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Introduction: Fluorescence in situ hybridization (FISH) is an essential ancillary study used to identify clinically aggressive subsets of large B-cell lymphomas that have MYC, BCL2 , or BCL6 rearrangements. Small-volume biopsies such as fine needle aspiration biopsy (FNAB) and core needle biopsy (CNB) are increasingly used to diagnose lymphoma and obtain material for ancillary studies such as FISH. However, the performance of FISH in small biopsies has not been thoroughly evaluated or compared to surgical biopsies., Methods: We describe the results of MYC, BCL2, and BCL6 FISH in a series of 222 biopsy specimens, including FNAB with cell blocks, CNBs, and surgical excisional or incisional biopsies from 208 unique patients aggregated from 6 academic medical centers. A subset of patients had FNAB followed by a surgical biopsy (either CNB or excisional biopsy) obtained from the same or contiguous anatomic site as part of the same clinical workup; FISH results were compared for these paired specimens., Results: FISH had a low hybridization failure rate of around 1% across all specimen types. FISH identified concurrent MYC and BCL2 rearrangements in 20 of 197 (10%) specimens and concurrent MYC and BCL6 rearrangements in 3 of 182 (1.6%) specimens. The paired FNAB and surgical biopsy specimens did not show any discrepancies for MYC or BCL2 FISH; of the 17 patients with 34 paired cytology and surgical specimens, only 2 of the 49 FISH probes compared (4% of all comparisons) showed any discrepancy and both were at the BCL6 locus. One discrepancy was due to necrosis of the CNB specimen causing a false negative BCL6 FISH result when compared to the FNAB cell block that demonstrated a BCL6 rearrangement., Discussion: FISH showed a similar hybridization failure rate in all biopsy types. Ultimately, MYC, BCL2 , or BCL6 FISH showed 96% concordance when compared across paired cytology and surgical specimens, suggesting FNAB with cell block is equivalent to other biopsy alternatives for evaluation of DLBCL or HGBCL FISH testing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Menke, Aypar, Bangs, Cook, Gupta, Hasserjian, Kong, Lin, Long, Ly, Menke, Natkunam, Ruiz-Cordero, Spiteri, Ye, Zadeh and Gratzinger.)

The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)-JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)-like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia., (© 2021 by The American Society of Hematology.)

We report 55 postchemotherapy resections of primary nonseminomatous mediastinal germ cell tumors with prominent vasculogenic features showing the formation of rudimentary to well-developed neoplastic vessels within primitive mesenchyme. These cases represented 25% of a cohort of 221 such specimens. The patients were 19 to 49 years old (mean, 28 y) and 98% had serological evidence of yolk sac tumor. The vasculogenic lesions, felt to represent a neoplastic reiteration of embryonic vasculogenesis in the splanchnic mesoderm of the yolk sac, were further subdivided into teratoma with vasculogenic stroma (n=9), vasculogenic mesenchymal tumor (VMT) (n=42, further classified into low grade [n=24] and high grade [n=18]), and angiosarcoma (n=4). The distinction of teratoma with vasculogenic stroma from VMT was based solely on the greater extent of VMT (exceeding 1 low power [×4 objective] microscopic field), with both categories showing a spectrum of vessels lined by atypical endothelium in a nonendothelial neoplastic stroma that often also generated vascular walls comprised of atypical smooth muscle. The angiosarcomas showed stratification of highly atypical endothelial cells or anastomosing vessels lined by nonstratified but cytologically similar endothelium. Immunohistochemical studies supported the generation of neoplastic vessels from the tumor stroma, most commonly by the development of stromal clefts showing reactivity for podoplanin, CD34, and occasionally ERG, followed by the gradual development from the clefts of thin-walled vessels that later became encircled by stromal cells showing smooth muscle differentiation by immunohistochemistry. Occasionally, round collections of stromal erythrocytes became surrounded by stromal cells to generate blood vessels. Fluorescence in situ hybridization showed chromosome 12p copy number increase in both the endothelial component and stromal component in 8/9 VMT cases and in 1/1 angiosarcoma. On follow-up, no patient with teratoma with vasculogenic stroma had evidence of a subsequent vascular tumor or sarcoma, whereas 8 of the 35 (23%) patients with VMTs (2 low grade and 6 high grade) and meaningful follow-up developed sarcoma (1 angiosarcoma, 2 rhabdomyosarcomas, and 5 not further characterized). The difference between low-grade and high-grade tumors was of borderline significance (P=0.058). Two of the 4 patients with angiosarcoma died of metastatic angiosarcoma, with the other 2 disease-free at 6.8 and 7 years. Compared with the 165 patients with follow-up and no vasculogenic lesions, there was a highly significant (P=4.3×10-5) association of any vasculogenic lesion with sarcomatoid tumors during the clinical course of VMT patients. In addition, 5/46 patients with follow-up and vasculogenic lesions (11%) died of either leukemia or myelodysplastic syndrome compared with 2 of 166 (1%) lacking them (P=0.0012). Three of the 5 patients had identifiable immature hematopoietic cells within their vasculogenic lesions, but 4 other VMT patients with these did not develop leukemia or myelodysplasia. We conclude: (1) vasculogenic lesions are frequent in postchemotherapy resections of primary mediastinal germ cell tumors with yolk sac tumor components; (2) they mostly consist of neoplastic vessels in a stroma that also generates neoplastic vascular walls of smooth muscle; (3) VMTs are associated with an increased incidence of sarcomas, even though most vasculogenic lesions in this context do not meet criteria for angiosarcoma; (4) the presence of vasculogenic lesions in postchemotherapy resections of primary mediastinal germ cell tumors place patients at increased risk for leukemia or myelodysplasia., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Cancer cell lines are not homogeneous nor are they static in their genetic state and biological properties. Genetic, transcriptional and phenotypic diversity within cell lines contributes to the lack of experimental reproducibility frequently observed in tissue-culture-based studies. While cancer cell line heterogeneity has been generally recognized, there are no studies which quantify the number of clones that coexist within cell lines and their distinguishing characteristics. We used a single-cell DNA sequencing approach to characterize the cellular diversity within nine gastric cancer cell lines and integrated this information with single-cell RNA sequencing. Overall, we sequenced the genomes of 8824 cells, identifying between 2 and 12 clones per cell line. Using the transcriptomes of more than 28 000 single cells from the same cell lines, we independently corroborated 88% of the clonal structure determined from single cell DNA analysis. For one of these cell lines, we identified cell surface markers that distinguished two subpopulations and used flow cytometry to sort these two clones. We identified substantial proportions of replicating cells in each cell line, assigned these cells to subclones detected among the G0/G1 population and used the proportion of replicating cells per subclone as a surrogate of each subclone's growth rate., (© The Author(s) 2019. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Anaplastic large cell lymphomas (ALCL) encompass several subgroups that differ in their clinical presentation, genetic features, and prognosis. We characterized the genetic subgroups of 74 patients with ALCL and correlated programmed death ligand 1 (PD-L1) protein expression and compared the densities and ratios of FOXP3+ T regulatory cells and CD8+ tumor-infiltrating lymphocytes (TILs) in tumor cells and the immune microenvironment. The subgroups included anaplastic lymphoma kinase (ALK)-positive (ALK+) ALCL and ALK-negative (ALK-) ALCL and DUSP22-rearranged and nonrearranged ALK- ALCL. None of our cases represented the TP63-rearrangement ALK- ALCL subgroup. Our results showed that ALK+ ALCL had a higher expression of PD-L1 in the tumor cells, in contrast to ALK- ALCL, which expressed high PD-L1 in tumor-associated macrophages (TAMs). DUSP22-rearranged ALK- ALCL lacked PD-L1 expression in the tumor cells and instead expressed PD-L1 only in TAMs. There was a significant positive correlation of PD-L1 expression between tumor and TAMs in ALK+ ALCL with a negative correlation in ALK- ALCL. Systemic ALCL subgroups had similar densities of CD8+ tumor-infiltrating lymphocytes and FOXP3 T regulatory cells, but differences were observed in the ratio of CD8/FOXP3. Our results suggest that alterations in tumor microenvironment and immune responses exist among systemic ALCL subgroups and these features may account for different clinical behavior and prognosis.

Undifferentiated malignancies (UMs) encompass a diverse set of aggressive tumors that pose not only a diagnostic challenge but also a challenge for clinical management. Most tumors in this category are currently treated empirically with nonspecific chemotherapeutic agents that yield extremely poor clinical response. Given that UMs are inherently genetically unstable neoplasms with the potential for immune dysregulation and increased neoantigen production, they are likely to be particularly amenable to immune checkpoint inhibitors, which target programmed cell death protein 1 (PD-1) or its ligands, PD-L1 and PD-L2, to promote T-cell antitumor activity. Aberrant expression of PD-L1 and, more recently, chromosomal 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) alterations can be used as biomarkers to predict responsiveness to checkpoint inhibitors. Here we evaluated 93 cases previously diagnosed as an "undifferentiated" malignancy and found that 56% (52/93) of UMs moderately to strongly express PD-L1 by immunohistochemistry (IHC). Concurrent CD274(PD-L1) and PDCD1LG2(PD-L2) fluorescence in situ hybridization (FISH) was performed on 24 of these cases and demonstrates a genetic gain at both loci in 62.5% of UMs. Genetic alterations at the CD274(PD-L1) and PDCD1LG2(PD-L2) loci were found to be completely concordant by FISH. Overall, we found that a significant proportion of UMs express PD-L1 and provide molecular support for using checkpoint inhibitors as a treatment approach for this class of tumors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mediastinal teratomas are enigmatic; those in children and women are almost invariably benign but in men they may be benign or malignant. There are few data on the chromosome 12p status of mediastinal germ cell tumors (GCT), whereas increased 12p copy number is virtually uniform in malignant testicular GCTs. We therefore studied chromosome 12p copy number in 34 diverse mediastinal GCTs and correlated the results with morphology and follow-up to gain insight into possible pathogenesis. Four prepubertal (below 12 y) children (3 females and 1 male), 7 postpubertal females (14 to 52 y) and 6 postpubertal males (12 to 40 y old) had pure, previously untreated teratomas; 15 were mature and 2 had low-grade immaturity. All lacked 12p copy number increase and cytologic atypia, and most (14/17) showed organoid morphology. On follow-up of 16, 1 died of postoperative complications and the remaining 15 were disease free (1 to 119 mo, mean: 39 mo). Eight postpubertal males (19 to 44 y old) had pure teratomas in postchemotherapy resections; 5/8 showed 12p copy number increase. All 8 had distinct cytologic atypia, with organoid morphology in 3. On follow-up, 6 were disease free after surgical resection (1.5 to 94 mo, mean 38 mo); 1 died of disease at 14.5 months, and 1 was alive with metastases at 176 months. Two postpubertal patients, 1 male (29 y) and 1 female (31 y), had teratoma with secondary somatic-type malignancies, with positive 12p copy number increase in the former but not the latter. The man's tumor occurred after chemotherapy and was a nonorganoid teratoma with primitive neuroectodermal tumor and malignant glioma; the woman's was a previously untreated organoid teratoma with an undifferentiated carcinoma component. The man died of disease (16 mo) and the woman was alive with metastases (27 mo). Seven patients had resections for mixed GCTs (4) or pure nonteratomatous tumors, all after chemotherapy; 5/7 had positive 12p copy number increase. The teratoma component of the 2 cases having one showed distinct cytologic atypia and lacked organoid morphology. On follow-up, 1 died of disease (5 mo), 2 were alive with disease (1, 1.5 mo), 3 were disease free (1 to 43 mo; mean: 18 mo), and 1 was alive with unknown status (31 mo). Our results support that mediastinal teratomas likely develop from 2 separate pathways. Those in children, women and some men arise as pure neoplasms from a nontransformed precursor cell and, therefore, lack 12p copy number increase, show no cytologic atypia, often have organoid morphology and are benign. Common 12p copy number increase, uniform atypia, infrequent organoid structures and malignant behavior support that pure teratomas after chemotherapy in postpubertal males derive from a malignantly transformed precursor cell. Interestingly, we identified organoid pancreatic differentiation only in the benign group and neuroglia more commonly in the malignant teratomas.

FLT3 fusions are associated with myeloid and lymphoid neoplasms with eosinophilia. We describe a patient presenting with clinicopathologic features of both chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) and systemic mastocytosis (SM). The bone marrow demonstrated a myeloproliferative neoplasm with eosinophilia and aggregates of atypical mast cells. Cytogenetic analysis revealed a t(13;14)(q12;q32), which was subsequently molecularly characterized as a novel TRIP11-FLT3 rearrangement. A KIT D816V mutation was also identified. The patient rapidly transformed to T-lymphoblastic leukemia/lymphoma and expired shortly after diagnosis. This is the fifth FLT3 fusion gene described in the literature; the presence of both myeloid and lymphoid neoplasms implicates involvement of an early hematopoietic progenitor by rearranged FLT3. We suggest that leukemias and lymphomas with FLT3 fusion genes exhibit similar clinicopathologic features to, and should be included in, the WHO category of "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2.", (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sexual differentiation across taxa may be due to genetic sex determination (GSD) and/or temperature sex determination (TSD). In many mammals, males are heterogametic (XY); whereas females are homogametic (XX). In most birds, the opposite is the case with females being heterogametic (ZW) and males the homogametic sex (ZZ). Many reptile species lack sex chromosomes, and instead, sexual differentiation is influenced by temperature with specific temperatures promoting males or females varying across species possessing this form of sexual differentiation, although TSD has recently been shown to override GSD in Australian central beaded dragons (Pogona vitticeps). There has been speculation that Australian Brush-turkeys (Alectura lathami) exhibit TSD alone and/or in combination with GSD. Thus, we sought to determine if this species possesses sex chromosomes. Blood was collected from one sexually mature female and two sexually mature males residing at Sylvan Heights Bird Park (SHBP) and shipped for karyotype analysis. Karyotype analysis revealed that contrary to speculation, Australian Brush-turkeys possess the classic avian ZW/ZZ sex chromosomes. It remains a possibility that a biased primary sex ratio of Australian Brush-turkeys might be influenced by maternal condition prior to ovulation that result in her laying predominantly Z- or W-bearing eggs and/or sex-biased mortality due to higher sensitivity of one sex in environmental conditions. A better understanding of how maternal and extrinsic factors might differentially modulate ovulation of Z- or W-bearing eggs and hatching of developing chicks possessing ZW or ZZ sex chromosomes could be essential in conservation strategies used to save endangered members of Megapodiidae.

Histiocytic sarcoma is rare and difficult to distinguish from histologic mimics such as myeloid sarcoma due to its relatively nonspecific immunoprofile. A subset of histiocytic sarcomas are clonally related to synchronous or metachronous follicular lymphomas. Interestingly, the histiocytic tumor component has been shown to harbor BCL2 gene translocations that are identical to those found in the lymphoma. We present one case of histiocytic sarcoma and initially occult follicular lymphoma in which detection of a BCL2 gene translocation helped support the diagnosis. We also provide follow-up regarding a previously published case of histiocytic sarcoma with IGH/BCL2 fusion gene in which the patient subsequently developed follicular lymphoma and, later, diffuse large B-cell lymphoma. Our findings suggest that BCL2 gene translocations are a recurrent feature of a distinct subset of histiocytic sarcomas that are associated with follicular lymphoma; the follicular lymphoma component may be clinically occult at the time of diagnosis. Testing for an IGH/BCL2 translocation should be considered in the diagnostic workup of difficult-to-characterize neoplasms with histiocytic/monocytic morphology and immunoprofile., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We sought to address the significance of isolated follicles that exhibit atypical morphologic features that may be mistaken for lymphoma in a background of reactive lymphoid tissue. Seven cases that demonstrated centroblast-predominant isolated follicles and absent BCL2 staining in otherwise-normal lymph nodes were studied. Four of seven cases showed clonal B-cell proliferations amid a polyclonal B cell background; all cases lacked the IGH-BCL2 translocation and BCL2 protein expression. Although three patients had invasive breast carcinoma at other sites, none were associated with systemic lymphoma up to 44 months after diagnosis. The immunoarchitectural features of these highly unusual cases raise the question of whether a predominance of centroblasts and/or absence of BCL2 expression could represent a precursor lesion or atypical reactive phenomenon. Differentiating such cases from follicular lymphoma or another mimic is critical, lest patients with indolent proliferations be exposed to unnecessarily aggressive treatment.

MYC translocations are a defining feature of Burkitt lymphoma and a group of diffuse large B-cell lymphoma (DLBCL) with inferior outcome. However, the clinical relevance of MYC gene rearrangement and its relationship with MYC protein expression has not been well characterized in lymphomas. Tissue microarrays containing 1214 lymphomas were successfully evaluated by immunohistochemistry using anti-MYC clone Y69 and a dual-color break-apart fluorescence in situ hybridization probe to detect MYC gene rearrangements. Aggressive B-cell lymphomas including Burkitt lymphoma and DLBCL showed the highest level of MYC protein staining defined as staining in >50% of lymphoma cells. A significant proportion of plasmablastic, B-lymphoblastic and T-lymphoblastic, and extranodal NK/T-cell lymphomas also showed staining in >50% of cells, whereas only occasional plasma cell myeloma, mantle cell lymphoma, and classical Hodgkin lymphoma showed a high level of staining. Small B-cell lymphomas, when positive, showed MYC protein in <50% of cells. In aggressive B-cell lymphomas, MYC rearrangement and MYC immunohistochemistry showed a high concordance rate; however, some DLBCL and all T-cell and NK-cell lymphomas with MYC protein expression lacked MYC gene rearrangements. Our results provide a baseline for MYC protein expression in lymphomas and indicate that its expression is not specific to lymphoma subtypes, cell lineage, or expected clinical behavior and is highly variable. In addition, MYC protein expression is not necessarily correlated with MYC gene rearrangements and suggests the need for caution in the interpretation of MYC immunohistochemistry in the differential diagnosis of lymphomas.

Importance: Spitz nevi are benign melanocytic proliferations that can sometimes be clinically and histopathologically difficult to distinguish from melanoma. Agminated Spitz nevi have been reported to arise spontaneously, in association with an underlying nevus spilus, or after radiation or chemotherapy. However, to our knowledge, the genetic mechanism for this eruption has not been described., Observations: We report a case of agminated Spitz nevi arising in a nevus spilus and use exome sequencing to identify a clonal activating point mutation in HRAS (GenBank 3265) (c.37G→C) in the Spitz nevi and underlying nevus spilus. We also identify a secondary copy number increase involving HRAS on chromosome 11p, which occurs during the development of the Spitz nevi., Conclusions and Relevance: Our results reveal an activating HRAS mutation in a nevus spilus that predisposes to the formation of Spitz nevi. In addition, we demonstrate a copy number increase in HRAS as a "second hit" during the formation of agminated Spitz nevi, which suggests that both multiple Spitz nevi and solitary Spitz nevi may arise through similar molecular pathways. In addition, we describe a unique investigative approach for the discovery of genetic alterations in Spitz nevi.

Background: : The typical algorithm for human epidermal growth factor-2 (HER2) testing is immunohistochemistry (IHC), followed by reflex HER2 fluorescence in situ hybridization (FISH) for HER2 IHC-ambiguous (2+) cases. At our institution, HER2 FISH testing is initially performed as part of routine breast cancer testing, with HER2 FISH-ambiguous (HER2:CEP17 ratio, 1.8 to 2.2) cases reflexed to HER2 IHC. This provides a unique dataset for lesions that may not routinely undergo FISH testing. The clinicopathologic characteristics of HER2 FISH-ambiguous cases are described., Design: : The electronic pathology database in our institution was searched for HER2 FISH-ambiguous cases from 2007 to December 2011. Review of clinical and pathologic characteristics was performed., Results: : Sixty cases from 60 patients were reported as HER2 FISH ambiguous. Reflex HER2 IHC testing was performed on all 60 cases, of which 26 were HER2 IHC negative (0 to 1+), 18 were HER2 IHC ambiguous (2+), and 16 were HER2 IHC positive (3+). Of the 46 HER2 FISH-ambiguous patients with available clinical records, 13 (32%) pursued anti-HER2 treatment (10 IHC 3+, 1 IHC 2+, 2 IHC 0 to 1+). All were grade II or III ductal carcinomas, with 1 grade III metaplastic carcinoma., Conclusions: : Reflex HER2 IHC testing after initially ambiguous HER2 FISH testing provides definitive HER2 status in a majority of cases (70%). However, a substantial percentage (30%) of HER2 FISH-ambiguous cases is also HER2 IHC ambiguous, suggesting an intermediate HER2 biology. Most HER2 FISH-ambiguous patients who received trastuzumab were HER2 IHC 3+, grade III, and had associated high-grade ductal carcinoma in situ. Although not statistically significant and with only minimal follow-up, no recurrences have occurred in those patients treated with trastuzumab (P=0.5754).

Despite numerous clinical trials over the past 2 decades, the overall survival for children diagnosed with diffuse intrinsic pontine glioma (DIPG) remains 9-10 months. Radiation therapy is the only treatment with proven effect and novel therapies are needed. Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the epidermal growth factor receptor and is expressed in many tumor types but is rarely found in normal tissue. A peptide vaccine targeting EGFRvIII is currently undergoing investigation in phase 3 clinical trials for the treatment of newly diagnosed glioblastoma (GBM), the tumor in which this variant receptor was first discovered. In this study, we evaluated EGFRvIII expression in pediatric DIPG samples using immunohistochemistry with a double affinity purified antibody raised against the EGFRvIII peptide. Staining of pediatric DIPG histological samples revealed expression in 4 of 9 cases and the pattern of staining was consistent with what has been seen in EGFRvIII transfected cells as well as GBMs from adult trials. In addition, analysis of tumor samples collected immediately post mortem and of DIPG cells in culture by RT-PCR, western blot analysis, and flow cytometry confirmed EGFRvIII expression. We were therefore able to detect EGFRvIII expression in 6 of 11 DIPG cases. These data suggest that EGFRvIII warrants investigation as a target for these deadly pediatric tumors.

Recent evidence suggests that trastuzumab, a monoclonal antibody which targets HER2, in combination with chemotherapy is a therapeutic option in patients with HER2-positive gastric or gastroesophageal junction cancer. Widely accepted guidelines for HER2 testing in gastric and gastroesophageal junction cancer have not been established. The purpose of this study was to analyze the incidence and patterns of HER2 expression in gastric and gastroesophageal junction cancer using a tissue microarray approach, which closely simulates small biopsies routinely tested for HER2. One hundred sixty-nine patients, including 99 primary gastric adenocarcinomas and 70 primary gastroesophageal junction carcinomas were analyzed for HER2 overexpression by immunohistochemistry and HER2 gene amplification by fluorescence in situ hybridization using scoring schemes proposed by both American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) and the results of the recently published Trastuzumab for Gastric Cancer (ToGA) trial. In our analysis, 19 adenocarcinomas were HER2 positive, defined as either a HER2/CEP17 ratio >2.2 and/or a 3+ HER2 immunohistochemistry score with either the ASCO/CAP or ToGA scoring schemes. Of the 19 HER2-positive adenocarcinomas, 8 (42%) exhibited a characteristic strongly intense basolateral membranous staining pattern which would be interpreted as negative (1+) using the accepted ASCO/CAP scoring scheme for HER2 assessment in breast carcinoma, but were correctly labeled as 3+ positive using the proposed ToGA scoring scheme. Of the 19 HER2-positive adenocarcinomas, 8 (42%) demonstrated heterogeneous HER2 protein expression by immunohistochemistry. Twelve of 99 (12%) gastric carcinomas were positive for HER2. Of these, HER2 was more often identified in intestinal-type adenocarcinomas (10 of 52, 19%) compared with diffuse (2 of 34, 6%) adenocarcinoma. Seven of 70 (10%) gastroesophageal junction carcinomas were positive for HER2 of which all were intestinal type (7 of 58, 12%). HER2 status or primary tumor site did not correlate with patient survival. Gastric and gastroesophageal junction adenocarcinomas typically display a characteristic basolateral membranous pattern of HER2 expression which is often heterogeneous rendering routine evaluation of HER2 status on small tissue samples challenging.

Translocations are key oncogenic events, and many rearrangements are characteristic for a specific malignancy. We present here a case of phenotypic precursor-B acute lymphoblastic leukemia (ALL), subsequently found to have both MYC and MLL translocations. Owing to the potential prognostic impact of these translocations, a novel treatment strategy was applied which merged precursor-B ALL, Burkitt-ALL, and "MLL-adapted" rationales. With the advent of expanding diagnostic panels and molecular therapeutic options, use of such adapted therapies for individualized treatment will undoubtedly continue to increase as we move toward pharmacogenomic-based approaches.

Chordomas are malignant neoplasms that typically arise in the axial spine and primarily affect adults. When chordomas arise in pediatric patients they are more likely to display unusual histological features and aggressive behavior. We noted the absence of SMARCB1/INI1 expression by immunohistochemistry in an index case of poorly differentiated chordoma of the sacrum, leading us to further examine SMARCB1/INI1 expression as well as that of brachyury, a highly specific marker of notochordal differentiation, in 3 additional poorly differentiated chordomas of the clivus, 10 typical chordomas, and 8 atypical teratoid/rhabdoid tumors (AT/RTs). All 4 poorly differentiated chordomas and all AT/RTs lacked nuclear expression of SMARCB1/INI1, while the 10 typical chordomas maintained strong nuclear SMARCB1/INI1 immunoreactivity. All 10 typical and 4 poorly differentiated chordomas expressed brachyury; all 8 AT/RTs were brachyury immunonegative. Cytogenetic evaluation utilizing FISH probes near the SMARCB1/INI1 locus on chromosome 22q was also performed in all of the poorly differentiated chordomas in this series. Three of the four poorly differentiated chordomas had evidence for deletion of this region by FISH. Analysis of the SMARCB1/INI1 gene sequence was performed using formalin-fixed paraffin-embedded tissue in all cases and no point mutations were observed. In summary, all poorly differentiated chordomas in this series showed the absence of SMARCB1/INI1 expression, and were reliably distinguished from AT/RTs, clinically by their characteristic primary sites of origin and pathologically by strong nuclear brachyury expression. Our findings reveal a likely role for SMARCB1/INI1 in a subset of chordomas with aggressive features.

Immunophenotypic identification of myeloid specific antigens is an important diagnostic tool in the management of patients with acute myeloid leukemia (AML). These antigens allow determination of cell of origin and degree of differentiation of leukemia blasts. AML with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is a relatively rare subtype of AML. The immunophenotypic characteristics of inv(3) AML patients are somewhat limited. We identified 14 new cases of hematological disorders with increased myeloid blasts carrying inv(3)(q21q26.2)/t(3;3)(q21;q26.2). Also, we identified another 13 cases previously published in the literature, where the immunophenotype of inv(3)(q21q26.2) was documented. As a group, patients with AML with inv(3)(q21q26.2) had high levels of early myeloid (CD13, CD33, CD117 and MPO) and uncommitted markers (CD34, HLA-DR and CD56) and a high rate of monosomy 7 in addition to the inv(3)(q21q26.2). Differential karyotype and expression of certain antigens were noted in patients with de novo AML with inv(3)(q21q26.2) vs. those with inv(3)(q21q26.2)-containing blasts., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Accurate assessment of Her-2/neu (erb-b2) status in breast carcinoma is essential for therapy planning. Clinical assays are targeted at protein overexpression (immunohistochemical analysis) or gene amplification (fluorescence in situ hybridization [FISH]). Cases with aberrant FISH signal patterns are problematic and may lead to underreporting of Her-2/neu amplification. We performed FISH with additional chromosome 17 probes, SMS (Smith-Magenis syndrome critical region) and RARA (retinoic acid receptor), on 7 cases with unusual Her-2/CEP17 (chromosome 17 centromere control probe) results to assess whether different measurements of chromosome 17 copy number might clarify the Her-2/neu amplicon status. Although the Her-2/CEP17 ratio scores were within normal range (<2.0), the Her-2/SMS or Her-2/RARA ratio revealed amplification of Her-2/neu in 5 of 7 cases. Immunohistochemical analysis demonstrated Her-2/neu protein overexpression in the same 5 cases only. We describe novel application of SMS/RARA FISH probes for assessing cases with complex Her-2/CEP17 FISH patterns. Such additional data, correlated with immunohistochemical analysis, may help guide therapy in patients with breast carcinoma.

Three patients diagnosed with acute myeloid leukemia (AML) with reciprocal 21q22/RUNX1(AML1) translocations involving chromosomes 1 and 4 were studied. Three novel RUNX1 translocation partner genes on 1q21.2 (ZNF687), 1p35 (YTHDF2), and 4q31.3 (SH3D19) were identified using a panhandle polymerase chain reaction and the 3' rapid amplification of cDNA ends method. The translocation events occurred between exons 3 and 7 of the RUNX1 gene. The partner gene breakpoints localized to the region in the partner gene with the highest Alu density, suggesting that Alus may contribute to the recombination events. Two out of three of the cases retained RUNX1's entire RUNT domain in the translocation, and RUNX1 mutations were absent in the fusion transcripts, confirmed by reverse transcription-polymerase chain reaction and sequencing analysis. SH3D19 encodes a cytoplasmic protein EBP known to suppress RAS-induced cellular transformation, which can be inhibited by nuclear recruitment. The t(4;21) created a hybrid RUNX1-EBP protein retaining RUNX1's DNA binding domain, which may result in nuclear localization of the chimeric protein and inhibition of EBP's RAS-suppressive functions. Future studies would be useful to further characterize these novel fusion protein products., ((c) 2006 Wiley-Liss, Inc.)

Background: The diagnosis and classification of lymphoma require correlation of morphologic, immunophenotypic, and molecular-cytogenetic studies. Fine-needle aspiration biopsy (FNAB) is a valuable diagnostic technique that allows material to be collected for these ancillary studies, and for morphologic evaluation., Methods: The authors report a series of seven cases clinically or morphologically suspicious for Burkitt lymphoma. Fluorescence in situ hybridization studies (FISH) for c-myc were performed on FNAB material and correlated with cytologic and immunophenotypic data., Results: Six of seven specimens were positive for c-myc rearrangement by FISH. However, only three of these cases represented Burkitt lymphoma, with one additional case of atypical Burkitt lymphoma. The other cases included diffuse large B-cell lymphoma, monomorphic posttransplant B-cell lymphoma, and an aggressive B-cell lymphoma, with the latter case negative for c-myc rearrangement by FISH. Of 2 non-Burkitt lymphoma specimens tested, 1 was positive for the immunoglobulin H/bcl-2 rearrangement, in addition to the c-myc rearrangement, suggesting transformation from a lower grade lymphoma., Conclusions: These cases illustrated the value of FNAB in the diagnosis of Burkitt lymphoma, as well as the importance of obtaining material for, and integrating results of, ancillary studies for the final diagnosis.

The chromosomal translocation t(9;14)(p13;q32) has been reported in association with lymphoplasmacytic lymphoma (LPL). Although this translocation involving the paired homeobox-5 (PAX5) gene at chromosome band 9p13 and the immunoglobulin heavy chain (IgH) gene at 14q32 has been described in approximately 50% of LPL cases, the actual number of cases studied is quite small. Many of the initial cases associated with t(9;14)(p13;q32) were actually low-grade B-cell lymphomas with plasmacytic differentiation other than LPL. Thus, we analyzed a series of low-grade B-cell lymphomas for PAX5 gene rearrangements. We searched records from the Department of Pathology, Stanford University Medical Center for low-grade B-cell lymphomas, with an emphasis on plasmacytic differentiation, that had available paraffin blocks or frozen tissue. We identified 37 cases, including 13 LPL, 18 marginal zone lymphomas (nodal, extranodal, splenic, and alpha-heavy chain disease), and 6 small lymphocytic lymphomas. A novel dual-color break-apart bacterial artificial chromosome probe was designed to flank the PAX5 gene, spanning previously described PAX5 breakpoints, and samples were analyzed by interphase fluorescence in situ hybridization. All cases failed to demonstrate a PAX5 translocation, indicating that t(9;14)(p13;q32) and other PAX5 translocations are uncommon events in low-grade B-cell lymphomas with plasmacytic differentiation. This study also confirms recent reports that found an absence of PAX5 rearrangements in LPL, suggesting the reassessment of PAX5 rearrangements in LPL.

Chromosome preparations currently provide the only direct view of the genome as a whole. Although molecular methods allow a more detailed analysis of specific regions of the genome, the study of genetics is not complete without an appreciation of the metaphase cell. The stimulated T cell system described in this unit is the most widely used means of obtaining large numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle in culture is described, combined with direct inhibition of chromosome condensation, to yield longer high-resolution prophase or prometaphase preparations. Such preparations are used for detailed analysis of microdeletions or subtle rearrangements, fine breakpoint analysis, and refined mapping. Microscope slide preparation of mitotic chromosomes from harvested cell culture suspensions is also explained in the support protocol.

Activation of the MDR1 (ABCB1) gene is a common event conferring multidrug resistance (MDR) in human cancers. We investigated MDR1 activation in MDR variants of a human sarcoma line, some of which express a mutant MDR1, which facilitated the study of allelic gene expression. Structural alterations of MDR1, gene copy numbers, and allelic expression were analyzed by cytogenetic karyotyping, oligonucleotide hybridization, Southern blotting, polymerase chain reaction, and DNA heteroduplex assays. Both chromosome 7 alterations and several cytogenetic changes involving the 7q21 locus are associated with the development of MDR in these sarcoma cells. Multistep-selected cells and their revertants contain three- to six-fold MDR1 gene amplification compared with that of the drug-sensitive parental cell line MES-SA and single-step doxorubicin-selected mutants. MDR1 gene amplification precedes the emergence of a mutant allele in cells that were coselected with doxorubicin and a cyclosporin inhibitor of P-glycoprotein (P-gp). Allele-specific oligonucleotide hybridization showed that the endogenous mutant allele was present as a single copy, with multiple copies of the normal allele. Reselection of revertant cells with doxorubicin in either the presence or the absence of the P-gp inhibitor resulted in exclusive reexpression of the mutant MDR1 allele, regardless of the presence of multiple wild-type MDR1 alleles. These data provide new insights into how multiple alleles are regulated in the amplicon of drug-resistant cancer cells and indicate that increased expression of an amplified gene can result from selective transcription of a single mutant allele of the gene., (Copyright Wiley-Liss, Inc.)

Chromosomal abnormalities are common causes of a variety of diseases, cancers, and malformation syndromes. Identification of chromosomal aberrations is important for counseling families about prognosis and reproductive risks with future pregnancies. However, limited resolution leads to the inability to detect small deletions, small insertions or duplications, and complex chromosomal rearrangements. Fluorescence-based assays, which have become possible because of sophisticated cloning technologies and improved sensitivity of antibody conjugates, enable the detection of subtle chromosomal changes beyond the resolution of classic cytogenetics. Such techniques have greatly expanded the diagnostic armamentarium available in the investigation of adolescents with mental retardation, malformations and many other disorders.

Purpose: The authors report the use of a cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP)-based chemotherapy regimen in treating six children with posttransplantation lymphoproliferative disorder (PTLD) that developed after solid organ transplantation., Materials and Methods: The chemotherapy regimen consisted of a 29-day induction with CHOP and then as many as 15 cycles of maintenance therapy using methotrexate and cytarabine alternating with vincristine, adriamycin, mercaptopurine, and prednisone., Results: All patients attained remission. One patient died of sepsis while in remission. Four of the five remaining patients have been followed-up in remission for as long as 8 years without losing the graft. One of the patients experienced relapse after completing therapy and subsequently died with disease., Conclusions: The authors conclude that pediatric patients with PTLD after solid organ transplantation that fails conservative management can be treated successfully with CHOP-based chemotherapy.

The AML1 (CBFA2) gene is the most frequent target of chromosomal rearrangements observed in human acute leukemia. These rearrangements include the commonly reported t(8;21)(q22;q22) or AML1/ETO fusion in AML-M2, the t(3;21)(q26;q22) or AML1 fusion with one of three genes, MDS1, EAP or EVI1, in therapy-related AML and MDS, as well as in blast crisis in CML and the t(12;21)(p13;q22) or TEL/AML1 fusion in B-cell ALL. In addition to the t(3;21), other AML1 translocations have also been reported in therapy-related MDS and AML, particularly after treatment with topoisomerase II inhibitors. AML1 gene rearrangements have also been observed less frequently with numerous other chromosomal partners. Here, we describe a patient with AML-M4 and a previously unreported rearrangement involving the AML1 locus and an unknown locus on the short arm of chromosome 1 at 1p32.

Spindle cell lipoma demonstrates a distinctive histologic appearance and characteristic clinical presentation. We recently observed two cases of solitary subcutaneous neoplasm of the foot with histologic features of spindle cell lipoma that in one case includes a minor component of the overlapping tumor, pleomorphic lipoma. Because the foot is an unusual location for these neoplasms, immunoperoxidase and cytogenetic studies were performed. In both cases, staining was strongly positive for CD34 and negative for smooth muscle actin. Cytogenetic studies from the tumor with a pleomorphic component revealed features consistent with a lipomatous neoplasm, but are otherwise diagnostically nonspecific. An analysis of the literature reveals that although CD34 immunoreactivity is characteristic of spindle cell lipoma and helps exclude nonlipomatous neoplasms, it does not clearly eliminate other well-differentiated lipomatous tumors. Accordingly, without the aid of classic tumor location, the diagnosis of the spindle cell/pleomorphic lipoma group relies primarily on histologic features, with supportive but not definitive information provided by immunoperoxidase and cytogenetic studies. Obscuring this issue, however, are the imprecise histologic distinction between these tumors and those of the atypical lipoma/atypical lipomatous tumor/ well-differentiated liposarcoma group and the nomenclature controversy that surrounds the latter group of neoplasms. Despite these obstacles, both groups of well-differentiated lipomatous tumors are clinically benign when subcutaneously located.

Normal human endothelial cells, like other somatic cells in culture, divide a limited number of times before entering a nondividing state called replicative senescence. Expression of the catalytic component of human telomerase, human telomerase reverse transcriptase (hTERT), extends the life span of human fibroblasts and retinal pigment epithelial cells beyond senescence without causing neoplastic transformation (Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S., and Wright, W. E. (1998) Science 279, 349-352; Jiang, X., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A., Wahl, G., Tlsty, T., and Chiu, C.-P. (1999) Nat. Genet. 21, 111-114). Here, we show that both human large vessel and microvascular endothelial cells also bypass replicative senescence after introduction of hTERT. For the first time, we report that hTERT expression in these life-extended vascular cells does not affect their differentiated and functional phenotype and that these cells maintain their angiogenic potential in vitro. Furthermore, hTERT(+) microvascular endothelial cells have normal karyotype, and hTERT(+) endothelial cell strains do not exhibit a transformed phenotype. Relative to parental cells at senescence, hTERT-expressing endothelial cells exhibit resistance to induction of apoptosis by a variety of different conditions. Such characteristics are highly desirable for designing vascular transplantation and gene therapy delivery systems in vivo.

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3(+)CD56(+) cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3(+) T cells (median 97%, range 90% to 99%). The CD3(+)CD56(+) subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4. Both the CD4(+) and CD8(+) subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 x 10(7) autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus-associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = . 03). This study shows that CIK cells, which are Ph chromosome-negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells., (Copyright 1998 by The American Society of Hematology)

Uncontrolled proliferation of acute myeloid leukemia (AML) cells is an important step during leukemogenesis. However, little is known about the mechanisms leading to growth autonomy. Studies using immortalized murine hematopoietic cell lines have suggested that autocrine production of growth factors, or the constitutive activation of molecules in growth factor signalling pathways, are involved. We have established six spontaneous factor-independent cell lines from the human growth factor-dependent TF-1 cell line. The factor-independent cells showed no detectable growth factor activity. Immunoblotting analyses of tyrosine phosphorylation, Raf-1 and extracellular signal-regulated kinase 2 (ERK-2) showed a similar pattern in all the cell lines including TF-1 cells. Furthermore, somatic-cell hybrids between TF-1 and the factor-independent cells grew in absence of growth factor. Taken together this data demonstrates that the factor independence in this system is dominant and suggests that the molecular event is located either downstream of the Raf-1 and MAP kinases pathway or on an alternative pathway. Finally, the karyotype analysis of one factor-independent cell line TF-1i1 and TF-1H- (G418 resistant, HAT sensitive TF-1 cells) and their hybrids demonstrated an unstable derivative chromosome [der(19) t(19;?) (q13.1;?)] which seemed to correlate with the factor-independence capacity. This model may help in our understanding of autonomous proliferation by human myeloid leukemias.

The authors performed immunohistochemical and cytogenetic studies in a 73-year old man with malignant angioendotheliomatosis. The patient was referred for evaluation of fever of unknown origin, hepatic failure, and neurologic deterioration. Examination of a muscle biopsy revealed numerous, noncohesive atypical mononuclear cells within small vessels. These cells stained positively with a pan-leukocyte marker CD45(PD7/26/16) and with a B-cell marker L26 but negatively with Factor VIII-related antigen, an endothelial cell marker. Peripheral blood obtained before chemotherapy was cultured and analyzed by the G-band method. A new translocation and numerous chromosomal aberrations were identified. The major cell line karyotype was 53,XY, +X, +5q?,-6, +i(6p), +7, -10, +11, -12, +12p-, +12p-, +18, +mar1, +mar2, t(1;3)(p22;p21),3q+,8p+. This is the first cytogenetic study performed in a case of malignant angioendotheliomatosis. Our findings demonstrate that the neoplastic cells in this disorder circulate in the peripheral blood and provide further evidence that malignant angioendotheliomatosis is a diffuse intravascular neoplasm of lymphoid origin. Furthermore, the authors conclude that this malignant lymphoproliferative disorder should be reclassified as a primary intravascular (angiotropic) lymphoma.

An unusual form of X chromosome aneuploidy, 47,XX,psu dic(X)(p11.2), was found during an evaluation for short stature of a prepubertal girl. Unlike 45,X, 47,XXX, 48,XXXX, and 49,XXXXX females, this patient is phenotypically normal except for her short stature, which appears to be unrelated to her chromosome abnormality. X chromosome inactivation studies disclosed inactivation (late replication) of one normal X and the abnormal X chromosome in all cells examined from this patient. Therefore, she is disomic for early-replicating distal Xp loci, found in inactivated X chromosomes, and thought to remain active after lyonization. These data suggest that the presence of three or more copies of the early-replicating, active Xp loci may be responsible for the cognitive deficits and other phenotypic abnormalities seen in and other phenotypic abnormalities seen in polysomy X females.

A human tumor cell line designated SU.86 has been established from a moderate-to-poorly differentiated pancreatic carcinoma of ductal origin specifically for adoptive immunotherapy studies. This line was characterized as to its ability to be lysed in vitro by autologous and allogeneic lymphokine-activated killer (LAK) and natural killer cells and to grow in nude mice. SU.86 has been growing continuously in cell culture for more than 100 passages since 22 September 1986. Transplantation orthotopically and heterotopically into athymic Swiss nude mice showed that tumor take was 100% in the orthotopic position when young (4 to 6 wk old) mice were used and 0% when adult (8 wk old) mice were used (P = 0.004). In the heterotopic position (subcutaneous), tumor take was 100% in neonate (2 to 3 wk old) and young mice and 50% in adults. The rate of tumor growth was inversely correlated with age (P less than 0.001). The histologic pattern is similar to that observed in most human pancreatic carcinomas with pseudoglandular structures and frequent mitotic figures. SU.86 has a doubling time of 77 h in vitro and produces carcinoembryonic antigen, 594 ng/10(6) cells in 3 d. Chromosomal analysis shows heterogeneity with two notable cell subpopulations. The cell line is moderately sensitive to lysis by LAK cells in a standard, 4-h chromium-51 release assay (35.4 +/- 4.0%). When grown together with LAK cells in vitro, it is lysed completely in culture in 8 to 15 d, depending on the serum concentration.

A boy with bilateral colobomas, preauricular pits, and developmental delay had a 46,XY,22q+ karyotype. His parents had normal chromosomes. The abnormality of 22q was interpreted as a de novo tandem duplication of 22q11.1----q11.2. Although no anal abnormality was identified, his manifestations are otherwise consistent with those of the cat-eye syndrome. Blood marker results and the levels of galactosidase-2, galactosidase-B and arylsulfatase-A, which are known to be coded on 22q, are normal.