynamic cs7 region of horizontally transferred inserts. Salmonella enterica subspecies I, responsible for most Salmonella infections in warm-blooded animals, carries a fimbrial gene cluster (saf) in this region as well as a regulatory gene (sinR). These genes are flanked by inverted repeats and are inserted in another laterally transferred region present in most members of Salmonella spp. encoding a putative invasin (pagN ). S. enterica subspecies I serovar Typhi, the Salmonella serovar that causes the most severe form of human salmonellosis, contains an additional insert of at least 8 kb in the sinR-pagN intergenic region harbouring a novel fimbrial operon (tcf ) similar to the coo operon encoding the CS1 fimbrial adhesin expressed by human-specific enterotoxigenic E. coli. It is suggested that the multiple insertions of fimbrial genes that have occurred in the cs7 region have contributed to phylogenetic diversity and host adaptation of Salmonella spp.

Ectopic breast tissue and breast-like sweat gland metaplasias: an overlapping spectrum of lesions.

There are many similarities in the morphology of benign and malignant lesions of the sweat glands and the breasts. The recently described cutaneous mammary-like sweat glands, also known as mixed sweat glands or apoeccrine glands, are also a likely source of selected proliferations that closely mimic those of the breast. We present three cases of breast-like lesions arising in the skin that demonstrate the ways in which the morphologic and pathologic continuum of the mammary glands, cutaneous mammary-like glands, and sweat glands can produce difficulties in precise diagnosis. The examples demonstrate that an anatomic location outside the milk line does not preclude the diagnosis of ectopic mammary tissue, and that lesions closely resembling those of the breast may also arise outside the milk line from conventional sweat glands or mixed sweat glands. The concept of homologous lesions of the breast, breast-like glands and sweat glands, in which morphology is partially mirrored by biochemical similarities, provides a perspective for classification of problematic cases of breast-like cutaneous lesions.

Expression of thin aggregative fimbriae promotes interaction of Salmonella typhimurium SR-11 with mouse small intestinal epithelial cells.

The factors that mediate binding of Salmonella typhimurium to small intestinal epithelial cells have not been fully characterized. In this paper we demonstrate that elimination of production of thin aggregative fiber by a transposon insertion within the gene encoding the subunit protein of the fiber reduced binding of S. typhimurium SR-11 to a conditionally immortalized proximal small intestinal epithelial cell line established from transgenic mice. This binding defect could be overcome by transcomplementation with a wild-type allele. The conditionally immortalized cell line should prove useful in identifying the epithelial cell receptor for bacterial attachment since expression of its bacterial binding activity can be induced by manipulating the line's proliferative status.

Development of a murine model of chronic Salmonella infection.

The invasive disease caused by Salmonella typhimurium in mice resembles the acute phase of human typhoid fever caused by Salmonella typhi, and experimental murine salmonellosis is a widely used experimental model for systemic salmonellosis. In this paper we demonstrate that murine S. typhimurium infection can also be used to model the development of the chronic carrier state that develops in humans after infection with S. typhi. We describe a virulent variant of S. typhimurium that has decreased expression of AgfA fibers under all environmental conditions studied and that causes a chronic carrier state in BALB/c mice after peroral inoculation. The chronic carrier state is associated with persistence of bacteria in the small intestine, spleen, and liver, and chronic infection continues despite the development of protective immunity to challenge with virulent Salmonella.