High prognostic impact of growth fraction parameters in advanced stage laryngeal squamous cell carcinoma.

One hundred and two cases of laryngeal squamous cell carcinoma, all treated in the same center with total or supraglottic laryngectomy, bilateral neck dissection and postoperative radiotherapy, were investigated with both Ki67 and MIB-1 monoclonal antibodies. The aim was to determine the prognostic impact of growth fraction markers in a homogeneous series of patients. All samples were stained with Ki67 monoclonal antibody on frozen sections, and with MIB-1 monoclonal antibody on paraffin sections, using the ABC immunoperoxidase method. The percentage of positive cells was compared in each case with the overall and disease-free survival, pathologic stage and histologic grading. The values obtained from Ki67 and MIB-1 counts were similar and highly correlated (r=0.90). Two groups of cases with low and high proliferation rate (59 and 43 respectively) were obtained by splitting up the whole series, on the basis of the median value; 84. 6% of the patients with high proliferation relapsed and/or died due to the tumor within two years from diagnosis whereas, at time of writing, 94% of the patients with low proliferation are alive and well (p<0.00001). No relation was found between growth fraction and histologic grading, pathologic stage (pT and pN) and site of the tumor. Only lymph node involvement was correlated with disease-free survival. Our results indicate that Ki67/MIB-1 index represents an independent variable to determine long-term prognosis in laryngeal squamous cell carcinomas. We recommend its use in diagnostic protocols, to distinguish high risk subsets of patients who might benefit from more aggressive treatments.

Chromosome 8, occupational exposures, smoking, and acute nonlymphocytic leukemias: a population-based study.

In a previous epidemiological study on acute myelocytic leukemia (M. M. Crane et al., Cancer Epidemiol. Biomark. Prev., 5: 639-644, 1996), clonal aberrations in chromosome 8 have been reported to be in excess in smokers and in workers exposed to paints. In that study, cytogenetics was performed after therapy. In our report, we describe a population-based survey on nonlymphocytic leukemias in northern Italy, in which 79 patients (acute myelocytic leukemia, myelodysplastic syndromes, or other nonlymphocytic leukemias) were studied before cytotoxic therapy. We found 9 aberrations involving chromosome 8 (six +8, two -8, and one translocation), whereas abnormalities involving chromosomes 5 and 7 occurred with a low frequency compared with previous studies. Aberrations involving chromosome 8 were associated with smoking (odds ratio, 6.3; 95% confidence interval, 0.9-42.3; among smokers of 10 or more cigarettes/day: odds ratio, 14.2; 95% confidence interval, 1.4-142.3); +8 aberrations were found in 1 of 24 nonsmokers and in 5 of 38 smokers. Three +8 aberrations were found in 22 subjects potentially exposed to solvents or polycyclic aromatic hydrocarbons. The low frequency of chromosome 5 and 7 aberrations in our population-based series (compared with other studies) can be attributed to the recruitment before cytotoxic therapies. Aberrations involving chromosome 8 (particularly +8) were associated with smoking habits. Chromosome 8 includes the c-myc oncogene.

Human herpesvirus type 7 in Hodgkin's disease.

Several lines of evidence have pointed to the involvement of a viral agent in the pathogenesis of Hodgkin's disease (HD). Therefore we investigated the presence of human herpesvirus type 7 (HHV-7) in 53 cases of HD by polymerase chain reaction (PCR), DNA in situ hybridization (ISH) and immunohistochemistry. HHV-7 DNA was frequently detected (68% of the cases) in HD biopsies by PCR independently of the histological type, whereas only 32% (P<0.05) of positive cases were found in 19 reactive lymph nodes. However, by applying the quantitative PCR technique, the majority of the samples showed a low level of viral load. Moreover, ISH for HHV-7 DNA was positive in a low number of small T lymphocytes and consistently negative in Hodgkin and Reed-Sternberg (HRS) cells, which appeared negative for HHV-7 also at immunohistochemistry. These results indicate that the high frequency of HHV-7 infection in HD: (i) is probably non-productive, (ii) mainly involves small lymphocytes belonging to the T-lineage, and (iii) is probably due to the recruitment of non-malignant reactive cells in HD tissue.

Significance of cell proliferation index in assessing histological prognostic categories in Hodgkin's disease. An immunohistochemical study with Ki67 and MIB-1 monoclonal antibodies.

BACKGROUND AND OBJECTIVE: In their review of the Rye histopathological classification of Hodgkin's disease, Bennett and coworkers have proposed that the nodular sclerosis (NS) type should be divided into two diagnostic categories on the basis of their clinical behaviour. In order to evaluate whether the proliferative activity of HD cells might correlate with histology in NS subtypes, we reviewed and re-evaluated cryostat and paraffin-embedded sections from 80 cases sent to our centre from 1986 to 1991. METHODS: In the present study, we investigated the growth cell fraction of 53 cases of Hodgkin's disease with nodular sclerosis by using Ki67 and MIB1 monoclonal antibodies to determine whether proliferative activity is associated with different pathological subtypes and prognostic categories. Eight cases with an interfollicular pattern and 19 with mixed cellularity were also investigated. The results in each group were compared to the others. RESULTS: The values of Ki67 and MIB1 were highly correlated (r = 0.88). In Hodgkin's disease with nodular sclerosis, two groups with significantly different growth fractions were morphologically identified: one with lymphocyte predominance and mixed cellularity subtypes, another composed of cases with variously extensive lymphocyte depletion. The figures were compared with those of interfollicular subtype, which fell into the first group, and of mixed cellularity type, in which the proliferative cell activity was significantly higher than in the second nodular sclerosis group. In all cases, Reed-Sternberg and Hodgkin cells accounted for the majority of the cell growth fraction, although a variable percentage of T-lymphocytes were also Ki67- or MIB1-positive. Taking the median value (15%) of MIB1 positive cells as a cut-off, a significant correlation (p = 0.05) was observed between MIB1 positivity and bulky disease, and a good trend (but not a significant relationship) between MIB1 and overall survival, disease-free survival, staging and the clinical response to therapy. INTERPRETATION AND CONCLUSIONS: Assessment of the growth cell fraction in Hodgkin's disease with different nodular sclerosis patterns provides biological support for the morphological reclassification of their degree of malignancy into two main groups with different impacts on the clinical parameters and a possible relation with the outcome of treatment.

Human herpesvirus 6 and Epstein-Barr virus in Hodgkin's disease: a controlled study by polymerase chain reaction and in situ hybridization.

Human herpesvirus-6 (HHV-6), a T-lymphotropic double-stranded DNA virus highly endemic in human populations, has been suggested to play a possible role in the development of lymphoid neoplasms, especially Hodgkin's disease. To investigate this point, we evaluated the presence and distribution of HHV-6 DNA by Southern blot, nested polymerase chain reaction, and in situ hybridization in a series of lymphoproliferative disorders including 73 Hodgkin's disease cases, 15 non-Hodgkin lymphomas, and 19 reactive lymph nodes. A high prevalence of HHV-6 infection was observed within the Hodgkin's disease category by polymerase chain reaction (38 of 52, 73%) and in situ hybridization (47 of 57, 82.4%); however, a similar prevalence was found in non-Hodgkin's lymphomas (10 of 15, 66.6%) and reactive lymph nodes (13 of 19, 68.4%). In no case did Southern blot detect viral DNA, suggesting that the neoplastic tissue contained a low number of HHV-6 copies. In situ hybridization showed that the HHV-6 positivity was restricted to lymphocytes, whereas Hodgkin and Reed-Sternberg cells were consistently negative. Immunohistochemical staining with specific monoclonal antibodies against viral structural proteins was also negative, indicating the absence of a productive infection. No relationship was observed between HHV-6 positivity and histological type, clinical parameters, and outcome of the disease. In the same series, a high proportion of cases (39 of 52, 75%) showed the presence of the Epstein-Barr virus (EBV) genome by polymerase chain reaction; In situ hybridization for Epstein-Barr-virus-encoded small RNA and immunohistochemical detection of latent membrane protein-1 gave similar results (73.6% of positive cases with both methods). In 54.9% of the cases, both sequences of HHV-6 and Epstein-Barr virus DNA were found, suggesting that a synergism of the two viruses may occur. However, the lack of detectable HHV-6 DNA in Reed-Sternberg and Hodgkin's cells seems to argue against such an interpretation. Based on these results, HHV-6 does not appear to play a specific role in the pathogenesis of Hodgkin's disease.

Biological heterogeneity of diffuse mixed small and large cell non-Hodgkin's lymphomas assessed by DNA flow cytometry and Ki67.

The cell proliferative activity of the clinico-pathologically heterogeneous non-Hodgkin's lymphomas (NHL) included in the intermediate grade F category of the Working Formulation (WF) was investigated. S-phase fraction with flow cytometry on cell suspensions, and Ki67 on frozen tissue sections were performed in 42 F NHL. An avidin-biotin immunocomplex method was used and 1000 cells from 10 representative fields were counted. DNA content, S-phase and Ki67 were also detected in 194 NHL covering the whole spectrum of the WF. DNA content anomalies were found in 52 of 194 NHL. Their incidence, like that of S-phase fraction and Ki67 positive cells, progressively increased from low- to high-grade. A linear correlation was found between Ki67 and S-phase (r = .59). Using the median value of proliferating cells obtained with both procedures as a cut off, two very different groups of lymphomas could be distinguished within a series of 42 F-intermediate NHL: with low and high proliferative cell activity (p < .0001) that were termed F(low) and F(high), respectively. A intermediate group was placed between them. It differed significantly from the others if Ki67 was used but only from the F(high) group if the S-phase fraction analysis was applied. No significant differences were seen when comparing F(low) with the single categories of low-grade NHL and F(high) with H high-grade NHL; no significant differences were found between F(high) and G, and between G and H categories. The existence of distinct groups of NHL in the F category, as defined by biological parameters assessing the cell proliferative activity, indicates that this category includes biologically heterogeneous lymphoma subtypes with different grades of aggressiveness. The results also indicate that the G intermediate category displays proliferation indices similar to those of H high grade category.

Immunoglobulin light chain restriction and clonal rearrangement in nodular paragranuloma.

B-cell clonality was demonstrated in a typical nodular paragranuloma case (NP) by both immunoglobulin (Ig) surface analysis and Ig genes rearrangement studies. On frozen sections, immunostaining for Ig light chain expression revealed a clear-cut predominance of Ig lambda-expressing cells, recognizable as both small lymphocytes and lympho-histiocytic (L&H) cells. Accordingly, molecular analysis of the Ig genes showed a monoclonal rearrangement of the lambda chain gene, although no specific pattern of heavy chain gene rearrangement could be detected by JH analysis. The C lambda rearranged band was identified with two different restriction enzymes, excluding the hypothesis of a genomic polymorphism. Furthermore, the C kappa gene was almost completely deleted, indicating that the developmental hierarchy of Ig genes rearrangement has been respected. The molecular pattern of the C lambda hybridizing band was consistent with monoallelic rearrangement of almost the entire DNA sample, indicating that clonal proliferation was not limited to L&H cells, but also involved surrounding lymphocytes. This finding is in keeping with the immunohistochemical evidence of a lambda light chain restriction on both L&H cells and small lymphocytes, pointing to a close relationship between these two cell types. Our results as a whole suggest that L&H cells and B lymphocytes share a common origin and may both be involved in clonal proliferation in NP.

Can Ki67 immunostaining predict response to radiotherapy in oral squamous cell carcinoma?

AIMS: To determine whether immunohistochemical evaluation of the abatement of proliferating cells after a first course of radiotherapy could predict the final response to treatment in oral squamous cell carcinoma (SCC). METHODS: Frozen sections from 31 cases of histologically confirmed oral SCC were stained with the monoclonal antibody Ki67 at diagnosis and after 10 Gy of radiotherapy. The percentage difference of Ki67 positive cells among the biopsy specimens taken at the beginning and after 10 Gy was correlated with the clinical response obtained at the end of the treatment and its significance determined. RESULTS: The percentage of Ki67 positive cells at diagnosis had no significant correlation with the final therapeutic result of radiotherapy. By contrast, the 32% difference of proliferating cells after 10 Gy of radiotherapy significantly differentiated responders from non-responders (p < 0.05). Furthermore, the abatement of the growth fraction after 10 Gy of radiotherapy was significantly correlated with the complete response (p < 0.01). CONCLUSIONS: These data show that the immunohistochemical evaluation of the abatement of Ki67 positive cells after 10 Gy of radiotherapy provides an independent variable of responsiveness to radiotherapy, allowing a reliable prediction of the final therapeutic result to be made.

Heterogeneous immunoglobulin gene rearrangement in a B-chronic lymphocytic leukemia progressing into non-Hodgkin lymphoma (Richter syndrome).

BACKGROUND: The relationship between chronic lymphocytic leukemia (CLL) and supervening non-Hodgkin lymphoma is debated, as is whether a particular genomic pattern is related to the emergence of the terminal lymphoma. To investigate these features, the molecular organization of the immunoglobulin (Ig) gene region in a case during both the B-CLL and Richter transformation phase was studied. METHODS: B-CLL and non-Hodgkin lymphoma cells were processed for Southern blot analysis of Ig heavy- and light-chain gene configuration. RESULTS: Molecular studies of B-CLL cells revealed the presence of a single Ig heavy-chain rearrangement with both kappa and lambda light-chain rearranged genes, which was consistent with the occurrence of multiple mutational events during the development of the B-CLL clone. Molecular analysis of the lymphoma DNA showed new Ig heavy- and kappa light-chain rearrangements in addition to the original ones related to the CLL phase, indicating that the lymphoma tissue consisted of two genotypically distinct populations of cells. CONCLUSIONS: On the basis of the overall molecular configuration, this heterogeneous pattern of Ig gene rearrangement was interpreted as an inherent genetic instability of the CLL clone, in which multiple mutational events allowed a selective pressure toward more aggressive subclones, resulting in the emergence of the terminal lymphoma.

Transient cytogenetic relapse in a Ph1-positive chronic myelogenous leukemia patient previously treated with alpha-interferon.

Therapy with alpha-interferon (IFN alpha) can suppress the Ph1-positive hemopoiesis in a percentage of patients with chronic myelogenous leukemia (CML). We used IFN alpha to treat a 30-year-old CML patient, characterized by favourable prognostic signs (such as low leukocytosis, absence of splenomegaly and no increase in bone marrow blasts) at diagnosis, and obtained a complete remission, as evaluated by Southern blot and cytogenetic analysis, after one year of treatment. However, the polymerase chain reaction (PCR) revealed the persistence of a minimal residual disease. The IFN alpha therapy was stopped and the hematological status remained stable until eighteen months later, when a cytogenetic analysis revealed the appearance of a clone characterized by t(9;22) and trisomy 8, accounting for 30% of bone marrow metaphases. This cell population spontaneously regressed in the following months, before any cytotoxic treatment. However, as leukemic cells, detected by PCR, were still present, the patient received a high dose chemotherapy, which induced the complete eradication of the Ph1-positive clone, as demonstrated by the absence of bcr-abl transcript at the PCR reaction. Molecular and cytogenetic remission persist one year later, without any further therapy.

Extra translocation +der(1q9p) is a prognostic indicator in myeloproliferative disorders.

An identical extra derivative chromosome resulting from a translocation between the long arm of chromosome 1 and the short arm of chromosome 9, +der(1q9p), has been observed in three patients with a myeloproliferative disorder. Two patients had polycythemia vera in transformation (erythroleukemia in one patient and refractory anemia in the second), whereas the third patient had myelofibrosis which later evolved into acute myelomonocytic leukemia. The two patients who developed overt leukemia did not receive any previous cytotoxic treatment. Non-isotopic in situ hybridization was performed in two patients, allowing for the localization of the breakpoints in 1q12 and 9q12. A similar rearrangement has been previously described in patients with polycythemia vera, either at diagnosis or in advanced stages of the disease. These data suggest that this chromosome abnormality may be consistently associated with myeloproliferative disorders showing a high propensity to transformation, which is not treatment related, and the finding of the +der(1q9p) may represent a poor prognostic sign when observed in the chronic phase.

Effect of recombinant human IL-3 on the mitotic index and karyotype of hemopoietic cells.

The proliferative induction by hemopoietic growth factors may provide a useful tool to improve the mitotic yield of hemopoietic cells, allowing a more accurate cytogenetic analysis in hematologic malignancies. For such a purpose, we studied the effects of the recombinant human IL-3 (rhIL-3) on the mitotic index and the karyotype of bone marrow cells from 14 patients with myelodysplastic (MDS) and myeloproliferative syndromes (MPS). The mitotic response to IL-3 of normal bone marrow samples was also evaluated. Total bone marrow cells were cultured for 24 to 72 hours either in presence or absence of rhIL-3. In most cases, IL-3--stimulated samples showed a considerably higher (4-70 times) mitotic index than unstimulated controls. Although a great patient-to-patient variability was observed, a common pattern of mitogenic response to IL-3 emerged among MPS, MDS, and normal cases. At 48 hours of incubation, the mean mitotic index from MPS and MDS cases stimulated with IL-3 was significantly higher (p less than 0.01) than unstimulated controls, whereas the mean mitotic increase from normal samples did not reach statistical significance (p greater than 0.1). Even though not statistically evaluable, a similar trend of response was observed at 24 and 72 hours of culture. Chromosome studies of MPS and MDS cases showed the same karyotype either in stimulated and unstimulated samples.

Trisomy 8 and an unbalanced t(5;17)(q11;p11) characterize two karyotypically independent clones in a case of idiopathic myelofibrosis evolving to acute nonlymphoid leukemia.

In a patient with idiopathic myelofibrosis (MFI) that had progressed to acute nonlymphoid leukemia (ANLL) after a long-lasting cytotoxic treatment, we observed two karyotypically independent cell populations, one showing trisomy of chromosome 8 as the only anomaly and one with an unbalanced translocation t(5;17)(q11) resulting in partial monosomy of 5q and 17p. The overall karyotypic configuration suggested that chromosome changes occurred as secondary events during the multistep process of leukemogenesis. The probable sequence of cytogenetic events in this patient and a review of the literature indicated that the t(5;17) may represent a therapy-induced abnormality nonrandomly related to the terminal phase of myeloid disorders.

Update on the prognostic implication of morphology, histology, and karyotype in primary myelodysplastic syndromes.

The prognostic value of the FAB classification, bone marrow histology, Bournemouth score, and chromosome findings was studied in 88 patients with primary myelodysplastic syndromes. The median survival for the whole group of patients was 22 months (RA 61.7 months, RARS 31.6 months, CMML 15.7 months, RAEB 10.3 months, and RAEBt 8.2 months). Chromosomal abnormalities were found in 37 of the 70 patients investigated (52%). Only the differences in survival between patients with complex versus normal karyotype were statistically significant (p = 0.02). The presence of small blastic cells, located away from the endosteal surface (abnormal localization of immature blasts or ALIP) appears to be a major prognostic factor in predicting the duration of survival and progression to ANLL, especially in the FAB subgroups RA and RARS. Median survival for the 22 ALIP- cases with RA/RARS was 65 months, compared with 31 months for the ALIP+ cases (p = 0.0006). Nine ALIP+ patients (53%) developed ANLL in contrast to 3 (13%) of the ALIP- cases (p = 0.008). By redefining ALIP and evaluating the number and characteristics of the accompanying cells, histological subtypes were distinguished correlating largely with the FAB subgroups. Our findings demonstrate the prognostic importance of bone marrow biopsy and quantifying the complexity of bone marrow chromosome changes. It should be helpful in evaluating current attempts to find effective treatment for patients with MDS.

Molecular cytogenetic analysis discloses complex genetic imbalance in a t(11;21) myelodysplastic syndrome.

Three cases of t(11;21)(q24;q11.2) myelodysplastic syndromes (MDS) showed karyotypic evolution resulting in the presence of two der(11)t(11;21) without normal chromosome 11 and with partial trisomy 21q. In one of these, we performed further molecular cytogenetic investigations which showed 1) that this rearrangement led to changes in the dosage and location of both c-ets 1 and c-ets 2 protooncogenes; and 2) that the presence of two 11q + chromosomes did not result from a nondisjunction, but that a second chromosome rearrangement had occurred. The final genetic imbalance resulting from this cytogenetic change involves at least hemizygosity for some sequences on the long arm of chromosome 11, including c-ets 1, plus trisomy for the most part of the long arm of chromosome 21, including c-ets 2.

Chromosome abnormalities involving heterochromatic regions in monocytic leukemia.

We report two cases of monocytic leukemia associated with cytogenetic changes involving the juxtacentromeric heterochromatin of different chromosomes. In a patient with chronic myelomonocytic leukemia (CMMoL) we describe a translocation t(1;9)(q12;q13) in which the duplicated derivative chromosome 9q + showed a huge centromeric C-band, derived by fusion of the heterochromatic regions of chromosomes 1 and 9. The constitutional karyotype showed two heterochromatin polymorphisms, 1qh + and inv(9qh). In the second case, an acute monoblastic leukemia was associated with an abnormally elongated juxtacentromeric heterochromatic region of chromosome 4 that was not constitutionally present.

Rearrangement of immunoglobulin and TCR genes in lymphoid blast crisis of Ph+ chronic myeloid leukaemia.

Clonal rearrangements of immunoglobulin heavy chain genes as well as both T cell receptor (TCR) delta and gamma genes were found in four cases of blast crisis of Ph+ chronic myeloid leukaemia with unequivocal B cell precursor (common) immunophenotype. In one case, the TCR beta chain gene was also rearranged. Although the developmental sequence of TCR delta, gamma and beta rearrangements in T lymphocytes appeared to be respected, a full phenotypic effect, characteristic of T cell was not observed in these otherwise typical 'common' blast cells. Cytogenetic analysis ruled out the occurrence of TCR rearrangement due to structural chromosome changes. A high incidence of unexpected TCR gene rearrangements has been previously reported in the de novo 'common' acute lymphoblastic leukaemias (ALL). Our cases of chronic myeloid leukaemia (CML) in lymphoid blast crisis show that genotypic similarities may exist between these two haematological entities.

5q- anomaly in lymphoid disorders.

Observations made in two patients and a review of the literature confirm the occurrence of a 5q- chromosome anomaly in lymphoproliferative disorders of both T and B cell type. Additional chromosome changes were invariably present and are of the "lymphoid" type. The chromosome morphology of the 5q- is indistinguishable from that found in myeloid disorders.

Multipotent stem cell involvement in megakaryoblastic leukemia: cytologic and cytogenetic evidence in 15 patients.

Cytologic and cytogenetic results obtained from patients fulfilling the FAB criteria for the diagnosis of acute nonlymphocytic leukemia (ANLL) of megakaryocytic lineage (ANLL-M7) are reported. Eleven cases were de novo ANLL-M7, of whom three presented with acute myelofibrosis. Four cases were megakaryoblastic transformations of chronic myelogenous leukemia (two cases), refractory anemia with excess of blasts (one case), and polycythemia vera (one case). Four patients showed a minority of granular blasts, with occasional Auer rods in one. Positive myeloperoxidase and/or sudan black-B stainings and CD13 positivity in these cases were consistent with the presence of a myeloid involvement. Morphologic evidence of associated myelodysplastic features was detected in all evaluable patients with de novo ANLL-M7. These cytologic findings indicate that ANLL-M7 may frequently represent a multilineage proliferation. Cytogenetic studies revealed -7/7q- and +8, alone or in combination with additional aberrations, in three cases each. Rearrangements involving bands 3q21 or 3q26 were seen in two patients and +21, as an additional aberration, in one. Other structural rearrangements all observed in a single patient were inv(16)(p13q22) at megakaryoblastic relapse with bone marrow eosinophilia, t(13;20)(q13 or 14;q11), del(20)(q11), and der(7)t(7;17)(p14;q22). Most breakpoints of these aberrations are located at bands frequently rearranged in malignant myeloid stem cell disorders. A review of 31 cases of the literature showed a frequent occurrence of -7/7q- and -5/5q- in ANLL-M7. Many of the chromosome aberrations so far described in ANLL-M7 appear to be shared by a spectrum of myeloid neoplasias and may be related to mechanisms conferring proliferative advantage to undifferentiated stem cells.