Evidence Of Aneuploidy Research Articles

Abstract A15: Clinico-pathologic and molecular characteristics of primary extranodal germinal-center B-cell like diffuse large B-cell lymphoma (EN GCB-DLBCL)

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease and in 30-40% cases can present or relapse at extranodal sites. Primary extranodal DLBCL (EN-DLBCL) is defined as disease presenting as clinically dominant extranodal masses with no or only minor nodal disease. EN-DLBCL is predominantly of the activated B-cell (ABC) and less commonly of germinal center B-cell (GCB) like DLBCL subtype. We aimed to identify genetic characteristics of EN GCB-DLBCL to gain insights into extranodal tropism and aggressive clinical behavior. Methods: Patient data was collected via retrospective chart review. Inclusion criteria consisted of pathologically confirmed diagnosis of GCB-DLBCL by the Hans algorithm, extra-nodal involvement at diagnosis or relapse, and availability of next-generation sequencing (NGS). Overall (OS) and progression-free survival (PFS) were analyzed using the Kaplan-Meier method. Results: Fifteen patients were identified. The median age at diagnosis was 64 years (range 33-89) and majority were men (62.5%). Two patients had purely nodal disease at presentation that relapsed at EN sites, while the remainder (13/15) had predominantly EN disease. EN sites included central nervous system [CNS] (n = 5), bone marrow (n = 4), epidural (n=4), bone (2), paranasal sinus (1), testes (1), stomach (n = 2), lung/pleura (n=2), and liver (n=3). Nine patients had disease in multiple EN sites. All patients had advanced stage disease and median IPI score was 3 (range 3-4). On FISH/cytogenetic analysis, two patients had MYC and four patients had BCL2 rearrangements. Copy gains in BCL6, BCL2 and MYC were seen in 2, 1 and 4 patients respectively. No patients had evidence of MYC and BCL2 and/or BCL6 rearrangements (double hit). TP53 deletion was seen in 1 patient. On NGS, prominent gene mutations identified were KMTD2 (8/15, 53%), TP53 (5/15, 33%), TNFRSF14 (4/15, 27%), and evidence of aneuploidy with abnormalities in chromosome 12 (5/15, 33%), chromosome 6 (4/15, 27%), and chromosome 8 (4/15, 27%), including both losses and gains of function. Notably, MYD88 (3/15, 20%), CARD11 (2/15, 13%), and IRF4 (2/15, 15%) mutations, representative of BCR/TLR4 pathway activation and NF-kB signaling were seen at initial presentation. All patients were treated with chemo-immunotherapies incorporating CNS prophylaxis and 2 patients received salvage CAR T-cell therapy. Median OS was 1.3 years and median PFS was 11.3 months. Nine patients (63%) progressed, with CNS relapses in 5 and of these, 7 patients died. Conclusions: This study shows that EN GCB-DLBCL is molecularly similar to EN ABC-DLBCL; specifically, activation of NF-kB pathway signaling through gene mutations involving the MCD (MyD88/CD79B) cluster is prominent, and this potentially enables extranodal tropism. The complex karyotype and TP53 mutations may contribute to chemoresistance and aggressive disease biology. Our results provide valuable molecular information and can aid in personalizing treatment strategies for this uncommon, aggressive DLBCL subset. Citation Format: Akshat Patel, Brian Lue, Kiran A Kumar, Farrukh T Awan, Jeffrey Gagan, Weina Chen, Praveen Ramakrishnan Geethakumari. Clinico-pathologic and molecular characteristics of primary extranodal germinal-center B-cell like diffuse large B-cell lymphoma (EN GCB-DLBCL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A15.

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse

The first 8 weeks of pregnancy is a critical time, with the majority of pregnancy losses occurring during this period. Abnormal chromosome number (aneuploidy) is a common finding in human miscarriage, yet is rarely reported in domestic animals. Equine early pregnancy loss (EPL) has no diagnosis in over 80% of cases. The aim of this study was to characterise aneuploidies associated with equine EPL. Genomic DNA from clinical cases of spontaneous miscarriage (EPLs; 14–65 days of gestation) and healthy control placentae (various gestational ages) were assessed using a high density genotyping array. Aneuploidy was detected in 12/55 EPLs (21.8%), and 0/15 healthy control placentae. Whole genome sequencing (30X) and digital droplet PCR (ddPCR) validated results. The majority of these aneuploidies have never been reported in live born equines, supporting their embryonic/fetal lethality. Aneuploidies were detected in both placental and fetal compartments. Rodents are currently used to study how maternal ageing impacts aneuploidy risk, however the differences in reproductive biology is a limitation of this model. We present the first evidence of aneuploidy in naturally occurring equine EPLs at a similar rate to human miscarriage. We therefore suggest the horse as an alternative to rodent models to study mechanisms resulting in aneuploid pregnancies.

Chromosome evolution in Bulbine glauca (Asphodelaceae or Xanthorrhoeaceae subfam. Asphodeloideae) indicates six species, not one

The genus Bulbine Wolf has ~130 species in Africa and only seven species in Australia. Base chromosome numbers in African and Australian Bulbine are x = 7 and x = 12, respectively. This raises the possibility of Australian Bulbine being of polyploidy origin or, alternatively African Bulbine may have evolved through dysploid reduction. Previous chromosome counts in Bulbine glauca (Raf.) E.M.Watson were predominantly 2n = 46, with aneuploid numbers of 2n = 45, 44, 48, 47, 40 and 41 also being encountered within and between populations. Novel chromosome counts are presented here for a wide sample of populations of B. glauca, occurring on volcanic and granite outcrops along the New South Wales and Victorian tablelands and on granite outcrops in Tasmania. Chromosome numbers for Bulbine crassa D.I.Morris and Duretto is 2n = 26. The modal chromosome count for B. glauca is confirmed as 2n = 46 with no evidence of aneuploidy. The species complex shows a variable asymmetric karyotype across its distribution, indicating karyotypic evolution involving structural rearrangements. There appear to be six distinctive groups within the complex based on karyomorphology. Evolutionary changes in both B. glauca s. lat. and B. crassa appear to have not involved recent polyploidy. Variation in chromosome morphology across the B. glauca complex is consistent with our morphological evidence of more than one species.

Chromosome studies in New Zealand species of Brachyscome (Asteraceae)

Chromosome numbers were determined for plants of five species and seven additional entities of uncertain taxonomic status of Brachyscome. We confirmed the chromosome numbers of several species but found new numbers in plants related to B. humilis, B. montana and B. radicata. Karyotype analysis showed a common karyotype in the diploids and suggested that the polyploids are autopolyploids. There is some evidence of aneuploidy in plants related to B. sinclairii and B. radicata. Karyotype analysis suggests that structural chromosome changes may have accompanied polyploidy in tetraploid B. humilis.

Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: A step towards exploring a potential use of matrix-induced cell therapy

Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and ‘chondrocytes-fibrin’ construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM + 10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM + 2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM + 10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs > FD2GFs > FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and ‘chondrocytes-fibrin’ constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, ‘chondrocytes-fibrin’ constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality ‘chondrocytes-fibrin’ constructs which may have potential use of matrix-induced cell implantation.

CREB Is Amplified in AML Blasts and Is Associated with an Increased Risk of Relapse and Decreased Event-Free Survival.

The cAMP Responsive Element Binding Protein (CREB) is a basic leucine zipper transcription factor that regulates cell proliferation and survival. Greater than 60% of AML patients have elevated CREB protein levels in the bone marrow. Transgenic mice that overexpress CREB in myeloid cells have monocytosis and splenomegaly. Bone marrow progenitor cells from CREB transgenic mice exhibit properties of transformed cells including immortalization and growth factor-independence in colony assays. Our results suggest that CREB acts as a proto-oncogene in hematopoietic cells. We first sought to understand the mechanism of CREB overexpression in AML blasts. Since both CREB protein and mRNA levels were increased in primary AML cells, we hypothesized that CREB could be amplified. We performed fluorescence in situ hybridization (FISH) on blast cells from 4 CREB overexpressing patients using a CREB specific BAC clone. The CREB gene has been previously localized to chromosome 2q32.3–q34. In blast cells from 3 out of 4 AML patients that overexpressed CREB, we detected 3 to 4 signals from one homolog and 1 signal from the other in over 100 interphase nuclei analyzed. However, in normal control lymphocytes in metaphase and CREB nonamplified bone marrow cells in interphase, the CREB BAC showed two clear and unambiguous signals. Three out of the 4 AML patients whose bone marrow overexpressed CREB had a normal karyotype with no evidence of aneuploidy or hyperdiploidy. These results indicate that in certain AML patients there are more than two copies of the CREB gene. To evaluate whether increased CREB expression predicts outcome in leukemia patients, Kaplan-Meier analysis was performed on data from 45 AML patients (14 age 18 or under and 31 over age 18). Time to relapse or persistent disease was markedly shorter in the 31 CREB overexpressing, i.e. CREB positive, patients in comparison to the 14 not expressing CREB (proportion with persistent disease or relapsing by 1 year 0.68 ± 0.10 vs. 0.23 ± 0.14, p<0.001 both before and after stratification on cytogenetics). Likewise, event-free survival was noticeably worse in the CREB positive group (p<0.003). The seven patients who showed persistent disease after initial induction therapy were all CREB positive, but excluding them from the analysis still yielded noticeably shorter relapse times for the CREB positive group (p=0.01). No variable other than CREB overexpression had a significant association with adverse time to relapse or event-free survival. Initial treatment, cytogenetics, AML subtype, age, gender, initial white blood count, absolute blast count and follow-up times were similar between the two CREB groups. Percent blasts at diagnosis (mean ± SD) were higher in the CREB overexpressing group (69 ± 28 vs 51 ± 24, p=0.02). However, neither stratification on blast percent nor on any other variable altered the significance of CREB as a predictor of initial adverse outcome. In contrast, overall survival tended to differ in the expected way among the cytogenetic prognostic groups (p=0.03), but without strong evidence for an association with CREB expression (p=0.35). Our results suggest that CREB expression may be an important independent predictor of remission duration and event-free survival in AML patients.

Evidence for aneuploidy and recombination in the human commensal yeast Candida parapsilosis

Isolates of Candida parapsilosis, including representatives of the three major sub-species groups, were screened for single nucleotide polymorphisms (SNPs) by sequencing five independent loci totaling 4 kb per isolate. Group I isolates were highly conserved and in some cases, group I alleles were found in group II and III strains. Unique alleles were also associated with groups II and III, consistent with earlier observations of intergroup divergence. There was no heterozygosity in any strain, and a FACS analysis demonstrated that for all three groups nuclei are variant in size, ranging from 0.5 to 1.0× the size of other diploid yeast genomes. This suggests that natural isolates of C. parapsilosis are aneuploid, with some isolates being essentially haploid. Taken collectively with the observation of group I alleles within group II and III strains, we propose that some form of recombination is occurring between groups.

Normal features of tissue-engineered auricular cartilage by flow cytometry and histology: patient safety

Background Cytokinetic abnormalities in DNA content, such as aneuploidy, haploidy, and tetraploidy, have been found to occur in human cartilaginous tumors. The high number of chondrocytes needed for tissue-engineered cartilaginous implants requires the cells to be passaged repeatedly. The theoretical risk of changes in the normal diploid state of these cells during their growth in vitro and after generation of tissue-engineered cartilage in vivo is not known. Materials and methods Auricular chondrocytes were obtained from 6 patients and cultured in vitro. Chondrocyte number was increased by repeated passaging. The passaged cells were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Fresh control chondrocytes along with the passaged cells and cells obtained from the tissue-engineered constructs were collected and compared for DNA content by flow cytometry. Results Flow cytometry demonstrated 100% diploidy with no evidence of aneuploidy, haploidy, or tetraploidy in all groups of cells. Histology of the tissue-engineered cartilage also showed no evidence of cellular atypia. Conclusion The number of human auricular chondrocytes can be increased by repeated passaging and passaged chondrocytes can be safely used for implantation to generate tissue-engineered constructs without a change in the normal diploid state of the cells. Histology of the cartilage generated showed normal features without atypia.

Normal features of tissue-engineered auricular cartilage by flow cytometry and histology: patient safety.

Cytokinetic abnormalities in DNA content, such as aneuploidy, haploidy, and tetraploidy, have been found to occur in human cartilaginous tumors. The high number of chondrocytes needed for tissue-engineered cartilaginous implants requires the cells to be passaged repeatedly. The theoretical risk of changes in the normal diploid state of these cells during their growth in vitro and after generation of tissue-engineered cartilage in vivo is not known. Materials and methods Auricular chondrocytes were obtained from 6 patients and cultured in vitro. Chondrocyte number was increased by repeated passaging. The passaged cells were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Fresh control chondrocytes along with the passaged cells and cells obtained from the tissue-engineered constructs were collected and compared for DNA content by flow cytometry. Flow cytometry demonstrated 100% diploidy with no evidence of aneuploidy, haploidy, or tetraploidy in all groups of cells. Histology of the tissue-engineered cartilage also showed no evidence of cellular atypia. The number of human auricular chondrocytes can be increased by repeated passaging and passaged chondrocytes can be safely used for implantation to generate tissue-engineered constructs without a change in the normal diploid state of the cells. Histology of the cartilage generated showed normal features without atypia.

OC060: The relationship between the severity of maternal obesity and fetal structural anomalies

Introduction: Choroids plexus cysts (CPC) are important for their association with fetal aneuploidy, particularly trisomy 18, which some authors described almost 20 years ago. In spite of numerous works through the years a clear-cut link between trisomy 18 and CPC has not been found. Methods: We analyzed 16 272 patients referred to our centre from January 1998 to December 2002. Only the patients with a first examination between 16 and 23 gestational weeks were included. Fetuses with a CPC were checked for either an amniocentesis (amnio) or a normal outcome at birth. From the group of CPC extracted, fetuses with associated anomalies were noted. From the group with associated anomaly a group with minor and a group with major was extracted. Results: From the total, 366 had CPC, that is 2.9%, 311 had no other associated anomalies; either the karyotype or the postnatal pediatric examination was normal, 55 of the patients had associated anomalies, from this group 12 fetus with present major anomaly were extracted and in two cases the amnio revealed two cases of trisomy 18. The other 10 cases had normal karyotype. The 43 fetuses with CPC and minor finding had either a normal amniocentesis or pediatric examination at birth. In addiction, in the period of study we identified overall 6 fetuses with karyotypically proven trisomy 18. In the 4 fetuses with trisomy 18, the presence of major anomalies in absence of CPC, strongly suggests the performance of a karyotype. Conclusion: In the isolated CPC there is not evidence of aneuploidy. If the isolated CPC had been aggressively pursued the total fetal loss would have been increased to 1.55 fetus assuming a commonly quoted risk of 1 : 200 miscarriage. Another reason is that the trisomy 18 is considered uniformly fatal, there is another important factor to consider the exposure of healthy population at the karyotype risk for recognized a very small population with a very poor prognosis.

OC059: Karyotyping of fetuses with isolated choroid plexus cyst or with associated minor marker is not justified in a low-risk population

Choroids plexus cysts (CPC) are important for their association with fetal aneuploidy, particularly trisomy 18, which some authors described almost 20 years ago. In spite of numerous works through the years a clear-cut link between trisomy 18 and CPC has not been found. We analyzed 16 272 patients referred to our centre from January 1998 to December 2002. Only the patients with a first examination between 16 and 23 gestational weeks were included. Fetuses with a CPC were checked for either an amniocentesis (amnio) or a normal outcome at birth. From the group of CPC extracted, fetuses with associated anomalies were noted. From the group with associated anomaly a group with minor and a group with major was extracted. From the total, 366 had CPC, that is 2.9%, 311 had no other associated anomalies; either the karyotype or the postnatal pediatric examination was normal, 55 of the patients had associated anomalies, from this group 12 fetus with present major anomaly were extracted and in two cases the amnio revealed two cases of trisomy 18. The other 10 cases had normal karyotype. The 43 fetuses with CPC and minor finding had either a normal amniocentesis or pediatric examination at birth. In addiction, in the period of study we identified overall 6 fetuses with karyotypically proven trisomy 18. In the 4 fetuses with trisomy 18, the presence of major anomalies in absence of CPC, strongly suggests the performance of a karyotype. In the isolated CPC there is not evidence of aneuploidy. If the isolated CPC had been aggressively pursued the total fetal loss would have been increased to 1.55 fetus assuming a commonly quoted risk of 1 : 200 miscarriage. Another reason is that the trisomy 18 is considered uniformly fatal, there is another important factor to consider the exposure of healthy population at the karyotype risk for recognized a very small population with a very poor prognosis.

Enlarged fetal gallbladder: prognostic importance for aneuploidy or biliary abnormality at antenatal US.

To test the strength of the association of cholecystomegaly with aneuploidy and biliary abnormality and to assess the prognostic importance of the detection of an enlarged fetal gallbladder at antenatal ultrasonography (US). Gallbladder size was prospectively evaluated during 842 consecutive second-and third-trimester US examinations in 775 fetuses. The area of the gallbladder was calculated on the image that depicted the maximal gallbladder size, and the actual gallbladder areas was compared with the gallbladder area expected on the basis of the gestational age. Fetuses with an enlarged gallbladder were followed up. Forty-three fetuses had an enlarged gallbladder (area more than 2 SDs above the mean for gestational age). Outcome was normal in 38 of the 39 fetuses who could be followed up. With the exception of a single baby with an isolated ventricular septal defect, which closed spontaneously, none of the babies with an enlarged fetal gallbladder had structural anatomic defects or evidence of aneuploidy or a biliary tract abnormality. Identification of an enlarged gallbladder at antenatal US does not appear to be associated with a substantially increased risk of chromosomal aneuploidy or biliary tract malformation.

Detection of aneuploidy in interphase nuclei from non-small cell lung carcinomas by fluorescence in situ hybridization using chromosome-specific repetitive DNA probes

Interphase fluorescence in situ hybridization (FISH) is particularly useful for detecting chromosome changes in tumors exhibiting a low mitotic index, as is the case in many human non-small cell lung carcinomas (NSCLCs). A panel of centromeric DNA probes specific for the autosomes 6, 7, 8, 9, 12, 17, and 18 was used to analyze 17 primary NSCLCs. Evidence for aneuploidy was obtained in all specimens. Gain of part or all of chromosome 7 was especially prominent, occurring in a large population of cells in each of 14 tumors (82%). Extra centromeric copies of chromosomes 6, 12, and 17 were also common, being observed in 9 to 11 cases each. Gain of chromosome 9 was infrequent (three tumors). In two cases, most of the nuclei had only a single chromosome 9 fluorescent signal. Karyotypic findings were available for six cases and were generally consistent with the FISH data. Both methods revealed considerable heterogeneity within individual tumors. NSCLC specimens from 26 males were assayed with a Y-specific centromeric sequence; loss of the Y was observed in 13 cases (50%). These investigations demonstrate the feasibility of interphase FISH for the successful analysis of numerical chromosome changes in NSCLCs.

Chromosomal constitution of mouse blastocysts derived from oocytes inseminated by multiple sperm insertion into the perivitelline space

Our purpose was to evaluate the rate of chromosomal aberrations in mouse blastocysts obtained after microinjection of multiple spermatozoa under the zona pellucida of mature oocytes. Without detecting the appearance of pronuclei, the microinjected mouse oocytes containing two polar bodies were cultivated to the blastocyst stage and then analyzed cytogenetically. A chromosome study was carried out in a total of 109 blastocysts derived after microinjection of motile spermatozoa into the perivitelline space. Fifty-five blastocysts (50.5%) exhibited normal diploid chromosome complements, 30 (27.5%) showed different forms of mosaicism, and 24 (22%) exhibited haploidy caused by parthenogenetic activation. Compared to in vivo and in vitro control groups there was a significant increase in the parthenogenesis and mosaic forms of embryos produced by micromanipulation (P < 0.001). A total of 360 well-spread metaphases of 103 blastocysts was analyzed to determine whether the micromanipulation procedure increased the chance of aneuploidy. Aneuploid numbers of chromosomes were absent in all the metaphases analyzed. Mosaicism and parthenogenesis appear to be increased significantly following microinjection of multiple spermatozoa under the zona pellucida of mouse oocytes, and there was no evidence of aneuploidy.

Isolated choroid plexus cysts in the second-trimester fetus: is amniocentesis really indicated?

Choroid plexus (CP) cysts have been associated with trisomy 18, although most fetuses with CP cysts are normal. Since many fetuses with trisomy 18 have other sonographic abnormalities, the necessity of obtaining a karyotype for all fetuses with isolated CP cysts remains controversial. The authors prospectively studied 234 second-trimester fetuses with sonographically discovered CP cysts. Two hundred twenty of them had no other sonographic findings. None of these 220 normal fetuses had evidence of aneuploidy at amniocentesis or an anomaly at birth. Fourteen fetuses had major anomalies detected in utero: 11 had trisomy 18, one had triploidy, and two had normal karyotypes but were structurally abnormal. While size and bilaterality of the CP cysts were not helpful in predicting aneuploidy, the meticulous anatomic survey of fetuses with CP cysts allowed successful identification of all aneuploid fetuses. These data show that the yield of abnormal karyotypes in fetuses with isolated CP cysts is low and may not justify the risk of amniocentesis.

Flow cytometric analysis of cellular deoxyribonucleic acid content of nine renal oncocytomas

The cellular deoxyribonucleic acid content of formalin-fixed, paraffin-embedded tissue from nine renal oncocytomas was determined using an Epics-C flow cytometer. The tissues were all from surgical specimens, and all neoplasms met stringent light and electron microscopic criteria for the diagnosis of oncocytoma. Thirteen samples of pure tumor, 5 of tumor and normal kidney, and 3 of normal kidney alone were analyzed. All oncocytomas had single, sharp G1 peaks with no evidence of aneuploidy, and this peak was similar to that of normal renal parenchyma. This was true even for three oncocytomas that had multiple major chromosomal anomalies on karyotyping. These data support the concept that renal oncocytomas are euploid neoplasms, consistent with their benign biologic behavior. The apparent discrepancy between the results of flow cytometry and karyotyping is discussed.

Flow cytometric DNA analysis of parathyroid tumors. Implication of aneuploidy for pathologic and biologic classification

The previous cytometric studies on parathyroid tumors have provided conflicting data regarding the relationship between DNA content and histopathology, resulting from differences in technical methods and data analysis. This study measured nuclear DNA of parathyroid tumors by flow cytometry in fresh material and determined whether DNA aneuploidy really assists in making a pathologic diagnosis of carcinoma or not. From May 1987 through April 1989, 65 consecutive patients operated on for primary hyperparathyroidism had DNA analysis of the freshly excised parathyroid tumors. Three of the patients had metastatic lesions of parathyroid carcinoma in the lung, cervical lymph nodes, and lung and mediastinal lymph nodes, respectively. Pathologic classifications of the lesions from the other 62 patients were 54 adenomas, four carcinomas, and four hyperplasias. In all the latter patients, hyperplasia was associated with a multiple endocrine neoplasia syndrome. Unequivocal evidence of aneuploidy was found in all of the metastatic lesions and 60% of the primary lesions of the carcinomas, in 9% of the adenomas and in 50% of the hyperplasias. Therefore, parathyroid carcinomas were more apt to be aneuploid than were adenomas (P = 0.0015, both-sided testing). In each of the cases of aneuploid hyperplasia, a small aneuploid peak was found. The high incidence of aneuploidy in patients with multiple endocrine neoplasia type 1 may indicate the presence of clonal heterogeneity of hyperplastic glands and the presence of an abnormal subset of cells that have malignant potential. Cell distribution analysis did not provide any significant information beyond ploidy level. In conclusion, DNA flow cytometric analysis of DNA ploidy patterns is a valuable adjunct to the histopathologic diagnosis of parathyroid neoplasms.

Use of Nurse Cultures to Select for Papaver somniferum Cell lines Capable of Enhanced Sanguinarine Production

Summary A nurse culture technique was used in which cell lines of Papaver somniferum capable of producing 12 % sanguinarine (dry weight basis) have been isolated. Cell aggregates composed of 4 to 16 cells were removed from suspension cultures and inoculated on a sterile cell support located on top of the actively growing callus. Approximately 25 % of the cultured aggregates developed into callus cultures, and nearly all of these were resuspended successfully. Up to a 30-fold range in the sanguinarine yields from these cell lines was observed, indicating that considerable cell variation was present in the cells of the original parental line. Evidence of aneuploidy was observed, which may explain this cellular variation. This report provides proof that aggregate selection is a valuable tool in increasing the yields of secondary metabolites in plant cell cultures.

Flow Cytometric Analysis of Tumour dna Profile Related to Response to Radiotherapy and Survival in Inoperable Lung Cancer

DNA flow cytometric analysis was done from 100 paraffin-embedded non-small cell (NSCLC) and 29 small cell lung carcinomas (SCLC). Most of the patients had locally advanced disease and all were considered inoperable and treated by radiotherapy. DNA aneuploidy was detected in 62 (62%) of the NSCLCs and in 20 (69%) of the SCLCs. Radiotherapy induced a slightly more extensive and prolonged tumour regression in DNA-aneuploid NSCLC with high S-phase fraction (SPF) than in those with low SPF and no evidence of aneuploidy. However, neither in NSCLC nor in SCLC were significant survival differences detected between DNA-diploid and DNA-aneuploid cases or between those with low ( 12%) S-phase fraction (SPF). Neither did the degree of differentiation have prognostic significance in this material.