Anwar N. Mohamed

Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells

The MCF10 series of cell lines was derived from benign breast tissue from a woman with fibrocystic disease. The MCF10 human breast epithelial model system consists of mortal MCF10M and MCF10MS (mortal cells grown in serum-free and serum-containing media, respectively), immortalized but otherwise normal MCF10F and MCF10A lines (free-floating versus growth as attached cells), transformed MCF10AneoT cells transfected with T24 Ha-ras, and premalignant MCF10AT cells with potential for neoplastic progression. The MCF10AT, derived from xenograft-passaged MCF10-AneoT cells, generates carcinomas in ∼25% of xenografts. We now report the derivation of fully malignant MCF10CA1 lines that complete the spectrum of progression from relatively normal breast epithelial cells to breast cancer cells capable of metastasis. MCF10CA1 lines display histologic variations ranging from undifferentiated carcinomas, sometimes with focal squamous differentiation, to well-differentiated adenocarcinomas. At least two metastasize to the lung following injection of cells into the tail vein; one line grows very rapidly in the lung, with animals moribund within 4 weeks, whereas the other requires 15 weeks to reach the same endpoint. In addition to variations in efficiency of tumor production, the MCF10CA1 lines show differences in morphology in culture, anchorage-independent growth, karyotype, and immunocytochemistry profiles. The MCF10 model provides a unique tool for the investigation of molecular changes during progression of human breast neoplasia and the generation of tumor heterogeneity on a common genetic background.

Cytogenetics of primary prostatic adenocarcinoma: Clonality and chromosome instability

We have examined 62 prostatic adenocarcinomas by conventional cytogenetic analysis. Most were primary cultures harvested in 14 days or less. The most consistent finding was a normal male diploid karyotype, found in 87% of all cells analyzed, and as the exclusive finding in 19 tumors. Nonrandom chromosomal changes included gain of chromosome 7 and loss of the Y chromosome. In addition, clonal gains of chromosomes 8, 12, and 18, and clonal losses of chromosomes 14 and 19 were noted in individual cases. Two structural clonal aberrations, a 9p+ in one case and a t(Y;22) (q11.2;p12) in another, were also seen. Ten of 62 cultures demonstrated chromosome instability, defined herein as nonclonal gain or loss of chromosomes in more than 10% of the metaphases examined from that culture. In those cases with nonclonal numerical aberrations, loss of chromosomes was more common than gain. The distribution of apparently random numeric abnormalities was similar to that of the clonal abnormalities in that the most frequent nonclonal gain was of chromosome 7 and the most frequent nonclonal loss was of the Y chromosome. Apparently random structural aberrations were observed in less than 1% of all analyzed cells. These included a 4p-,del(3)(q13), and t(1;11). The extent of apparently random aneuploidy suggests that chromosome instability characterizes cultured prostatic adenocarcinomas. An increase in the frequency of nonclonal aberrations may be an indicator of tumor origin in a predominantly diploid cell population. The coexistence of clonally aberrant, nonclonally aberrant, and normal diploid cells in culture may reflect heterogeneity of prostate tumors in vivo.

Chromosomal analyses of 52 cases of follicular lymphoma with t(14;18), including blastic/blastoid variant.

We have identified 52 patients of follicular lymphoma (FL) with t(14;18)(q32;q21). Histologically, the lymphomas were placed into six groups according to their cellular composition and growth pattern. Chromosome analysis revealed that all cases but one had additional secondary chromosomal abnormalities. The most frequent numerical aberrations were gains of chromosomes 7 (38%), X (36%), 5 (15%), 12 (15%), 18/der(18)t(14;18) (25%), and 21 (15%). Structural abnormalities of chromosome 1 were seen in 19 tumors (36%) affecting both arms with breakpoints clustered at 1p36. Other structural abnormalities included partial deletions of 6q, 10q, and 13q. Breakpoint at 8q24 was seen in four cases. The chromosome aberrations were correlated with the morphological subtypes of follicular lymphoma. Gain of chromosome 7 appeared to be associated with follicular large cell lymphoma. The incidence of trisomy 5 and 12, and 13q- was higher in follicular lymphoma with aggressive histological features than in low-grade lymphoma. In addition, complexity of the karyotype and high degree of polyploidy increased with the grade. The most valuable cytogenetic markers in the t(14;18) lymphomas are those involving 8q24 which was found exclusively in the blastic/blastoid variant FL. Therefore, chromosome analysis in relation to histologic pattern of follicular lymphoma can provide additional information in predicting tumor evolution and transformation to a higher-grade malignancy.

Evaluation of MYC and chromosome 8 copy number in breast carcinoma by interphase cytogenetics.

We used fluorescence in situ hybridization (FISH) to determine MYC and chromosome 8 copy number on whole nuclear imprint preparations of 24 breast carcinomas, seven benign breast samples, and two phyllodes tumors. None of the benign tissues and neither of the phyllodes tumors demonstrated an increased copy number for MYC or chromosome 8, which was defined as greater than two signals in > 10% of nuclei. In contrast, 22 of 24 carcinomas demonstrated an increased MYC copy number. The modal numbers of MYC copies/nucleus were 0–2 in seven cases (29%), 3–5 in seven cases (29%), 6–9 in five cases (21%), and >9 in five cases (21%). An increased chromosome 8 copy number was observed in 21 of 22 carcinomas with MYC gain, and the modal number of signals/nucleus was either identical to (n = 14; 64%) or less than (n = 8; 36%) the number of MYC copies. The number of MYC copies correlated with cellular DNA content, as determined by using flow cytometry. In peridiploid tumors (DNA index 0.9–1.2; n = 7), the MYC copy numbers/nucleus were 0–2 in five cases and 3–5 in two cases. In contrast, the modal MYC copy numbers/nucleus among the 11 hyperdiploid tumors (DNA index 1.3–1.9) were 0–2 in one case, 3–5 in four cases, 6–9 in five cases, and >9 in one case. All three tetraploid/hypertetraploid carcinomas exhibited >9 MYC copies/nucleus. We conclude that an increased MYC copy number, as detected by using interphase cytogenetics, is extremely frequent in human breast carcinomas. However, in most cases, MYC gene duplication is probably secondary to polysomy of chromosome 8 and/or genomic endoreduplication (i.e., DNA aneuploidy). Genes Chromosom. Cancer 18:1–7, 1997.

Blastic natural killer cell lymphoma/leukemia: A report of seven cases

Only a few blastic natural killer (NK) cell leukemias and lymphomas have been reported. As such, the clinicopathologic spectrum of this disease is incompletely understood. We report 7 cases of blastic NK cell lymphoma/leukemia. All patients were men, 5 white and 2 Arab American. All cases exhibited blastic morphologic features and were CD3- and CD56+ with germline T-cell receptor genes. Five cases were CD4+ and involved the skin. Both CD4- cases never involved the skin. Other markers of mature NK cells such as CD16, CD57, and TIA-1 were expressed infrequently. Three cases were CD33+. One CD33+ case had a clonal rearrangement of the immunoglobulin heavy chain gene. Skin and lymph nodes were involved most often, with frequent evolution to a leukemic phase. Initial responses to therapy were achieved in most patients, but the tumors invariably recurred.

CD56+/CD4+ Lymphomas and Leukemias Are Morphologically, Immunophenotypically, Cytogenetically, and Clinically Diverse

CD56, a neural adhesion molecule, is a marker of natural killer (NK) lymphocytes as well as a subgroup of CD8+ T cells. Normal lymphocytes with a CD56/CD4 phenotype are scarce. Physiologic increases may occur in patients with immunosuppression, chronic inflammation, and autoimmune disorders. We report 4 cases of lymphomas/leukemias with the unusual CD56/CD4 phenotype. Two were of T-cell and 2 of true NK-cell origin. The T-cell lymphomas had large granular lymphocyte morphologic features and splenomegaly. One patients had a benign course; the other died within months of the leukemia diagnosis. The 2 NK cell lymphomas had blastic morphologic features, initially involved skin, and had a very aggressive clinical course; 1 patient died of acute leukemia, and 1 had recurrence after bone marrow transplantation. Cytogenetic analyses did not show a consistent pattern of abnormalities. The NK lymphoma with acute leukemia had a t(2;5) but was CD30- and anaplastic lymphoma kinase negative. Although CD56+/CD4+ lymphomas/leukemias are a heterogeneous group, there may be a distinct subgroup of NK lymphoblastoid lymphomas of the skin, judging from our cases, as well as those previously reported.

Establishment of a Human Pancreatic Tumor Xenograft Model : Potential Application for Preclinical Evaluation of Novel Therapeutic Agents

Adenocarcinoma of the pancreas is currently the fifth leading cause of death in the United States. It remains generally incurable by available treatment modalities. We report here on the characterization of a permanent pancreatic cell line (KCI-MOH1), established as a xenograft in severe combined immune deficient (SCID) mice, from a 74 year-old African American male patient diagnosed with pancreatic cancer. Sections from paraffin-embedded tumors excised from SCID mice revealed typical adenocarcinoma of the pancreas. Karyotypic analysis of cultured cells derived from tumors grown in SCID mice revealed a male karyotype with multiple clonal aberrations: 42, XY, add (3)(p11.2), der(7) t(7;12) (p22;q12), -10, -12, add (14)(p11), -18, add (20)(q13)-22/84, idemx2. Immunostaining of KCI-MOH1 tissues shows strong expression of p53 and p21 proteins. The xenograft model was established by transplanting the KCI-MOH1 cells subcutaneously (s.c.) in SCID mice. When the s.c. tumor was transplanted in vivo to other SCID mice, the success rate was 100%, with a doubling time of 8.5 days. The SCID mouse xenograft model was used to test the efficacy of selected standard chemotherapeutic drugs (taxol, gemcitabine, 5-fluorouracil, and Ara-C) and novel biological agents (Bryostatin 1 and Auristatin-PE). Results show that gemcitabine, Ara-C, and Bryostatin 1 were active against KCI-MOH1. The xenograft described herein can be used as an animal model to facilitate the development of novel therapeutic agents against human pancreatic cancers.

Differential growth factor responses of epithelial cell cultures derived from normal human prostate, benign prostatic hyperplasia, and primary prostate carcinoma

Because of a lack of information of the optimum nutritional requirements, epithelial cells derived from normal human prostate and prostate tumors have been difficult to propagate in vitro, which hinders research in prostate carcinogenesis. In an effort to establish optimum nutritional conditions and differences in growth characteristics of normal human prostate (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA), we have compared the effects of several growth factors on cell proliferation and elucidated growth properties of low passage epithelial cells derived from NP, BPH, and PCA of an African‐American patient. Primary and low passage cultures were propagated in serum‐free keratinocyte basal medium (KBM) supplemented with insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (BPE; 50 μg/ml), cholera toxin (10 ng/ml), and antibiotics. Almost all NP, BPH, and PCA cells were positive for cytokeratins and prostate‐specific antigen (PSA). The NP, BPH, and PCA cells were essentially diploid and lacked mutations in c‐K‐ras and c‐Ha‐ras oncogenes, and p53 tumor suppressor gene. However, they exhibited progressively accelerating growth parameters. The population doubling times of NP, BPH and PCA were 51 hr, 37 hr, and 29 hr, respectively; their saturation densities were 2.9 × 104/cm2, 3.3 × 104/cm2, and 7.2 × 104/cm2, respectively. The NP and BPH cells required all of the growth factors in the medium, as deletion of any one of the above factors strongly inhibited their growth. The PCA cells, however, were independent of EGF and hydrocortisone. PC‐3, an established human prostate cancer cell line, was independent of the growth factors tested. Fetal bovine serum (FBS) inhibited the growth of NP, BPH and PCA cells. In contrast, FBS stimulated the growth of the PC‐3 cells in a concentration‐dependent manner. These results indicate that in the absence of any apparent karyotype alterations and mutations in c‐K‐ras, c‐Ha‐ras and p53 genes, epithelial cells derived from NP, BPH, and PCA exhibit significant differences in their growth properties and responses to growth factors. These variations may represent early changes involved in prostate cancer, while gene mutations and cytogenetic alterations occur in advanced and/or metastatic tumors.

Blastic mantle cell lymphoma associated with Burkitt-type translocation and hypodiploidy

Two elderly men with stage IV mantle cell lymphoma (MCL) rapidly developed a leukaemic phase with unusually high blast counts that proved fatal. One lymph node biopsy showed diffuse MCL, the other blastic morphology. In addition to t(11;14), there were t(8;14) and t(1;19) in case 1 and dup(8)(q24q13) in case 2. Fluorescence in situ hybridization revealed genomic fusion of IgH/MYC genes in case 1 and an extra copy of C‐MYC gene in case 2. The genomic alteration of C‐MYC oncogene is probably implicated in the blastic transformation and aggressive behaviour of the disease.

Isochromosome 8q formation is associated with 8p loss of heterozygosity in a prostate cancer cell line.

In advanced prostate cancer, loss of chromosomal regions on 8p is frequently associated with gain of 8q. We studied the gross chromosomal abnormalities associated with 8p loss of heterozygosity (LOH) in the prostate tumor cell line 1542 CP3Tx. The cell line was previously established from a primary prostatic adenocarcinoma by immortalization with a recombinant retrovirus carrying the E6 and E7 genes of human papilloma virus type 16. Allelotyping studies demonstrated LOH at multiple markers on 8p.