PAX5 P80R-mutated B-cell acute lymphoblastic leukemia with transformation to histiocytic sarcoma: clonal evolution assessment using NGS-based immunoglobulin clonality and mutation analysis.

Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.

How we do: optimizing bone marrow biopsy logistics for sign-out within 2 days.

Since the introduction of fast diagnostic tracks in many areas of oncology, the traditional processing of bone marrow biopsies (BMB), requiring either resin embedding or lengthy fixation and decalcification, is due to an upgrade. Thanks to a growing number of new commercially available tissue processors, microwave-enhanced processing is becoming a standard tool in the pathology laboratory, allowing rapid fixation and decalcification of BMB with preserved morphology and antigens. In this short report, we describe the use of a commercially available EDTA-based decalcification fluid (USEDECALC, Medite, Orlando, USA) in combination with the LOGOS J (Milestone, Bergamo, Italy), a closed microwave-enhanced tissue processor, for overnight fixation, decalcification, and paraffin impregnation of the BMB. This allows next-day reporting without impaired morphology or immunohistochemistry, and even improved DNA quality of the BMB.

A 20-year population-based study on the epidemiology, clinical features, treatment, and outcome of nodular lymphocyte predominant Hodgkin lymphoma.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a subtype of Hodgkin lymphoma characterized by a unique clinical and histological presentation. Because of the rare nature of this disease, few large-scale studies are available. We conducted a cohort study in which patients were identified in the Netherlands Cancer Registry in the Southeast of the Netherlands between 1990 and 2010. Of these patients, we collected all clinical characteristics and re-reviewed pathologic material to confirm NLPHL diagnosis. Seventy-three histologically confirmed cases of NLPHL were analyzed with a median follow-up of 65 months (range 4-257 months). Median age at diagnosis was 43 years (range 1-87); 84.9 % of the patients were male; B symptoms were present in 5.5 %; and stage I/II disease was most common (75.4 %). Patients were primarily treated with radiotherapy (50.7 %), chemotherapy (26 %), combined modality (radiotherapy and chemotherapy) (11 %), or surgical excision with careful watch-and-wait (12.3 %). Relapses occurred in seven patients (9.6 %) after a median of 26 months (21-74 months). Six patients (8.2 %) developed histologic transformation to large cell lymphoma. Five patients (6.8 %) died during follow-up due to progression of NLPHL (n = 1), histologic transformation (n = 2) and intercurrent deaths (n = 2). The estimated 10-year overall survival was 94.0 % and the 10-year progression-free survival 75.8 %. Our study confirms the distinct characteristics of NLPHL with a relatively good long-term prognosis. It may be possible to reduce treatment intensity in early stage NLPHL without affecting long-term outcome.

Young age and a positive family history of colorectal cancer are complementary selection criteria for the identification of Lynch syndrome.

Families at high risk for Lynch syndrome can effectively be recognised by microsatellite instability (MSI) testing. The aim of the present study is to compare the effectiveness of a MSI test for the identification of Lynch syndrome in patients selected by a pathologist mainly based on young age at diagnosis (MSI-testing-indicated-by-a-Pathologist; MIPA), with that of patients selected by a clinical geneticist mainly based on family history (MSI-testing-indicated-by-Family-History; MIFH). Patients with a Lynch syndrome associated tumour were selected using MIPA (n=362) or MIFH (n=887). Germline DNA mutation testing was performed in 171 out of 215 patients (80%) with a MSI positive tumour. MSI was tested positive in 20% of the MIPA-group group compared to 16% in the MIFH-group (P=0.291). In 91 of 171 patients with MSI positive tumours tested for germline mutations were identified as Lynch syndrome patients: 42% in the MIPA-group and 56% in the MIFH-group (P=0.066). Colorectal cancer (CRC) or endometrial cancer (EC) presenting at an age below 50 years would have led to the diagnosis of Lynch syndrome in 89% of these families (CRC below 50 years: 88% and EC below 50 years: 12%). Families detected by MIPA were characterised more often by extracolonic Lynch syndrome associated malignancies, especially EC (P<0.001). Our results indicate that recognition of Lynch syndrome by CRC or EC below 50 years is as effective as a positive family history. Families from patients selected by individual criteria more often harbour extracolonic Lynch syndrome associated malignancies.

PCR clonality detection in Hodgkin lymphoma.

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein-Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.

[Recognition of congenital endometrial carcinoma: the importance of family history and investigation of microsatellite instability in the tumour]. / Herkennen van erfelijk endometriumcarcinoom: het belang van familieanamnese en onderzoek naar microsatellietinstabiliteit in de tumor.

Endometrial cancer diagnosed at a relatively young age or in a patient with a medical history of colorectal cancer may be indicative of Lynch syndrome. Four women, aged 43, 60, 41 and 54 respectively, with a family history of endometrial or colorectal neoplasm were examined for microsatellite instability (MSI) in tumour tissue with positive results. Subsequently, a mutation was found in one of the DNA mismatch repair genes. Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC), is caused by a germline mutation in a mismatch repair gene and is an autosomal dominant disorder that is characterized by the development of carcinoma of the endometrium and colorectum at a relatively young age. Until recently, recognition of Lynch syndrome was mainly based on an, often incomplete, family history, but today the presence of MSI in tumour tissue can be used to identify patients at risk for Lynch syndrome. A pathologist can contribute to identifying a patient at risk for Lynch syndrome by initiating MSI testing when: (a) endometrial cancer is diagnosed under the age of 50, (b) a combination of endometrial cancer and colorectal cancer is diagnosed under the age of 70.

Patients with an unexplained microsatellite instable tumour have a low risk of familial cancer.

The cancer risk is unknown for those families in which a microsatellite instable tumour is neither explained by MLH1 promoter methylation nor by a germline mutation in a mismatch repair (MMR) gene. Such information is essential for genetic counselling. Families suspected of Lynch syndrome (n = 614) were analysed for microsatellite instability, MLH1 promoter methylation and/or germline mutations in MLH1, MSH2, MSH6, and PMS2. Characteristics of the 76 families with a germline mutation (24 MLH1, 2 PMS2, 32 MSH2, and 18 MSH6) were compared with those of 18 families with an unexplained microsatellite instable tumour. The mean age at diagnosis of the index patients in both groups was comparable at 44 years. Immunohistochemistry confirmed the loss of an MMR protein. Together this suggests germline inactivation of a known gene. The Amsterdam II criteria were fulfilled in 50/75 families (66%) that carried a germline mutation in an MMR gene and in only 2/18 families (11%) with an unexplained microsatellite instable tumour (P<0.0001). Current diagnostic strategies can detect almost all highly penetrant MMR gene mutations. Patients with an as yet unexplained microsatellite instable tumour likely carry a different type of mutation that confers a lower risk of cancer for relatives.

Very low prevalence of germline MSH6 mutations in hereditary non-polyposis colorectal cancer suspected patients with colorectal cancer without microsatellite instability.

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by mutations in one of the mismatch repair genes MLH1, MSH2, MSH6, or PMS2 and results in high-level microsatellite instability (MSI-high) in tumours of HNPCC patients. The MSI test is considered reliable for indicating mutations in MLH1 and MSH2, but is questioned for MSH6. Germline mutation analysis was performed in 19 patients with an MSI-high tumour and absence of MSH2 and/or MSH6 protein as determined by immunohistochemistry (IHC), without an MLH1 or MSH2 mutation, and in 76 out of 295 patients suspected of HNPCC, with a non-MSI-high colorectal cancer (CRC). All 295 non-MSI-high CRCs were analysed for presence of MSH6 protein by IHC. In 10 patients with an MSI-high tumour without MSH2 and/or MSH6 expression, a pathogenic MSH6 mutation was detected, whereas no pathogenic MSH6 mutation was detected in 76 patients with a non-MSI-high CRC and normal MSH6 protein expression. In none of the 295 CRCs loss of MSH6 protein expression was detected. The prevalence of a germline MSH6 mutation is very low in HNPCC suspected patients with non-MSI-high CRC. Microsatellite instability analysis in CRCs is highly sensitive to select patients for MSH6 germline mutation analysis.

FISH analysis of MALT lymphoma-specific translocations and aneuploidy in primary cutaneous marginal zone lymphoma.

Primary cutaneous marginal zone lymphomas (PCMZL) share histological and clinical characteristics with mucosa-associated lymphoid tissue (MALT) lymphomas suggesting a common pathogenesis. A number of recurrent structural and numerical chromosomal aberrations have been described in MALT lymphoma, but their incidence in PCMZL is largely unknown, as is their relation with clinical and pathological data. In this study, the incidence of t(11;18)(q21;q21), t(1;14)(p22;q32), two different t(14;18)(q32;q21), involving either IGH/MALT1 or IGH/BCL2, and numerical aberrations of chromosomes 3, 7, 12 and 18 were analysed in 12 patients with PCMZL, with follow-up of up to 10 years. Nuclei were isolated from paraffin wax sections for dual-colour interphase fluorescence in situ hybridization (FISH) using various probe sets either flanking or spanning the involved genes. T(14;18)(q32;q21), with breakpoints in IGH and MALT1, was found in three cases. All three had partly monocytoid histological appearances and lacked blastic transformation. An additional trisomy of chromosome 3 was detected in one of these cases. Trisomy 18 was present in two lymphomas without monocytoid morphology. No definite correlation was seen with any clinical feature, including Borrelia serology. Neither t(11;18)(q21;q21), nor t(1;14)(p22;q32) or any other translocation involving IGH, BCL10, MALT1, BCL2 and API2, amplification or deletion of chromosomal region 11q21, 18q21, 1p22, and 14q32 was detected. These results indicate that a subgroup of PCMZL with partly monocytoid morphology is genetically related to MZL at other extranodal sites.

Lack of correlation between numbers of circulating t(14;18)-positive cells and response to first-line treatment in follicular lymphoma.

In follicular lymphoma, the t(14;18) status of the peripheral blood and bone marrow analyzed by polymerase chain reaction (PCR) is assumed to correlate with disease activity in patients with relapsed disease. The clinical significance of quantitating circulating lymphoma cells by real-time PCR is reported in patients on first-line treatment. Thirty-four consecutive patients with previously untreated follicular lymphoma and detectable t(14;18)-positive cells in pretreatment peripheral blood samples were monitored. All patients were treated with standard chemotherapy in combination with interferon alfa-2b. Before and after induction therapy, blood samples were taken for quantitative analysis of t(14;18). At presentation, a median of 262 t(14;18)-positive cells per 75,000 normal cells was found (range, 1-75 000). Patients with lower numbers of circulating tumor cells more frequently had bulky disease (P =.02). Seventy-nine percent of the patients responded clinically to treatment. In 22 of 28 patients, including 4 patients in whom treatment had failed clinically, the number of circulating t(14;18)-positive cells decreased to undetectable or low levels after therapy. In the remaining responding patients, circulating tumor cells persisted after therapy. These quantitative data on circulating t(14;18)-positive cells call into question the usefulness of molecular monitoring of the blood in a group of patients with follicular lymphoma uniformly treated with a noncurative first-line regimen. T(14;18)-positive cells decreased in peripheral blood after treatment, irrespective of the clinical response. Therefore, the significance of so-called molecular remission should be reconsidered in follicular lymphoma. (Blood. 2001;98:940-944)

A case of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma presenting with spontaneous splenic rupture: an extremely unusual presentation.

In a 22-year-old male with a 10-day history of fever, painful swelling in the left groin, and abdominal complaints, emergency surgery was performed because of spontaneous splenic rupture. At histology, a cellular infiltrate of intermediate-sized atypical lymphocytes was seen in the splenic white pulp, staining for T-cell markers. In addition, CD30 and anaplastic lymphoma kinase 1 (ALK) were diffusely positive, thus, representing a case of anaplastic large cell lymphoma (ALCL), T-cell, ALK-positive, small cell monomorphic variant. ALK-positive ALCL patients generally bear a much better prognosis than patients with T-cell lymphomas, unspecified, or ALK-negative ALCL. Therefore, besides the very unusual clinical presentation, this case highlights the importance of immunostaining for CD30 and ALK in all T-cell lymphomas. This report is the first extensive description of ALK-positive ALCL involvement of the spleen.

Short- and long-term normal tissue damage with photodynamic therapy in pig trachea: a fluence-response pilot study comparing Photofrin and mTHPC.

The damage to normal pig bronchial mucosa caused by photodynamic therapy (PDT) using mTHPC and Photofrin as photosensitizers was evaluated. An endobronchial applicator was used to deliver the light with a linear diffuser and to measure the light fluence in situ. The applied fluences were varied, based on existing clinical protocols. A fluence finding experiment with short-term (1-2 days) response as an end point showed considerable damage to the mucosa with the use of Photofrin (fluences 50-275 J cm(-2), drug dose 2 mg kg(-1)) with oedema and blood vessel damage as most important features. In the short-term mTHPC experiment the damage found was slight (fluences 12.5-50 J cm(-2), drug dose 0.15 mg kg(-1)). For both sensitizers, atrophy and acute inflammation of the epithelium and the submucosal glands was observed. The damage was confined to the mucosa and submucosa leaving the cartilage intact. A long-term response experiment showed that fluences of 50 J cm(-2) for mTHPC and 65 J cm(-2) for Photofrin-treated animals caused damage that recovered within 14 days, with sporadic slight fibrosis and occasional inflammation of the submucosal glands. Limited data on the pharmacokinetics of mTHPC show that drug levels in the trachea are similar at 6 and 20 days post injection, indicating a broad time window for treatment. The importance of in situ light dosimetry was stressed by the inter-animal variations in fluence rate for comparable illumination conditions.

Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination.

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.

Damage to tumour and brain by interstitial photodynamic therapy in the 9L rat tumour model comparing intravenous and intratumoral administration of the photosensitiser.

In the 9L rat brain tumour model the damage to tumour and normal brain by photodynamic therapy after intratumoural photosensitizer administration (intratumoural PDT) was studied. Twenty four rats received an intratumoural injection of 4 or 40 mm3 haematoporphyrin derivative (HpD, 5 mg ml-1), followed by interstitial irradiation with 20 Joule (J) (630 nm) 5 h later. For comparison, seven rats were treated with 20 Joule 24 h after an intravenous injection of 10 mg kg-1 HpD (intravenous PDT). With the chosen PDT parameters there was no important difference between the damaged areas produced by intratumoural PDT or intravenous PDT. No selective tumour kill was observed. Even though normal brain tissue was heavily damaged, vital tumour parts were still present. Intravenous PDT caused extensive diffuse damage to small blood vessels in tumour and surrounding normal brain. Intratumoural PDT was characterised by an infiltration of polymorphonuclear cells into damaged tissue, dilatation of larger blood vessels and gross haemorrhage. These results suggest an immediate vascular shutdown in the intravenous approach, while in the intratumoural approach the vasculature remained patent initially. Because of the severe side effects observed, the use of HpD seems not advisable for intratumoural PDT of brain tumours.

5-Aminolevulinic acid induced endogenous porphyrin fluorescence in 9L and C6 brain tumours and in the normal rat brain.

A new approach in photodynamic therapy is the use of endogenous porphyrins for sensitisation of tumours to light. The induction of endogenous porphyrins after intravenous injection of 5-aminolevulinic acid (ALA, 200 mg kg-1) was studied in 23 rats, bearing intracranial 9L or C6 tumours. After 0, 2, 4, 6, 8, and 22 hours the rats were sacrificed and the fluorescence distribution of endogenous porphyrins was studied in brain tissue sections with a standard fluorescence microscope and a confocal laser scanning microscope. The role of blood-brain barrier disruption on porphyrin production was studied in 2 rats with a cryo-lesion of the cortex. Additionally, 9L and C6 tumour cell cultures were incubated with ALA for 8 hours in vitro. Fluorescence was measured with a fluorescence spectrophotometer in cell cultures and in the brain sections. Porphyrins were detected in vitro in the tumour cells from 2 hours onwards and ex vivo in the tumour sections mainly from 2 to 8 hours, by 22 hours porphyrin fluorescence had almost disappeared. The contralateral brain showed low fluorescence levels between 2 and 6 hours after ALA administration. At the site of the cryo-lesions low fluorescence was measured 6 hours after ALA administration. The 9L tumours fluoresced homogeneously, with a sharp demarcation towards normal brain tissue. Fluorescence in the C6 tumours was patchy, with a poorly fluorescing edge. In both tumour models fluorescence was also detected in brain surrounding the tumour and sometimes in contralateral white matter and ventricle ependyma and pia mater. The slight increase of porphyrin fluorescence in the normal brain of tumour bearing rats, compared to the absence of this in rats without a tumour, was attributed to transport by bulk flow of porphyrins made in the tumours, and possibly also of circulating porphyrins or ALA leaking from the tumour vessels.

Photodynamic therapy in AIDS-related cutaneous Kaposi's sarcoma.

For evaluating the role of photodynamic therapy (PDT) in the local treatment of acquired immune deficiency syndrome (AIDS)-related cutaneous Kaposi's sarcoma (KS), nine treatments were performed in eight human immunodeficiency virus-positive homosexual men. The patients received 2 mg Photofrin/kg and either 120 J/cm2 (n = 5) or 70 J/cm2 (n = 4) laser light (630 nm). A total of 83 lesions were evaluable for response with a follow-up of 3-8 months. The overall response rates by patient for all treated lesions were 50-100% (120 J/cm2) and 83.3-90.3% (70 J/cm2), with a median duration of 3 months (range, 2-6 months). Tumors located at the head had higher response rates than those at the trunk or extremities (p = 0.005 and p - 0.015 respectively). The size of the KS showed a negative relationship with the probability of complete response (p = 0.047). Local and general side effects occurred, including pain, blisters, temperature increase, muscle stiffness, and severe edema. The cosmetic result was unsatisfactory because of a high prevalence of scars and long-lasting hyperpigmentation. Although the response rates of PDT are high, light dose of 70-120 J/cm2 cannot be recommended in the treatment of cutaneous KS in combination with 2 mg/kg Photofrin because of severe side effects and unsatisfactory cosmetic result.

Fluorescence localization in tumour and normal brain after intratumoral injection of haematoporphyrin derivative into rat brain tumour.

In the intracerebral 9L rat gliosarcoma, the spatial distribution of the photosensitizer haematoporphyrin derivative (HpD) was studied after intratumoral injection. The fluorescence volume was measured in histological sections from 10 min up to 5 days after injection. Complete sensitization of the tumours could not be achieved by slow stereotactical injection of 4 mm3 HpD (mean HpD fluorescence volume, 13 +/- 11 mm3). Larger parts of the tumour could be loaded with HpD (39 +/- 23 mm3, p = 0.0001) by increasing the injection velocity and the volume to 50 mm3. Again, complete sensitization of the tumours was not achieved during a time scale of 5 days after intratumoral injection. Although the fluorescence volume did not change significantly with time, it was influenced by the injection site within the tumour. Injection of HpD within 1 mm from the tumour border resulted in significantly smaller fluorescence volumes in the tumour than injection into the tumour centre. Large injection volumes caused an increased leakage of HpD to normal brain, leading to the loss of selectivity of photosensitizer content and the occurrence of dark toxicity of normal brain while the tumours still appeared vital.