Pain Control, Glucose Control, and Quality of Life in Patients With Chronic Pancreatitis After Total Pancreatectomy With Islet Autotransplantation: A Preliminary Report.

BACKGROUND: Total pancreatectomy (TP) is offered as a last treatment option for pain relief in patients with chronic pancreatitis. Concurrent islets autotransplantation (TP-IAT) may improve glucose control. METHODS: We analyzed results in 20 recent patients who underwent TP-IAT at The University of Chicago. The median observation period was 28 months (2-38). Data were collected prospectively then analyzed retrospectively. RESULTS: The number of patients requiring opioids daily for pain control decreased from 16 (80%) prior to surgery to 2 (13%) 1 year after, with only 1 (6.5%) patient experiencing persistent phantom pancreatic pain. Opioid requirements decreased from a median 56.3 (0-240) morphine equivalent dose to 5 (0-130) on day 75 and to 0 (0-30) at 1-year follow up. Five patients (25%) completely stopped insulin support prior to day 75 while maintaining hemoglobin A1c of 5.9% (5-6.3). Eight (53%) patients were insulin free at 1 year with A1c of 6% (5.5-6.8) and a similar rate persisted in next 2 years. For the remaining patients, the more islet function that was preserved, the less insulin they required and A1c was closer to optimal. Quality of Life (QoL) measured by SF36 Physical (PCS) and Mental (MCS) Component Score improved on day 75 (P < .001) and maintained improvement later on. Both PCS and MCS improved regardless of whether patient requires insulin support or not. CONCLUSIONS: Improvements of QoL with pain resolution and good glucose control can be achieved after TP-IAT in properly selected patients with CP and intractable pain, regardless of patient insulin support status.

External Validation of the Newly Developed BETA-2 Scoring System for Pancreatic Islet Graft Function Assessment.

BACKGROUND: BETA-2 score using a single fasting blood sample was developed to estimate beta-cell function after islet transplantation (ITx) and was validated internally by a high ITx volume center (Edmonton). The goal was to validate BETA-2 externally, in our center. METHODS: Areas under receiver operating characteristic curves (AUROCs) were obtained to see if beta score or BETA-2 would better detect insulin independence and glucose intolerance. RESULTS: We analyzed values from 48 mixed meal tolerance tests (MMTTs) in 4 ITx recipients with a long-term follow-up to 140 months (LT group) and from 54 MMTTs in 13 short-term group patients (ST group). AUROC for no need for insulin support was 0.776 (95% confidence interval [CI] 0.539-1, P = .02) and 0.922 (95% CI 0.848-0.996, P < .001) for beta score and 0.79 (95% CI 0.596-0.983, P = .003) and 0.941 (95% CI 0.86-1, P < .001) for BETA-2, in LT and ST groups, respectively, and did not differ significantly. In LT group BETA-2 score ≥ 13.03 predicted no need for insulin supplementation with sensitivity of 98%, specificity of 50%, positive predictive value (PPV) of 93%, and negative predictive value (NPV) of 75%. In ST group the optimal cutoff was ≥13.63 with sensitivity of 92% and specificity, PPV, and NPV 82% to 95%. For the detection of glucose intolerance BETA-2 cutoffs were <19.43 in LT group and <17.23 in ST group with sensitivity > 76% and specificity, PPV, and NPV > 80% in both groups. CONCLUSION: BETA-2 score was successfully validated externally and is a practical tool allowing for frequent and reliable assessments of islet graft function based on a single fasting blood sample.

Oncogenic activity of amplified miniature chromosome maintenance 8 in human malignancies.

Miniature chromosome maintenance (MCM) proteins play critical roles in DNA replication licensing, initiation and elongation. MCM8, one of the MCM proteins playing a critical role in DNA repairing and recombination, was found to have overexpression and increased DNA copy number in a variety of human malignancies. The gain of MCM8 is associated with aggressive clinical features of several human cancers. Increased expression of MCM8 in prostate cancer is associated with cancer recurrence. Forced expression of MCM8 in RWPE1 cells, the immortalized but non-transformed prostate epithelial cell line, exhibited fast cell growth and transformation, while knock down of MCM8 in PC3, DU145 and LNCaP cells induced cell growth arrest, and decreased tumour volumes and mortality of severe combined immunodeficiency mice xenografted with PC3 and DU145 cells. MCM8 bound cyclin D1 and activated Rb protein phosphorylation by cyclin-dependent kinase 4 in vitro and in vivo. The cyclin D1/MCM8 interaction is required for Rb phosphorylation and S-phase entry in cancer cells. As a result, our study showed that copy number increase and overexpression of MCM8 may play critical roles in human cancer development.

MCM7 amplification and overexpression are associated with prostate cancer progression.

The genomic DNA profiles of prostate cancers with aggressive features were compared to the profiles of matched normal DNA to identify genes that are selectively amplified in the cancer cells. One of the identified genes, MCM7, which is a component of the DNA replication licensing complex, has been studied extensively both at the DNA and protein levels in human prostate tissues. Approximately half of the prostate cancer specimens studied showed MCM7 gene amplification, and 60% of the aggressive prostate cancer specimens had increased MCM7 protein expression. Amplification or overexpression of MCM7 was significantly associated with relapse, local invasion and a worse tumor grade. Constitutive expression of MCM7 in a human prostate cancer cell line, DU145, resulted in markedly increased DNA synthesis and cell proliferation compared to vector-only controls, and an increased cell invasion in vitro. Indeed, MCM7 overexpression produced primary tumors 12 times larger than vector-only controls and resulted in a rapid demise of mice bearing those tumors. These studies implicate MCM7, and the DNA replication licensing gene family, in prostate cancer progression, growth and invasion.

Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers.

Prostate cancer is one of the leading causes of cancer-related deaths for men in the United States. Like other malignancies, prostate cancer is underscored by a variety of aberrant genetic alterations during its development. Although loss of heterozygosity or allelic loss is frequently identified among prostate cancers, few genes have been identified thus far as critical to the development of invasive prostate cancers. In this report, we used the recently developed technology, the "differential subtraction chain," to perform a genome-wide search for sequences that are deleted in an aggressive prostate cancer. Among the deleted sequences, we found that one sequence was deleted in >50% of prostate cancers we tested. We mapped this sequence to chromosome 4q25 by screening the Genebridge 4 hamster radiation panel with primers specific to this probe, and subsequently identify a 54-kb minimal common deletion region that contains the sequence encoding myopodin. Sequence analysis indicates that myopodin shares significant homology with synaptopodin, a protein closely associated with podocyte and neuron differentiation. Further study shows that frequent complete or partial deletions of the myopodin gene occurred among invasive prostate cancer cases (25 of 31 cases, or 80%). Statistical analysis indicates that deletion of myopodin is highly correlated with the invasiveness of prostate cancers, and thus may hold promise as an important prognostic marker for prostate cancers.

Involvement of 2p23 in pulmonary inflammatory pseudotumors.

Pulmonary inflammatory pseudotumors (IP) are rare mesenchymal proliferations that have a polymorphic histology and an unpredictable biologic behavior. The histologic spectrum of IP has led to uncertainty as to whether this tumor has a reactive or neoplastic pathogenesis. Reports of extrapulmonary IP have identified clonal chromosomal aberrations involving 2p23 in the region of the ALK gene. Using fluorescence in situ hybridization with a probe flanking the ALK gene at 2p23 and immunostaining for the ALK gene product, we studied formalin-fixed, paraffin-embedded tissues of pulmonary IP and found a subset (33%) with 2p23 aberrations. We suggest that chromosomal rearrangements and ALK immunostaining may be helpful in the diagnosis of a group of pulmonary IP and should be investigated as a potential tool for predicting their future biologic behavior. An association with anaplastic large-cell lymphoma was also observed. HUM PATHOL 32:428-433.

Comparative genomic hybridization of hepatocellular carcinoma: correlation with fluorescence in situ hybridization in paraffin-embedded tissue.

BACKGROUND: Proto-oncogene MYC, mapped to chromosomal band 8q24 and the genes for hepatocyte growth factor (HGF at 7q21) and its receptor, MET, at chromosomal band 7q31, have an important role in the biology and growth of normal and neoplastic liver. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies have reported frequent abnormalities of chromosomes 1 and 8 in hepatocellular carcinomas (HCCs) of various clinical and pathological stages. Chromosome 7 involvement is reported to be less frequent. MATERIALS AND METHODS: Frozen tissue from 17 HCCs was used for CGH analysis and sections of corresponding formalin-fixed, paraffin-embedded HCC tissue were used for dual-color FISH with locus-specific (LSI-cMYC for chromosome 8q24 and LSI D7S486 for chromosome 7q31) and centromeric probes, CEP8 (8p11.1-q11.2) and CEP7 (7p11.1-q11.2) (Vysis, Inc, Downers Grove, IL). This study intended to determine the pattern of chromosomal aberrations in early-stage (incidental) HCC and large surgically resected HCC, and also compared the efficiency and usefulness of the two cytogenetic methods. RESULTS: CGH showed abnormalities on chromosomes 1q, 5q, 7q, 8q, 9, 10, 13q, 15, 16, 17p, 18q, 19, 20, 21, 22, and X. Gains of 8q were noted in 50% of the HCCs, including five cases of incidental HCCs by CGH. Increase in copy numbers of MYC detected by FISH was noted in 25% of tumors that had shown 8q gains by CGH and in five cases with no chromosome abnormalities noted by CGH. Three cases with 7q31 copy number abnormalities were found by FISH in addition to those detected by CGH. CONCLUSION: Combined use of CGH and FISH may provide important information about early and/or primary genetic changes in the development of HCC.

Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH studies.

Chromosome translocations have been known to affect disjunction of chromosomes unrelated to the translocation in the mouse and in Drosophila. However, in humans, an interchromosomal effect in chromosome translocations has not been demonstrated. The availability of techniques that allow the study of nondisjunction in sperm cells has permitted us to evaluate the possibility of an interchromosomal effect in male translocation heterozygotes. In this study, multicolor fluorescence in situ hybridization was used to determine levels of disomy for the clinically relevant chromosomes X, Y, 13, 18, and 21 in 332,858 spermatozoa from nine reciprocal translocation heterozygotes and nine controls with normal karyotypes. The specific translocations studied were as follows: t(10;12)(p26.1;p13.3), t(2;18)(p21;q11.2), t(3;19)(p25;q12), t(5;8)(q33;q13), t(11;22)(q23;q11), t(3;4)(p25;p16), t(8;9) (q24.2;q32), t(10;18)(q24.1;p11.2), and t(4;10)(q33;p12.2). Comparisons of disomy rates between carriers and controls were performed by using the Mann-Whitney test. Our results showed that the rates of sex chromosome hyperhaploidy were similar in controls (0.21%) and in translocation carriers (0.19%). Similarly, the frequencies of disomy for chromosomes 13, 18, and 21 did not differ significantly between controls and carriers (0.05% versus 0.08%, 0.07% versus 0.03%, and 0.14% versus 0.20%, respectively). Sex chromosome nondisjunction was more common than nondisjunction of chromosomes 13 and 18 both in controls (P=0.0057) and in carriers (P=0.0008). Similarly, the rates of chromosome disomy for chromosome 21 were higher than those for chromosomes 13 and 18 in both controls (P=0.0031) and translocation carriers (P=0.0057). In our study, the excess of chromosome 21 disomy versus disomy of the other autosomes was more pronounced in carriers than in controls. Thus, although the difference of disomy 21 between controls and carriers was not statistically significant, it is worthy of attention.

Multicolor fluorescence in situ hybridization analysis of the spermatozoa of a male heterozygous for a reciprocal translocation t(11;22)(q23;q11).

A reciprocal translocation between chromosomes 11 and 22 is a site-specific translocation that has been seen in many families with no common ancestry. This translocation is of particular interest because balanced carriers have a 0.7-3.7% risk of having children with the supernumerary der(22), resulting from a 3:1 segregation. We have used a three color fluorescence in situ hybridization (FISH) with specific DNA probes to determine the chromosome segregation pattern of a male carrier of a translocation t(11;22)(q23;q11). The probes selected included a centromeric marker for chromosome 11, a marker closely linked to the centromere of chromosome 22, and a third probe distal to the translocation breakpoint of chromosome 22. The results showed that 3:1 segregation is preferential in this patient, with 40.1% of spermatozoa belonging to this segregation type. Alternate segregation followed with 27.4% of analyzed spermatozoa; 17.6% resulted from adjacent 1 and 12.5% resulted from adjacent 2 segregation. We detected 0.5% of presumably diploid spermatozoa. Complementary adjacent 1 products were observed at statistically different frequencies (P = 0.02). Complementary adjacent 2 products without recombination in the interstitial segments were also seen at different frequencies (P = 0.002). In 3:1 segregation, the products containing one chromosome were observed more frequently than those with three chromosomes (P = 0.0001). The 24,+der(22) gamete was seen more frequently than all of the other gametes combined which had 24 chromosomes resulting from 3:1 segregation. The results of this study demonstrate that in this t(11;22) carrier, 3:1 segregation is preferential but not exclusive.

Meiotic products of a Klinefelter 47,XXY male as determined by sperm fluorescence in-situ hybridization analysis.

The meiotic segregation of 24 spermatozoa obtained from a 47,XXY male is described. Three-colour fluorescence in-situ hybridization with probes for chromosomes X, Y and 18 was used. Five spermatozoa carried an X chromosome, seven carried a Y, six had an XY gonosomal complement, five were missing the sex chromosome and one spermatozoon was presumably diploid with an XX/1818 complement. Our results support the hypothesis that XXY cells are able to complete meiosis. In this patient, the percentage of spermatozoa with an abnormal number of sex chromosomes increased from 1/6 (17%) among spermatozoa with normal morphology to 11/18 (61%) in spermatozoa with abnormal morphology.

Meiotic products of two reciprocal translocations studied by multicolor fluorescence in situ hybridization.

The sperm products of two male carriers of reciprocal translocations were studied by fluorescence in situ hybridization (FISH) using a combination of three probes for each translocation. One patient carried a t(2;18)(p21;q11.2), the other a t(8;9)(q24.2;q32). The probes selected included a centromeric marker for each chromosome involved in the translocation plus a third probe distal to the translocation breakpoint of one of the translocation chromosomes. This assay identifies alternate, adjacent 1, adjacent 2, and 3:1 types of meiotic products. It allows the identification of recombination events and also estimation of the frequency of diploidy. For the t(2;18), the frequency of normal and balanced sperm and of adjacent 1, adjacent 2, and 3:1 products was 43.6%, 29. 8%, 10.5%, and 12.8%, respectively. Similar segregation patterns had been reported for this donor by direct sperm karyotyping of sperm cells. For the t(8;9), the frequency of normal and balanced sperm and of adjacent 1, adjacent 2, and 3:1 products was 44.4%, 41%, 3.1%, and 9.4%, respectively. The frequency of complementary adjacent 1 products was statistically different in both the t(2;18) (P < 0. 0001) and the t(8;9) (P < 0.0001) carrier. When the number of adjacent 2 products with one translocation chromosome (regardless of normal or derivative) was compared to the number of adjacent 2 products with the second translocation chromosome (again, regardless of normal or derivative), no statistical difference was noted for either the t(2;18) (P = 0.32) or the t(8;9) (P = 0.69). Recombination events within the interstitial segment of chromosome 2 were statistically higher than those seen in chromosome 18 (P < 0. 0001), whereas in chromosomes 8 and 9, recombination in the interstitial segments was similar (P = 0.64). The rate of diploidy was similar in both the t(2;18) (0.5%) and the t(8;9) (0.6%). Thus, FISH provides chromosome information on the sperm products produced by translocation carriers, although it cannot provide an assessment of the full chromosome complement of the spermatozoon.

A Ph-negative chronic myeloid leukemia patient with a non-classical BCR-ABL rearrangement characterized by fluorescence in situ hybridization.

A patient with chronic myeloid leukemia (CML), a normal karyotype and a BCR-ABL rearrangement is presented. Southern blot analysis detected the rearrangement, whereas RT-PCR with b2a2 and b3a2 primers did not. Fluorescence in situ hybridization (FISH) with an ABL probe (9q34.2) and an Mbcr probe (22q11) showed ABL and BCR signals on chromosome 22. Subsequent FISH studies with cosmids mapping to 9q34.3 showed normal hybridization patterns to chromosome 9, suggesting an interstitial insertion of ABL containing DNA sequences into chromosome 22 in this patient. The lack of reciprocal translocation sequences was investigated with RT-PCR, primers a1b and c7. The absence of ABL-BCR gene expression in this and other patients described in the literature with this subtype of Ph-negative CML, does not seem to have an impact on the clinical course of the disease.

The meiotic segregation pattern of a reciprocal translocation t(10;12)(q26.1;p13.3) by fluorescence in situ hybridization sperm analysis.

The meiotic segregation of chromosomes 10 and 12 was analyzed in a male heterozygous for a reciprocal translocation, t(10;12)(q26.1;p13.3), using fluorescence in situ hybridization (FISH). Centromeric specific probes that detect alpha satellite sequences of chromosomes 10 and 12 were used. A total of 10,049 spermatozoa were analyzed. The frequencies of alternate/adjacent 1, adjacent 2, and 3:1 modes of segregation were: 84.25, 10.95%, and 4.42%, respectively. Diploidy was present in 0.23% of spermatozoa. Similar segregation patterns have been reported for this donor by direct karyotyping of sperm cells. FISH is a valuable technique for studying meiotic segregation patterns in that larger samples can be studied in a relatively short time. However, it does not provide information on the full chromosome complement of the spermatozoon.

An analysis of human sperm chromosome aneuploidy.

A sperm chromosome analysis of 24 men with normal or balanced karyotypes was carried out to study the frequency of sperm chromosome aneuploidy. A total of 3,446 human sperm complements (36-315 per donor) was analyzed after in vitro penetration of hamster eggs. Two sets of donors were studied at two different centers in the United States (center 1) and Spain (center 2). The frequencies of hyperhaploidy and hypohaploidy in control donors were similar between center 1 (1.9% vs. 7.7%) and center 2 (1.8% vs. 10.3%). In carrier donors there were no significant differences between the two centers in the frequency of hyperhaploidy (0.8% vs. 1.9%), but that of hypohaploidy was significantly higher in center 2 (11.0%) than in center 1 (4.6%). A significant excess of hypohaploid complements, as compared to hyperhaploid complements, was found in both centers in both control and carrier donors. The sex ratio was similar in both centers and did not differ significantly from a 1:1 sex ratio. The larger chromosomes in the complement (1, 2, 3, 4, 5, 7, and 10) presented a significantly lower frequency of hypohaploidy, while some of the smaller chromosomes (13, 19, and 21) showed a higher frequency of hypohaploidy than expected. Chromosome 21 and the sex chromosomes showed an increase in the percentage of hyperhaploidy, as compared to other chromosomes, that was close to statistical significance (P = 0.08). Our results reflect a preferential loss of small chromosomes during slide preparation and suggest that chromosome 21 and the sex chromosomes could be more frequently involved in aneuploidy.

An analysis of human sperm chromosome breakpoints.

Sperm chromosome analysis of 19 sperm donors with either normal or balanced karyotypes was carried out in order to explore the nature of sperm chromosome structural aberrations. A total of 2,389 cells (range 36-298/donor) were karyotyped after in vitro penetration of hamster eggs. The median percentage of sperm structural aberrations was 9.3% (SD +/- 4.7; range 0%-17.8%), with a total of 247 breakpoints, of which 220 could be characterized fully. Two sets of donors were studied in two different centers: center 1 (United States) and center 2 (Spain). The frequencies of nonrejoined and rejoined chromosome-type aberrations were very similar between center 1 and center 2: 83.6% and 10.0%, and 75.0% and 10.3%, respectively. Chromatid-type aberrations were more frequent in center 2 (14.7%) than in center 1 (6.4%) (P = .037). Chromosome 4 had less than the expected number of breakpoints (P < .001). A positive significant correlation was found between sperm breakpoints reported in this study and sites of balanced chromosome de novo rearrangements detected at prenatal diagnosis and reported in the literature (P = .0001).

Segregation analysis of four translocations, t(2;18), t(3;15), t(5;7), and t(10;12), by sperm chromosome studies and a review of the literature.

We examined the meiotic segregation patterns of 444 sperm cells belonging to four reciprocal translocation carriers, t(2;18)(p21;q11.2), t(3;15)(q26.2; q26.1), t(5;7)(q13; p15.1), and t(10;12)(q26.1;p13.3). For the t(2;18) carrier, the frequencies of alternate, adjacent-1, adjacent-2, and 3:1 segregations were 41.9%, 35.2%, 14.4%, and 8.4%, respectively. For the t(3;15) carrier, the segregation pattern was 48% alternate, 36% adjacent-1, 12% adjacent-2, 2% 3:1, and 2% 4:0. One cell was the result of a 4:0 segregation. For the t(5;7) heterozygote, the corresponding segregation frequencies were 40.2%, 26.2%, 16.6%, and 17.0%. This translocation heterozygote showed a higher number of 3:1 segregations than adjacent-2 segregations, which is unusual. The t(10;12) segregations were 61.1%, 26.3%, 6.9%, and 5.6%. The percentages of chromosome abnormalities unrelated to the translocation ranged from 0% to 0.6% for aneuploidy and from 5.5% to 10.9% for structural abnormalities. These frequencies are within the ranges for control donors. Sperm chromosome data from the literature on the segregation of 30 reciprocal translocations were reviewed.

The segregation of a translocation t(1;4) in two male carriers heterozygous for the translocation.

We examined the meiotic segregation pattern of a t(1;4)(p36.2;q31.3) reciprocal translocation in two male cousins heterozygous for the translocation. The wife of subject 1 had four recognized spontaneous abortions and two carrier daughters, and the wife of subject 2 had three recognized spontaneous abortions and no live-born children. The results showed that subject 1 had an imbalance rate of 54% and subject 2 had an imbalance rate of 61% with respect to the translocation. This was not statistically different (P = 0.3174) and the 95% confidence intervals overlapped for each segregation type. The sex ratio of X- and Y-bearing sperm was not statistically different than the expected 50%. The rate of structural abnormalities was 11.3% in subject 1 and 17.8% in subject 2. Both of these values were above the range of control subjects in our lab, but only subject 2's value fell outside the 95% confidence interval for the control population.

Cytogenetic studies in human sperm.

Sperm chromosome studies were performed in seven males. One of them had a history of exposure to lysergic acid (LSD) although he was free of the drug for 1 year before the study began. Sixteen ejaculates provided a total of 555 fully analyzable sperm cells. The overall frequency of hyperhaploid sperm cells was 2% and that of structural abnormality 3.6%. The most common structural abnormality was chromosome breaks followed by small chromosome fragments of unknown origin. Three chromosome breakpoints, 10q25, 2q21, and 9q21, were involved twice in different chromosome or chromatid type aberrations. Two of these, 10q25 and 2q21, correspond to chromosomal locations known as common fragile sites.