A codon 891 exon 15 RET proto-oncogene mutation in familial medullary thyroid carcinoma: a detection strategy.

A ser891ala RET proto-oncogene mutation has been previously discovered in a single kindred with familial medullary thyroid carcinoma (FMTC). An additional two probands with this mutation and with medullary thyroid carcinoma (MTC) without any other manifestations of MEN 2 have been identified. In one of thse families, two other individuals also had the mutant sequence and FMTC. Analysis of both cases showed cosegregation of the mutation and MTC. To facilitate detection of this mutation, a primer was engineered which creates a Hha I recognition site in the presence of the mutant sequence. As a result, this codon 891 exon 15 mutation can be identified with a restriction enzyme digestion.

Mutational analysis of the GDNF/RET-GDNFR alpha signaling complex in a kindred with vesicoureteral reflux.

Glial cell line-derived neurotrophic factor (GDNF) mediates signaling across the cell membrane by interaction with the RET-GDNFR alpha receptor complex. We identified a family in which one member had medullary thyroid carcinoma (MTC) and four members had vesicoureteral reflux (VUR). Knowledge that mutations in the RET proto-oncogene cause MTC and studies documenting genitourinary abnormalities in RET or GDNF knockout mice led us to examine the GDNF/RET-GDNFR alpha signaling complex in this family. RET and GDNF were excluded as the causative VUR gene by haplotype and sequence analysis. The GDNFR alpha gene was mapped to chromosome 10q25-26 by radiation hybrid techniques and was eliminated as the causative gene by haplotype analysis and sequencing of cDNA from an obligate carrier. Sequencing identified a 15-nucleotide deletion in GDNFR alpha mRNA, which was found to code for a single exon; analysis of several cell types revealed an identical mRNA form, indicating that this variant is a product of alternative RNA processing. We conclude that GDNFR alpha maps to 10q25-26 and that its RNA transcript is alternatively processed. Mutation abnormalities in the GDNF/RET-GDNFR alpha signaling system do not cause VUR in this family.

Relevance of RET proto-oncogene mutations in sporadic medullary thyroid carcinoma.

Analysis of peripheral blood or tumor DNA samples from 101 patients with apparent sporadic medullary thyroid carcinoma (MTC) was performed to assess the frequency of RET proto-oncogene mutations in this patient population. Peripheral blood and/or tumor DNA was amplified by polymerase chain reaction. DNA sequence or restriction enzyme analysis was performed to detect mutations of RET proto-oncogene codons 609, 611, 618, 620, 634, 768, and 918. Six of 101 patients with apparent sporadic MTC had peripheral blood DNA mutations more commonly associated with hereditary MTC. In 4 patients, these mutations led to the identification of previously unrecognized kindreds. The remaining 2 patients were examples of de novo mutations. A codon 918 mutation was found in 14 of 57 (approximately 25%) tumor DNA samples. Mutations were not identified in the remaining patients. In this large cancer center population, approximately 6% of patients with sporadic MTC carry peripheral blood DNA mutations, either inherited or de novo, more commonly associated with MEN 2A or familial MTC. Seven additional gene carriers were identified as a direct result of these studies, a 2-fold multiplying effect. We conclude routine application of RET proto-oncogene testing should be included in all cases of apparent sporadic MTC.

Treatment of uveal melanoma metastatic to the liver: a review of the M. D. Anderson Cancer Center experience and prognostic factors.

BACKGROUND: Liver metastasis develops in approximately two-thirds of patients with recurrent uveal melanoma. Despite therapy, the median survival of those with liver metastasis is 5 to 7 months. The recognition of a grave prognosis associated with liver metastasis has led to evaluation of new modalities of therapy, including the use of regional therapies such as intrahepatic arterial chemotherapy and either embolization or chemoembolization of hepatic metastases. In this study, the results of an institutional experience over the past 2 decades are reviewed and prognostic factors that affect survival from the time the liver metastasis is diagnosed are assessed. METHODS: In this study of 201 patients with uveal melanoma involving the liver who were treated at M. D. Anderson Cancer Center between 1968 and 1991, the authors restrospectively reviewed the cases and compared the results of systemic therapies, hepatic intra-arterial chemotherapies, and chemoembolization of liver metastases. Cox's multivariate analysis and stepwise logistic regression were then computed to determine significant prognostic variables. RESULTS: The systemic therapies produced a response rate of less than 1%. Chemoembolization was the most effective treatment, inducing responses in 36% of patients. Survival curves were calculated using the life-table method of Kaplan and Meier. Patient- and tumor-related characteristics were examined and their relation to on survival from the time of diagnosis of liver metastasis was determined. Levels of serum alkaline phosphatase, total bilirubin, and lactic dehydrogenase plus response to treatment showed a strong relation to survival. In contrast, univariate analysis showed that patient age and gender, metastasis free interval, presence of extrahepatic metastasis, and type of therapy for liver metastasis did not influence survival. Multivariate stepwise regression analysis identified serum alkaline phosphatase and metastasis free interval as the main independent prognostic factors for survival after liver metastasis diagnosis. CONCLUSIONS: Of the three modalities of therapy used for choroidal melanoma metastatic to the liver, only chemoembolization using cisplatin-based regimens produced a meaningful response rate. Information from this analysis can be used to predict the outcome of patients with uveal melanoma metastatic to the liver. Patients with metastatic ocular melanoma confined to the liver should be treated with chemoembolization and should not be included in chemotherapy trials designed for cutaneous melanoma.

Clinical use of molecular information in the management of multiple endocrine neoplasia type 2A.

One hundred and ninety-seven members of 28 kindreds with multiple endocrine neoplasia type 2A (MEN 2A) were screened for RET proto-oncogene exon 10 and 11 mutations. Seventy-one known affected individuals had mutations of codons 609, 618, 620 or 634, whereas 53 unaffected individuals had no abnormalities. Nineteen out of 54 individuals of unknown status, mostly children, had RET mutations. Four of these children had thyroidectomy based on this analysis and were found to have C-cell abnormalities. We identified one false negative mutation analysis because of a codon 691 polymorphism. We conclude that RET mutational analysis is a cost-effective and accurate method for determination of gene carrier status in MEN 2A.

High-stage endometrioid carcinoma of the ovary. Prognostic significance of pure versus mixed histologic types.

We retrospectively reviewed 80 cases of endometrioid carcinoma of the ovary: 68 pure endometrioid tumors and 12 predominantly endometrioid carcinomas (> 50%) mixed with either papillary serous or undifferentiated carcinoma. Each group had 11 cases of stage III or IV tumors, which were studied to determine whether the overall prognosis was affected by grade, histology, number of mitoses, residual tumor after surgery, and patient's age. Prognosis was significantly affected only by a mixed histologic pattern. The 5- and 10-year survival rates and the median survival time for pure endometrioid carcinomas were much better than those for mixed endometrioid carcinomas (63% and 45% and > 86 months versus 8% and 0% and 18 months, respectively). Recurrent tumors in cases of mixed endometrioid carcinoma were pure serous or undifferentiated carcinomas, whereas those in cases of pure endometrioid carcinoma were either endometrioid or high-grade carcinoma. Our results show that the presence of even a small component of serous or undifferentiated carcinoma in an otherwise predominantly endometrioid carcinoma significantly affects the prognosis. Thus pathologists should thoroughly sample all endometrioid carcinomas, especially high-stage tumors, to ensure that no serous or undifferentiated component is present.

Therapy of relapsed or refractory low-grade follicular lymphomas: factors associated with complete remission, survival and time to treatment failure.

Patients with relapsed low-grade follicular lymphomas (LGFL) frequently respond to subsequent therapy and can have long survival, but are rarely cured. Factors associated with complete remission (CR) rate, length of survival, and time to treatment failure (TTF) after relapse are not well known. We assessed such factors by multivariate analysis in a retrospective review of 95 patients with relapsed LGFL treated with investigational chemotherapy regimens at our institution. The CR rate after therapy was 22%; the likelihood of achieving CR was inversely associated with the number of previous treatment failures (P less than 0.001) and serum LDH level (P less than 0.05). Both the presence of constitutional symptoms and a history of more than two previous treatment failures were associated with shortened survival and TTF. Hemoglobin level was also significantly associated with survival. Prognostic models for survival and TTF were derived to define patient groups with different projected outcomes after therapy for relapsed disease. The results of this study can be used to select patients for new investigational treatments and to evaluate the outcome of such therapies.

Neuroendocrine tumors of the nasal cavity: an ultrastructural and morphometric study of 24 cases.

A series of 24 small cell nasal tumors with neuroendocrine differentiation was studied by electron microscopy, and the numbers of secretory granules and cell junctions were assessed. To investigate the relationship between the extent of dendrite formation and the behavior of the tumors, the size of the tumor cells and the area occupied by dendritic processes were determined for each tumor by morphometric analysis performed on low-magnification electron micrographs. A positive correlation was demonstrated between the dendritic area index and the survival of the patients (P = 0.017). Neither the number of secretory granules nor the frequency of cell junctions was prognostically significant.

Evidence for somatic cell expression of the c-mos protein [corrected].

The c-mos protein has been found to be enriched in germ cells of male mice, as described in a recent report from this laboratory (Herzog et al., Oncogene 3, 225, 1988). We report on further studies which indicate that the c-mos protein (a 41 to 43 kDa protein termed p43c-mos) is expressed in somatic tissues of mice and in cells grown in culture. In testes of mice, germ cell fractions have increased levels of p43c-mos relative to other cells of the testes. However, non-germ cells harbor significant levels of p43c-mos, as judged by comparison of testes from normal mice to those with mutations that affect the germ cell content of the testes. Thus, homozygous S1, at, and the W/Wv mutant mice are sterile due to severe deficiencies of germ cells. Such mice had only an estimated 50%-60% reduction in p43c-mos as judged by western immunoblotting using two different site-directed anti-mos antibodies. Similarly, X/X-sex reversed mice in which germ cells die after 10 days of age had only an 85% reduction of p43c-mos in mice 35 days of age. Thus, the germ cell content of testes did not correlated with p43c-mos levels in this tissue. Direct analyses of non-germ cells derived from mouse testes confirmed these findings, since Sertoli and Leydig cell lines grown in culture expressed p43c-mos. In addition, tissues such as kidney, liver, spleen and brain were found to contain p43c-mos. Surprisingly, mouse NIH3T3 cells were found to express significant levels of the c-mos protein based upon immunoblotting and one-dimensional peptide mapping experiments performed with both anti-mos antibodies. The concentration of the c-mos protein was not affected by expression of viral mos proteins. We conclude that the c-mos protein is enriched in male germ cells, but p43c-mos is also expressed in significant amounts in somatic tissues and in fibroblastic cells grown in culture.

Analysis of risk factors for fatal hemorrhage during induction therapy of patients with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL), an uncommon subtype of acute myelogenous leukemia (AML), has a high incidence of early hemorrhagic death during induction therapy; however, patients with APL surviving induction and obtaining complete remission have a good prognosis, with a longer remission duration than other subtypes of AML. We sought to determine if changes in selected clinical and laboratory coagulation parameters during induction therapy were significantly associated with fatal hemorrhage in that week, or were predictive of fatal hemorrhage in the following week. The first six weeks of induction therapy were studied in 65 patients, during which time 23 patients (35%) died from hemorrhagic complications. Time intervals analyzed were week 1, week 2, weeks 3 and 4, and weeks 5 and 6. There was no significant difference in the prevalence of severe hypofibrinogenemia (less than 150 mg%), prolongation of the prothrombin time (greater than 14 s), or presence of infection between patients who sustained fatal hemorrhage versus those who did not, for any given time period. The most significant parameter was the inability to obtain a posttransfusion platelet count of over 40,000/microliters in at least one half of the platelet transfusions administered in that week (p less than 0.001). This last parameter was the only variable that was also significantly associated with an increased risk of fatal hemorrhage for the following week (p less than 0.005). Clinical trials investigating management of coagulopathy in APL should evaluate the effect of therapy on response to platelet transfusion as well as on other laboratory parameters.

Evaluation of drugs for elimination of leukemic cells from the bone marrow of patients with acute leukemia.

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4-hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.

T-cell involvement in benign phase chronic myelogenous leukemia.

T cells from the peripheral blood of patients with chronic myeloid leukemia (CML) were cultured with phytohemagglutinin and T-cell growth factor (TCGF) in agar culture. These T-cell colonies were pooled and expanded further in liquid culture with TCGF and then simultaneously analysed for the E-rosette receptor with the monoclonal antibody OKT11 and for the presence of the Philadelphia (Ph1) chromosome. OKT11 analysis showed these populations to be composed 99.5% or more of T cells. In four of the seven patients the T-cell suspension showed 7/50 (14%), 3/36 (8%), 2/34 (6%), and 4/44 (9%) Ph1 metaphases. Furthermore, Ph1 metaphases were demonstrated in T-cell cultures in two patients when bone marrow metaphases simultaneously showed 90 and 100% Ph1 negative metaphases secondary to human leukocyte interferon therapy or combination chemotherapy. A minority of T cells in benign phase CML have the Ph1 abnormality despite reduced number of Ph1 metaphases in bone marrow from therapy.